二苯乙烯苷对阿霉素心脏毒性的体内外保护作用及机制
|
摘要
第一部分二苯乙烯苷对阿霉素心脏毒性的体内外保护作用及机制
阿霉素(DOX)是一种高效广谱的葸环类抗肿瘤抗生素，心脏毒性是阿霉素临床应用的主要不良反应之一，因出现剂量依赖性的严重心力衰竭而使它的应用受到限制。DOX所致心脏毒性的机制至今仍未完全阐明，但有许多证据表明其与产生的大量自由基密切相关。2，3，5，4’-四羟基二苯乙烯-2-O-β-D葡萄糖苷(2，3，5，4’-tetrahydroxystilbene-2-O-β-D-glucoside，THSG)是何首乌的活性单体，体外实验研究表明THSG具有较强的抗氧化活性。已有的研究也证实THSG能够降低阿尔茨海默病(Alzheimer's Disease)模型小鼠和缺血再灌注损伤模型小鼠组织中的过氧化物水平。THSG的化学结构与白藜芦醇相似，后者具有显著的保护受损心肌的作用。所以我们推测THSG对DOX导致的心脏毒性具有保护作用。
论文将分别以体外培养的乳鼠心肌细胞和DOX诱导的小鼠急性心脏毒性模型为对象探讨THSG的保护作用及机制。
§1 THSG对小鼠急性阿霉素心脏毒性的保护作用及机制
1．THSG对急性阿霉素心脏毒性的保护作用
方法：一次性腹腔注射DOX 15mg／kg诱导急性心脏毒性模型，给予THSG 0．1g／kg灌胃一天1次，连续7天。观察THSG对小鼠阿霉素心脏毒性的保护作用。
结果：①体重和生存率：处理前各组小鼠体重无明显差别。处理第7天，DOX组小鼠体重(21．8±3．6 g)和空白对照组(25．6±2．6 g，P＜0．01)比较明显减轻；给予THSG(0．1g／kg·d)治疗后没有增加造模小鼠的体重(22．5±2．8 g，P＞0．05 compared with DOXalone)。处理7天后，3组小鼠心脏指数(心脏重量与体重的比值)分别为空白对照组：4．5±0．6mg/g；DOX组：4．3±0．7mg/g；THSG组：4．3±0．4 mg/g，统计学分析均无明显差异。对照组20只动物全部存活，DOX处理7天后动物存活70％，给予THSG治疗后动物存活率升高为85％(P＞0．05，log rank test)。②组织学观察：正常对照组小鼠心肌纤维完整，排列整齐，无出血和水肿。DOX模型组可见明显的心肌纤维断裂甚至消失，心肌间质水肿并伴有局灶性心内膜下出血。而THSG治疗组和DOX模型组相比心肌纤维受损程度明显降低。
2．THSG对急性阿霉素心脏毒性小鼠血清LDH和CK水平的影响
方法：生物化学法测定血清LDH和CK的水平
结果：DOX处理后第七天，血清中LDH水平从空白对照组3．87±0．40 U／ml上升到6．00±0．27 U／ml (P＜0．01)。经过THSG(0．1 g／kg·d)治疗后，LDH水平显著降低，为4．86±0．20U／ml(P＜0．05)。血清中CK水平从正常组1．55±0．49 U／ml上升到模型组3．02±0．28U／ml (P＜0．01)。经过THSG(0．1g／kg·d)治疗后CK水平降至1．94±0．4U／ml，与DOX单独处理组比较差异有显著性(P＜0．05)。
3．THSG对急性阿霉素心脏毒性小鼠心肌组织中MDA和GSH含量的影响
方法：生物化学方法测定心肌组织中MDA和GSH的含量
结果：DOX处理后第七天，MDA含量从空白对照组1．15±0．30 nmol／mg protein上升到4．17±0．91 nmol／mg protein(P＜0．01)。THSG(0．1g／kg·d)可以显著抑制MDA的升高，其数值为2．32±0．76 nmol／mg protein(P＜0．01)。DOX处理后第七天，GSH含量从正常组24．43±6．57mg/g protein下降到模型组13．65±3．46 mg/g protein(P＜0．01)。THSG(0．1g／kg·d)可以升高GSH含量，为18．31±3．27 mg/g protein，与DOX组比较差异有显著性(P＜0．01)。
§2 THSG对阿霉素损伤心肌细胞的保护作用及机制
1．THSG对阿霉素引起的心肌细胞存活减少的影响
方法：MTT法分析心肌细胞存活率。
结果：空白对照组细胞存活率为100％，THSG300μM作用24h后细胞存活率为101．50±5．84％，与空白对照组比较差异无统计学意义。加入DOX 1μM作用24小时后，细胞存活率下降为72．68±8．52％(P＜0．01)。THSG 3，10，30，100，300μM可剂量依赖性削弱DOX导致的细胞存活率下降，其值分别为76．09±10．24％，81．37±9．84％，83A3±11．47％，91．21±7．54％，95．24±10．87％。除3μM THSG组与DOX单独处理组比较差异无统计学意义外，其余均有显著性(P＜0．05，P＜0．01)。
2．THSG对阿霉素引起的细胞凋亡的影响
方法：TUNEL法检测细胞凋亡。
结果：空白对照组凋亡阳性细胞百分率为4．57±+0．52，给予DOX 1μM作用24小时后，细胞凋亡率明显增加为32．55±3．47％(P＜0．01)。THSG10，30，100，300μM可剂量依赖性减少DOX导致的细胞凋亡，其凋亡阳性细胞百分率分别为26．53±3．27％，20．12±2．64％，13．34±8．64％，10．48±1．57％，与DOX单独处理组比较，差异均有统计学意义(P＜0．05，P＜0．01)。
3．THSG对阿霉素导致的心肌细胞线粒体膜电位降低的影响
方法：采用荧光标记法检测线粒体膜电位。
结果：正常对照组细胞伸展良好并呈现出红色或橙色荧光，定量分析显示其红绿荧光比例为14．52±3．86％。给予DOX 1μM作用24小时后，心肌细胞多数固缩为圆形并显示绿色荧光，红绿荧光比例为0．61±0．16％(P＜0．01，compared with control)，提示细胞线粒体膜电位降低。THSG10，30，100，300μM可剂量依赖性减轻DOX导致的心肌细胞线粒体膜电位的降低，其荧光比例分别为1．48±0．34％，2．32±0．46％，3．70±0．87％，5．35±1．25％，除10μM THSG组与DOX单独处理组比较差异无统计学意义外，其余均有显著性(P＜0．01)。
4．THSG对阿霉素引起的活性氧自由基增多的影响
方法：采用荧光标记法检测氧自由基。
结果：细胞内氧自由基经标记后呈现绿色荧光。定量分析数据用各组细胞绿色荧光面积和正常对照组细胞荧光面积的百分率来表示。正常对照组为100％，给予DOX 1μM作用24小时后荧光比值为337．56±45．56％(P＜0．01，compared with control)，提示阿霉素导致心肌细胞氧自由基增多。THSG 30，100，300μM可剂量依赖性减轻DOX导致的氧自由基增多，其荧光比例分别为265，65±23，46％，227．70±2657％，184．17±34．52％，与DOX单独处理组比较，差异均有统计学意义(P＜0．05，P＜0．01)。
5．THSG对阿霉素引起的凋亡相关蛋白表达改变的影响
方法：用Western blot检测caspase-3，Bcl-2和Bax蛋白表达。
结果：①DOX 1μM诱导24h后，caspase-3活化较正常对照组明显增加，且17kDa条带灰度明显深于正常对照组。caspase-3(17kDa)灰度值与内参β-actin灰度值的比值从正常对照组0．08±0．01上升到1．31±0．27，并有显著性差异(P＜0．01)。THSG30，100，300μM可以剂量依赖性抑制DOX导致的caspase-3蛋白的激活，其17kDa条带灰度值与内参β-actin灰度值的比值分别为0．80±0．14、0．42±0．06、0．12±0．13，与DOX单独处理组比较差异均有显著性(P＜0．05，P＜0．01)。②DOX 1μM诱导24h后，Bcl-2蛋白表达量较正常对照组明显增加，Bcl-2蛋白灰度值与内参β-actin灰度值的比值从正常对照组0．42±0．04上升到0．88±0．09，并有显著性差异(P＜0．05)。THSG30，100，300μM可以剂量依赖性地使DOX导致的Bcl-2蛋白表达量的增高更加显著，其灰度比值分别为0．99±0．10，1．37±0．08，1．64±0．12，除30μM THSG组与DOX单独处理组比较差异无统计学意义外，其余均有显著性(P＜0．05，P＜0．01)。③DOX 1μM诱导24h后，Bax蛋白表达量较正常对照组明显增加，Bax蛋白灰度值与内参β-actin灰度值的比值从正常对照组0．64±0．07上升到1．09±0．12，差异有显著性意义(P＜0．05)。THSG 30，100，300μM可以剂量依赖性抑制DOX引起的Bax蛋白表达量增加，其灰度比值分别为0．95±0．08，0．75±0．06，0．66±0．05，除30μM THSG组与DOX单独处理组比较差异没有统计学意义外，其余均有显著性(P＜0．05，P＜0．01)。
6．THSG对阿霉素导致的心肌细胞胞内钙水平增高的影响
方法：加载荧光探针后用激光共聚焦检测细胞胞内钙水平[Ca~(2+)]_i。
结果：加入1μM DOX后，心肌细胞胞内钙水平快速增高并在5min后持续上升，5min时其荧光强度与基础值的比值为2．33±0．43。THSG 30μM预孵育2h可以显著抑制DOX导致的胞内钙水平增高，并使这种增高在2min内进入平台期，5min时其荧光强度与基础值的比值为1．33±0．07。THSG 100、300μM预孵育2h几乎能够使胞内钙水平在DOX加入后保持不变，5min时其荧光强度与基础值的比值分别为1．09±0．04、1．06±0．02。所有THSG组与DOX单独处理组比较均有统计学差异(P＜0．01)。
结论：
①THSG对DOX诱导的小鼠急性心脏毒性有保护作用，且作用与抑制DOX引起的氧化损伤有关。
②THSG能够减少DOX诱导的细胞凋亡，而且作用与减少细胞线粒体膜电位下降，抑制caspase-3激活，增加Bcl-2及减少Bax的蛋白表达有关。
③THSG抑制DOX引起的活性氧自由基增多。
④THSG能够抑制DOX诱导的心肌细胞胞内钙浓度增高。
第二部分体外培育牛黄镇痛作用的电生理机制研究
牛黄是名贵中药，《本草纲目》记载：其“清心解毒、凉肝息风”，主治惊厥抽搐、癫痫发狂、咽喉肿痛等症。以牛黄为主要成分的牛黄千金散、牛黄解毒丸等经典中成药主要用于解热、镇痛，具有良好的临床疗效。已证实，牛黄及培植牛黄均能显著降低醋酸所致的小鼠疼痛反应。关于牛黄镇痛的作用机制，除与抗炎止痛有关外，推测也可能涉及对痛觉信息传入的直接干预作用。天然牛黄药源匮乏、价格昂贵。国家药品监督管理部门自1972年陆续批准了3个牛黄代用品：人工牛黄、培植牛黄、体外培育牛黄。体外培育牛黄(calculus bovis sativus，CBS；曾译名in vitro cultivated calculusbovis，ICCB)是模拟体内胆结石形成的原理和生化过程，在体外牛胆汁内培育的牛胆红素钙结石，其性状、结构、成分、含量、药效及疗效等与天然牛黄一致。批准文号国药准字20010075，已收载入2005年药典(一部)。
实验以CBS为对象，以蟾蜍坐骨神经干和三叉神经节细胞为标本，观察CBS对复合动作电位及电压依赖性总钠通道电流(I_(Na)-T)、TTX不敏感型钠通道电流(I_(Na)-TTX-r)及电压依赖性钙通道电流(I_(Ca))的影响，探讨牛黄镇痛作用的电生理机制，以证实其阻滞痛觉信息传导的推测。
1．CBS对蟾蜍坐骨神经干复合动作电位的影响
方法：复合动作电位记录法
结果：0．4 mg·mL~(-1) CBS能轻度抑制蟾蜍坐骨神经干复合动作电位的幅度，30 min时使动作电位由给药前的2．68±0．26 mV下降为给药后的2．55±0．29 mV，抑制率为5．0±2．4％(n=7，P＞0．05)。4 mg·mL~(-1) CBS可时间依赖性的显著抑制动作电位，给药后10 min、20 min和30 min时，对动作电位的抑制率分别为21．3±10．3％、36．8±14．2％、42．5±17．6％(n=8，P＜0．01)，用任氏液冲洗10～20 min可使动作电位部分恢复。同样条件下，给予0．02％利多卡因10 min、20 min和30 min时，动作电位分别下降26．8±14．9％、48．0±19．1％和60．7±14．2％(n=6，P＜0．05)。溶剂对照组对动作电位无明显影响。
2．CBS对大鼠三叉神经元总钠通道电流(I_(Na)-T)的影响
方法：全细胞膜片钳记录
结果：溶剂对照对于TRG细胞I_(Na)-T无明显影响。CBS能够剂量依赖性的抑制TRG细胞的总钠电流幅值，0．2、2、20μg·mL~(-1) CBS可使神经元钠电流幅值分别减少25．9±5．7％、45．0±7．6％和55．8±7．8％(n=6，P＜0．01)。20μg·mL~(-1) CBS使I_(Na)由给药前的12．2±0．7 nA减少至给药后的5．4±0．9 nA，但冲洗后电流未见明显恢复(5．6±1．0 nA，P＞0．05)，提示药物作用难以逆转。以I_(Na)幅值对应各测试电位作图，得I_(Na) I-V曲线。各测试电压下，CBS使I_(Na)-T幅值减小，给药前后最大激活电位及激活阈值电位无改变。根据所记录到的I_(Na)，用Boltzmann函数拟合比较激活曲线发现：给药前半数激活电压V_(1/2)=-19．0±10．6 mV，k=4．3±1．7，给予20μg·mL~(-1) CBS后V_(1/2)=-24．5±10．3 mV，k=4．9±2．5(n=6，P＞0．05)，表明20μg·mL~(-1) CBS对I_(Na)-T的激活动力学无明显影响。
3．CBS对大鼠三叉神经元TTX不敏感型钠通道电流(I_(Na)-TTX-r)的影响
方法：全细胞膜片钳记录
结果：给药前I_(Na)-TTX-r幅值为10．2±0．2 nA，给予溶剂对照后为9．9±0．4 nA(n=5，P＞0．05)，表明溶剂对TRG细胞I_(Na)-TTX-r无明显影响。CBS可剂量依赖性的抑制TRG细胞I_(Na)-TTX-r幅值，0．2、2、20μg·mL~(-1) CBS可使TRG细胞I_(Na)-TTX-r幅值分别减少8．1±2．0％、28±5．2％和39．3±5．7％(n=6，P＜0．01)。20μg·mL~(-1) CBS使I_(Na)-TTX-r幅值由给药前的11．4±1．4 nA减少至给药后的6．9±0．6 nA，冲洗后电流同样未见明显恢复(6．8±0．8 nA，P＞0．05)。I_(Na)-TTX-r给药前后的I-V曲线显示，在各测试电压下CBS可使I_(Na)-TTX-r幅值减小，对I-V曲线的形状无明显影响。比较给药前后电流的激活曲线发现：给药前半数激活电压V_(1/2)=-24．1±0．2 mV，k=1．6±0．1，给予20μg·mL~(-1) CBS后V_(1/2)=-23．0±1．4 mV，k=1．6±0．2(n=6，P＞0．05)，表明20μg·mL~(-1) CBS对TRG细胞I_(Na)-TTX-r的激活时间过程无明显影响。
4．CBS对大鼠三叉神经元细胞电压依赖性钙通道电流(I_(Ca))的影响
方法：全细胞膜片钳记录
结果：0．05％溶剂对照对于TRG细胞I_(Ca)无明显影响。CBS能够剂量依赖性的抑制TRG细胞电压依赖性钙电流幅值，0．2、2、20μg·mL~(-1) CBS可使神经元钙电流幅值分别减少303±4．7％、41．9±3．6％和56．7±6．8％(n=6，P＜0．01)。20μg·mL~(-1) CBS使I_(Ca)由给药前的5．7±0．9 nA减少至给药后的2．5±0．4 nA，但冲洗后电流未见明显恢复(2．4±0．6 nA，P＞0．05)，提示药物作用难以逆转。I_(Ca) I-V曲线显示，各测试电压下，CBS使I_(Ca)幅值减小，给药前后最大激活电位及激活阈值电位无改变。比较给药前后电流的激活曲线发现：给药前半数激活电压V_(1/2)=-15．5±3．2m V，k=3．4±0．8，给予20μg·mL~(-1) CBS后V_(1/2)=-12．3±4．6 mV，k=4．5±0．6(n=6，P＞0．05)，表明20μg·mL~(-1) CBS对I_(Ca)的激活动力学无明显影响。比较给药前后I_(Ca)的稳态失活曲线发现：给药前半数失活电压V_(1/2)=-36．3±4．5 m V，k=21．4±5．4，给予20μg·mL~(-1) CBS后V_(1/2)=-33．5±5．8mV，k=18．5±4．3(n=6，P＞0．05)，表明20μg·mL~(-1) CBS对I_(Ca)的失活动力学无明显影响。
结论：
CBS对电压依赖性钠电流和钙电流的阻滞作用可能是其镇痛作用的机制之一。
Part 1Protective effect of THSG on cardiotoxicity induced bydoxorubicin in vitro and in vivo
Doxorubicin (also named adriamycin) is an anthracycline antibiotic that has beenused for more than 30 years for the treatment of a wide variety of cancers. However, severecardiomyopathy and heart failure have been observed in DOX-treated cancer patient, whichlimits the clinical chemotheraphy. It has been proposed that doxorubicin-inducedcardiomyopathy is at least partially caused by increased oxidant production in the heart, andthere is a great deal of supportive evidence for this hypothesis.Tetrahydroxystilbene-glucoside(THSG) was the antioxidative component of Polygonummultiflorum Thunb and it showed strong antioxidant activity in vitro. Previous researcheselucidated THSG could diminish peroxidation level of brain in Alzheimer's Disease modelmice and ischemia-reperfusion model mice. Structurally, THSG belongs to hydroxystilbene,and its structure is similar to that of resveratrol extracted from red wine which hassignificantly protective effects on cardiomyocyte injury. Based on the previous reportsthat THSG protects normal tissues against oxidative stress, we envisioned a possibility thatTHSG might protect heart from DOX injury.
In the present study we used neonate rattus cardiomyocytes in vitro and acutemouse model of DOX-induced cardiotoxicity to examine the protective effect of THSG.
§1. Protective effects and mechamism of THSG on DOX induced actue cardiotoxicityin mice
1. Protective effects of THSG on DOX-induced cardiotoxicity in mice
Method: A single DOX (15 mg/kg) was intraperitoneally injected to the animals to inducedthe actue cardiotoxicity, THSG (0.1g/kg·d) were administered with intragastric injection(p.o.) for 7 days continuously. Then it was observed whether THSG have protective effectson DOX-induced cardiotoxity in mice.
Results:①Animal body weight and survival: There was no difference on body weightamong 3 groups at day 1. On the 7th day after DOX administration, animals' body weightswere reduced(21.8±3.6 g) by DOX treatment compared with control(25.6±2.6 g,P＜0.01). Treatment with THSG did not increase body weights of animals (22.5±2.8 g,P＞0.05 compared with DOX alone). There was no difference in the heart weight/bodyweight ratio among the 3 groups(4.5±0.6mg/g in control group, 4.3±0.7mg/g in DOXgroup and 4.3±0.4 mg/g in THSG group). The survival rate of animals in the control groupwas 100%. DOX treated animals had a compromised survival rate (70%), and there was atrend of increased survival in THSG treated animals (85%) compared with DOX treatedgroup (P＞0.05, log rank test).②Histological findings : Normal heart histologicalfindings were seen in the control group.On the other hand, there were histological changesin DOX group by the presence of marked interstitial oedema, focal subendocardial bleed,cardiac muscle fibre destruction or loss, which was qualitatively recognized as DOX-inducecardiac damage. The lesions were significantly reduced in the groups treated with THSGcompared to DOX group.
2. Effects of THSG on serum LDH and CK in DOX administrated mice
Method: Serum LDH and CK were investigated with biochemical method.
Results: On the 7th day after DOX adminstration, serum LDH increased from 3.87±0.40U/ml of normal control group to 6.00±0.27 U/ml of the DOX group (P＜0.01). However,THSG (0.1g/kg) could prevent the increase of serum LDH induced by DOX, with the value4.86±0.20U/ml (P＜0.05, compared with DOX alone). Serum CK increased from 1.55±0.49 U/ml of control group to 3.02±0.28U/ml of the DOX alone administrationgroup (P＜0.01). THSG administration could inhibit the increase of serum CK induced byDOX, with the value of 1.94±0.4U/ml (P＜0.05, compared with DOX alone)。
3. Effects of THSG on MDA and GSH in DOX administrated mice
Method: MDA and GSH were investigated with biochemical method.
Results: On the 7th day after DOX adminstration, MDA content increased from 1.15±0.30nmol/mg protein of normal control group to 4.17±0.91 nmol/mg protein of the DOX group(P＜0.01). However, THSG (0.1g/kg) could significantly prevent the increase of MDAinduced by DOX, with the value 2.32±0.76 nmol/mg protein (P＜0.01, compared withDOX alone). GSH content decreased from 24.43±6.57mg/g protein of control group to13.65±3.46 mg/g protein of the DOX alone administration group(P＜0.01). THSGadministration could inhibit the decrease of GSH induced by DOX, with the value of18.31±3.27 mg/g protein (P＜0.01, compared with DOX alone).
§2 Protective effects of THSG on DOX induced cardiomyocyte lesion
1. Effects of THSG on viability of DOX treated cells
Method: Cell viability was determinated by MTT assay.
Results: Cell viability of control group was 100%. DOX markedly affected cell survival asthe rate of cell survival after exposure to 1μM DOX for 24h was 72.68±8.52%(P＜0.01),while in the presence of THSG(3,10,30,100,300μM), cell viability was significantlyenhanced in a concentration-dependent manner with the values of 76.09±10.24 %, 81.37±9.84 %, 83.43±11.47 %, 91.21±7.54 %, 95.24±10.87 % respectively. Except 3μMTHSG group, 10-300μM THSG groups had significant difference compared with the DOXalone group (P＜0.05, P＜0.01).
2. Effects of THSG on DOX-induced apoptosis
Method: Cell apoptosis was investigated by TUNEL assay.
Results: The rate of apoptotic cells of control group was 4.57±0.52%. DOX markedlyincreased the rate of positive cells after exposure to 1μM DOX for 24h with the value was32.55±3.47% (P＜0.01), while in the presence of THSG(10,30,100,300μM), the rate ofapoptotic cells was significantly decreased in a concentration-dependent manner with thevalues 26.53±3.27%,20.12±2.64 %,13.34±8.64 %,10.48±1.57 % respectively. Allgroups had significant difference compared with the DOX alone group (P＜0.05, P＜0.01).
3. Effects of THSG on DOX-induced loss of mitochondrial membrane potential
Method: Mitochondrial membrane potential was investigated by fluorescence probe.
Results: Nearly all cells were well spread and exhibited red or orange fluorescence in anuntreated culture, Quantitative analysis showed ratio of JC-1 aggregates/monomericwas14.52±3.86 %. In contrast, a culture treated with DOX for 24h demonstrated multiplerounded cells, a majority of which fluoresce green exclusively with the value of 0.61±0.16% (P＜0.01, compared with control), indicating loss of mitochondrial membrane potential.A culture treated with DOX in the presence of THSG(10,30,100,300μM) appeared similarto untreated control, with spread cells exhibiting red or orange fluorescence with thevaluesl.48±0.34%, 2.32±0.46 %,3.70±0.87%,5.35±1.25% respectively. Except 10μMTHSG group, 30-300μM THSG groups had significant difference compared with DOXgroup (P＜0.01).
4. Effects of THSG on DOX-mediated ROS generation
Method: ROS were investigated by fluorescence probe.
Results: Quantitative results (green fluorescence area) are expressed as % of values foundin cells untreated. The value was increased from 100% of control group to 337.56±45.56 % after exposure of 1μM DOX for 24h (P＜0.01), indicated that DOX stimulatedsignificant increase of ROS level. A culture treated with DOX in the presence of THSG(30,100,300μM) could inhibited DOX-mediated ROS generation with the values 265.65±23.46 %, 227.70±26.57%, 184.17±34.52%. All groups had significant differencecompared with the DOX alone group (P＜0.05, P＜0.01).
5. Effects of THSG on apoptosis-related proteins expression
Method: Protein expressions were investigated by Western blot.
Results:①Treatment of the cells with 1μM DOX for 24h induced the cleavage ofprocaspase-3(32 kDa) to its 17 kDa subunits. Caspase-3(17 kDa)/β-actin density ratio wassignificantly increased from normal control group 0.08±0.01 to DOX group 1.31±0.27(P＜0.01). THSG (30,100,300μM) could inhibit caspase-3 protein activation indose-dependent manner, and density ratio were 0.80±0.14,0.42±0.06,0.12±0.13. Allthese THSG groups had significant difference from DOX alone group (P＜0.05, P＜0.01) .②DOX treated for 24h markly increased Bcl-2 protein expression. Bcl-2/β-actin densityratio was significantly increased from normal control group 0.42±0.04 to DOX group0.88±0.09 (P＜0.05). THSG (30,100,300μM) could dose-dependently make this potentiallyadaptive response more outstanding, and density ratio were 0.99±0.10, 1.37±0.08,1.64±0.12. Except 30μM THSG group, 100 and 300μM THSG groups had significantdifference from DOX alone group (P＜0.05, P＜0.01).③DOX treated for 24h marklyincreased Bax protein expression. Bax/β-actin density ratio was increased from normalcontrol group 0.64±0.07 to DOX group 1.09±0.12 (P＜0.05). THSG (30,100,300μM) coulddose-dependently inhibit the increase of Bax protein expression, and density ratio were0.95±0.08, 0.75±0.06, 0.66±0.05. Except 30μM THSG group, 100 and 300μM THSGgroups had significant difference from DOX alone group (P＜0.05, P＜0.01).
6. Effects of THSG on DOX-induced increase of [Ca~(2+)]_i
Method: [Ca~(2+)]_i was investigated by fluorescence probe.
Results: Treatment of cardiomyocytes with DOX greatly increased level of [Ca~(2+)]_i. TheCa~(2+) increase was very quick and did not reach plateau at 5 min. Quantitative analysisshowed fluorescence intensity after treatment of DOX /basal value was 2.33±0.43.Pretreatment of THSG inhibited DOX-induced increase of [Ca~(2+)]_i. It reached plateau atapproximately 2 min with pretreatment of 30μM THSG and the ratio was 1.33±0.07.Treatment of 100 and 300μM THSG nearly maintained basal [Ca~(2+)]_i after DOX was addedwith the value 1.09±0.04 and 1.06±0.02. All these THSG groups had siginificant differencefrom DOX group (P＜0.01).
Conclusions:
1. THSG had protective effects on acute cardiotoxicity induced by DOX in mice, and itseffects may be associated with inhibiting oxidative damage by DOX.
2. THSG could inhibit DOX-induced apoptosis and it may be involved that it preventedDOX-mediated loss of mitochondrial membrane potential, caspase-3 activation andchanges of Bax and Bcl-2 protein expression.
3. THSG could inhibit DOX-mediated ROS generation.
4. THSG could inhibit the increase of [Ca~(2+)]_i induced by DOX.
Part 2
The electrophysiological mechanism of calculus bovissativus on analgesic effect
Calculus bovis has been used for thousand years as the Chinese traditional medicine.The classical Chinese patent medicine NiuHuang JieDu Wan and NiuHuang QianJing Shanwhose major ingredient were calculus bovis had antipyretic and analgesic effect andapproved for clinical use. Previous reports had verified that calculus bovis and culturalcalculus bovis could relieve pain induced by acid in mice. We presumed that themechanism of calculus bovis on analgesic effect was not only involved itsanti-inflammatory effect but also the directly intervention of afferent pain sense. In order toresolve the shortage raw material of calulus bovis, the national management of drugsurveilance had validated in succession three substitutes since 1972: artificial bezoar,cultural calulus boris and calulus bovis stativus (CBS).CBS is prepared in ox bile bysimulating the biochemical process of bilirubin calcium formation and by employingbioengineering techniques. It has same quality as that of natural calculus bovis in terms ofproperties, structure, component and content.
In the present study we used sciatic nerve trunk of toads and cultured adult rattrigeminal ganglion neurons to examine the effect of CBS on action potential, currents ofvoltage-dependent sodium channel and voltage-dependent calcium channel.
1. The effect of CBS on action potential of sciatic nerve trunk of toads
Method: The action potential of sciatic nerve trunk of toads was recorded by RM6240BDpolygraph.
Results: 0.4 mg·mL~(-1) CBS could solfly decrease the action potential of satatic nerve trunkof toads. The amplitude of action potential was decreased from 2.68±0.26 mV to 2.55±0.29 mV of CBS treatment for 30min. The inhibitory rate was 5.0±2.4% (n=7, P＞0.05). 4mg·mL~(-1) CBS could significantly decrease the action potential of satatic nerve trunk oftoads in time-dependent manner. The inhibitory rates were 21.3±10.3%, 36.8±14.2%,42.5±17.6% respectively when CBS treated for 10, 20 and 30min (n=8, P＜0.01), washed byRinger solution for 30 min could recovery the action potential partly. Under the samecondition, treated satatic nerve trunk of toads with 0.02% lidocaine for10, 20 and 30min,the inhibitory rates of action potential were 26.8±14.9 %, 48.0±19.1%, 60.7±14.2 %respectively (n=6, P＜0.05). Dissolvant control had no effect on action potential.
2. The effect of CBS on total currents of voltage-dependent sodium channel (I_(Na)-T).
Method: whole cell patch clamp record.
Results: The amplitude of total currents before treatment was 12.1±0.5 nA and the valuewas 12.1±0.6 nA (n=5, P＞0.05) after 0.05% dissolvent treatment, indicated that thedissolvent had no effect on I_(Na)-T. CBS could dose-dependently inhibit the total currents ofvoltage-dependent sodium channel on trigeminal ganglion neurons (TRG). The inhibitoryrates of 0.2, 2 and 20μg·mL~(-1) CBS on I_(Na)-T were 25.9±5.7%, 45.0±7.6%, 55.8±7.8%respectively (n=6, P＜0.01). Treatment with 20μg·mL~(-1) CBS decreased the currents from12.2±0.7 nA to 5.4±0.9 nA and washing could not make it recovery(5.6±1.0 nA,P＞0.05), indicated that the drug action was inreversible. Current-voltage (I-V) relation ofI_(Na)-T under different concentration showed CBS had no effect on maximum activationpotential and threshold of activation potential. Conductance-voltage (G-V) relation showedno significant change after 20μg·mL~(-1) CBS treatment (control: V_(1/2)=-19.0±10.6 mV,k=4.3±1.7; 20μg·mL~(-1) CBS:V_(1/2)=-24.5±10.3 mV, k=4.9±2.5 (n=6, P＞0.05)), indicatedthat CBS had no effect on activation kinetics of I_(Na)-T.
3. The effect of CBS on TTX-resistant currents of voltage-dependent sodium channel(I_(Na)-TTX-r).
Method: whole cell patch clamp record.
Results: The dissolvent had no effect on I_(Na)-TTX-r. CBS could dose-dependently inhibitthe TTX-resistant currents of voltage-dependent sodium channel on TRG. The inhibitoryrate of 0.2, 2 and 20μg·mL~(-1) CBS on I_(Na)-TTX-r were 8.1±2.0%, 28±5.2%, 39.3±5.7%respectively(n=6, P＜0.01). Treatment with 20μg·mL~(-1) CBS decreased the currents from11.4±1.4 nA to 6.9±0.6 nA and washing could not make it recovery(6.8±0.8 nA,P＞0.05), indicated that the drug action was inreversible. Current-voltage (I-V) relation ofI_(Na)-TTX-r under different concentration showed CBS had no effect on maximum activationpotential and threshold of activation potential. Conductance-voltage (G-V) relation showedno significant change after 20μg·mL~(-1) CBS treatment.( control: V_(1/2)= -24.1±0.2 mV,k=1.6±0.1; 20μg·mL~(-1) CBS: V_(1/2)= -23.0±1.4 mV, k=1.6±0.2 (n=6, P＞0.05)), indicatedthat CBS had no effect on activation kinetics of I_(Na)-TTX-r.
4. The effect of CBS on the currents of voltage-dependent calcium channel (I_(Ca)).
Method: whole cell patch clamp record.
Results: The amplitude of the currents before treatment was 5.3±0.8 nA and the value was5.1±0.6 nA (n=6, P＞0.05) after treated with 0.05% dissolvent, indicated that the dissolventhad no effect on I_(Ca). CBS could dose-dependently inhibit the currents of voltage-dependentcalcium channel on TRG. The inhibitory rate of 0.2, 2, 20μg·mL~(-1) CBS on I_(Ca) were30.3±4.7%, 41.9±3.6% and 56.7±6.8% respectively (n=6, P＜0.01). Treatment with 20μg·mL~(-1) CBS decreased the currents from 5.7±0.9 nA to 2.5±0.4 nA and washing couldnot make it recovery(2.4±0.6 nA, P＞0.05). Current-voltage (I-V) relation of I_(Ca) underdifferent concentration showed CBS had no effect on maximum activation potential andthreshold of activation potential. Conductance-voltage (G-V) relation showed no significantchange after 20μg·mL~(-1) CBS treatment( control:V_(1/2)=-15.5±3.2 mV, k=3.4±0.8; 20 μg·mL~(-1) CBS:V_(1/2)= -12.3±4.6 mV, k=4.5±0.6 (n=6, P＞0.05)), indicated that CBS had noeffect on activation kinetics of I_(Ca). Steady-state inactivation curves showed no significantchange after 20μg·mL~(-1) CBS treatment( control:V_(1/2)= -36.3±4.5 mV, k=21.4±5.4; 20μg·mL~(-1) CBS: V_(1/2)= -33.5±5.8 mV, k=18.5±4.3 (n=6, P＞0.05)), indicated that CBS hadno effect on inactivation kinetics of I_(Ca).
Conclusion:
The inhibitory effect of CBS on the voltage-dependent sodium and calcium currents maycontribute to its relieving pain.
阿霉素(DOX)是一种高效广谱的葸环类抗肿瘤抗生素，心脏毒性是阿霉素临床应用的主要不良反应之一，因出现剂量依赖性的严重心力衰竭而使它的应用受到限制。DOX所致心脏毒性的机制至今仍未完全阐明，但有许多证据表明其与产生的大量自由基密切相关。2，3，5，4’-四羟基二苯乙烯-2-O-β-D葡萄糖苷(2，3，5，4’-tetrahydroxystilbene-2-O-β-D-glucoside，THSG)是何首乌的活性单体，体外实验研究表明THSG具有较强的抗氧化活性。已有的研究也证实THSG能够降低阿尔茨海默病(Alzheimer's Disease)模型小鼠和缺血再灌注损伤模型小鼠组织中的过氧化物水平。THSG的化学结构与白藜芦醇相似，后者具有显著的保护受损心肌的作用。所以我们推测THSG对DOX导致的心脏毒性具有保护作用。
论文将分别以体外培养的乳鼠心肌细胞和DOX诱导的小鼠急性心脏毒性模型为对象探讨THSG的保护作用及机制。
§1 THSG对小鼠急性阿霉素心脏毒性的保护作用及机制
1．THSG对急性阿霉素心脏毒性的保护作用
方法：一次性腹腔注射DOX 15mg／kg诱导急性心脏毒性模型，给予THSG 0．1g／kg灌胃一天1次，连续7天。观察THSG对小鼠阿霉素心脏毒性的保护作用。
结果：①体重和生存率：处理前各组小鼠体重无明显差别。处理第7天，DOX组小鼠体重(21．8±3．6 g)和空白对照组(25．6±2．6 g，P＜0．01)比较明显减轻；给予THSG(0．1g／kg·d)治疗后没有增加造模小鼠的体重(22．5±2．8 g，P＞0．05 compared with DOXalone)。处理7天后，3组小鼠心脏指数(心脏重量与体重的比值)分别为空白对照组：4．5±0．6mg/g；DOX组：4．3±0．7mg/g；THSG组：4．3±0．4 mg/g，统计学分析均无明显差异。对照组20只动物全部存活，DOX处理7天后动物存活70％，给予THSG治疗后动物存活率升高为85％(P＞0．05，log rank test)。②组织学观察：正常对照组小鼠心肌纤维完整，排列整齐，无出血和水肿。DOX模型组可见明显的心肌纤维断裂甚至消失，心肌间质水肿并伴有局灶性心内膜下出血。而THSG治疗组和DOX模型组相比心肌纤维受损程度明显降低。
2．THSG对急性阿霉素心脏毒性小鼠血清LDH和CK水平的影响
方法：生物化学法测定血清LDH和CK的水平
结果：DOX处理后第七天，血清中LDH水平从空白对照组3．87±0．40 U／ml上升到6．00±0．27 U／ml (P＜0．01)。经过THSG(0．1 g／kg·d)治疗后，LDH水平显著降低，为4．86±0．20U／ml(P＜0．05)。血清中CK水平从正常组1．55±0．49 U／ml上升到模型组3．02±0．28U／ml (P＜0．01)。经过THSG(0．1g／kg·d)治疗后CK水平降至1．94±0．4U／ml，与DOX单独处理组比较差异有显著性(P＜0．05)。
3．THSG对急性阿霉素心脏毒性小鼠心肌组织中MDA和GSH含量的影响
方法：生物化学方法测定心肌组织中MDA和GSH的含量
结果：DOX处理后第七天，MDA含量从空白对照组1．15±0．30 nmol／mg protein上升到4．17±0．91 nmol／mg protein(P＜0．01)。THSG(0．1g／kg·d)可以显著抑制MDA的升高，其数值为2．32±0．76 nmol／mg protein(P＜0．01)。DOX处理后第七天，GSH含量从正常组24．43±6．57mg/g protein下降到模型组13．65±3．46 mg/g protein(P＜0．01)。THSG(0．1g／kg·d)可以升高GSH含量，为18．31±3．27 mg/g protein，与DOX组比较差异有显著性(P＜0．01)。
§2 THSG对阿霉素损伤心肌细胞的保护作用及机制
1．THSG对阿霉素引起的心肌细胞存活减少的影响
方法：MTT法分析心肌细胞存活率。
结果：空白对照组细胞存活率为100％，THSG300μM作用24h后细胞存活率为101．50±5．84％，与空白对照组比较差异无统计学意义。加入DOX 1μM作用24小时后，细胞存活率下降为72．68±8．52％(P＜0．01)。THSG 3，10，30，100，300μM可剂量依赖性削弱DOX导致的细胞存活率下降，其值分别为76．09±10．24％，81．37±9．84％，83A3±11．47％，91．21±7．54％，95．24±10．87％。除3μM THSG组与DOX单独处理组比较差异无统计学意义外，其余均有显著性(P＜0．05，P＜0．01)。
2．THSG对阿霉素引起的细胞凋亡的影响
方法：TUNEL法检测细胞凋亡。
结果：空白对照组凋亡阳性细胞百分率为4．57±+0．52，给予DOX 1μM作用24小时后，细胞凋亡率明显增加为32．55±3．47％(P＜0．01)。THSG10，30，100，300μM可剂量依赖性减少DOX导致的细胞凋亡，其凋亡阳性细胞百分率分别为26．53±3．27％，20．12±2．64％，13．34±8．64％，10．48±1．57％，与DOX单独处理组比较，差异均有统计学意义(P＜0．05，P＜0．01)。
3．THSG对阿霉素导致的心肌细胞线粒体膜电位降低的影响
方法：采用荧光标记法检测线粒体膜电位。
结果：正常对照组细胞伸展良好并呈现出红色或橙色荧光，定量分析显示其红绿荧光比例为14．52±3．86％。给予DOX 1μM作用24小时后，心肌细胞多数固缩为圆形并显示绿色荧光，红绿荧光比例为0．61±0．16％(P＜0．01，compared with control)，提示细胞线粒体膜电位降低。THSG10，30，100，300μM可剂量依赖性减轻DOX导致的心肌细胞线粒体膜电位的降低，其荧光比例分别为1．48±0．34％，2．32±0．46％，3．70±0．87％，5．35±1．25％，除10μM THSG组与DOX单独处理组比较差异无统计学意义外，其余均有显著性(P＜0．01)。
4．THSG对阿霉素引起的活性氧自由基增多的影响
方法：采用荧光标记法检测氧自由基。
结果：细胞内氧自由基经标记后呈现绿色荧光。定量分析数据用各组细胞绿色荧光面积和正常对照组细胞荧光面积的百分率来表示。正常对照组为100％，给予DOX 1μM作用24小时后荧光比值为337．56±45．56％(P＜0．01，compared with control)，提示阿霉素导致心肌细胞氧自由基增多。THSG 30，100，300μM可剂量依赖性减轻DOX导致的氧自由基增多，其荧光比例分别为265，65±23，46％，227．70±2657％，184．17±34．52％，与DOX单独处理组比较，差异均有统计学意义(P＜0．05，P＜0．01)。
5．THSG对阿霉素引起的凋亡相关蛋白表达改变的影响
方法：用Western blot检测caspase-3，Bcl-2和Bax蛋白表达。
结果：①DOX 1μM诱导24h后，caspase-3活化较正常对照组明显增加，且17kDa条带灰度明显深于正常对照组。caspase-3(17kDa)灰度值与内参β-actin灰度值的比值从正常对照组0．08±0．01上升到1．31±0．27，并有显著性差异(P＜0．01)。THSG30，100，300μM可以剂量依赖性抑制DOX导致的caspase-3蛋白的激活，其17kDa条带灰度值与内参β-actin灰度值的比值分别为0．80±0．14、0．42±0．06、0．12±0．13，与DOX单独处理组比较差异均有显著性(P＜0．05，P＜0．01)。②DOX 1μM诱导24h后，Bcl-2蛋白表达量较正常对照组明显增加，Bcl-2蛋白灰度值与内参β-actin灰度值的比值从正常对照组0．42±0．04上升到0．88±0．09，并有显著性差异(P＜0．05)。THSG30，100，300μM可以剂量依赖性地使DOX导致的Bcl-2蛋白表达量的增高更加显著，其灰度比值分别为0．99±0．10，1．37±0．08，1．64±0．12，除30μM THSG组与DOX单独处理组比较差异无统计学意义外，其余均有显著性(P＜0．05，P＜0．01)。③DOX 1μM诱导24h后，Bax蛋白表达量较正常对照组明显增加，Bax蛋白灰度值与内参β-actin灰度值的比值从正常对照组0．64±0．07上升到1．09±0．12，差异有显著性意义(P＜0．05)。THSG 30，100，300μM可以剂量依赖性抑制DOX引起的Bax蛋白表达量增加，其灰度比值分别为0．95±0．08，0．75±0．06，0．66±0．05，除30μM THSG组与DOX单独处理组比较差异没有统计学意义外，其余均有显著性(P＜0．05，P＜0．01)。
6．THSG对阿霉素导致的心肌细胞胞内钙水平增高的影响
方法：加载荧光探针后用激光共聚焦检测细胞胞内钙水平[Ca~(2+)]_i。
结果：加入1μM DOX后，心肌细胞胞内钙水平快速增高并在5min后持续上升，5min时其荧光强度与基础值的比值为2．33±0．43。THSG 30μM预孵育2h可以显著抑制DOX导致的胞内钙水平增高，并使这种增高在2min内进入平台期，5min时其荧光强度与基础值的比值为1．33±0．07。THSG 100、300μM预孵育2h几乎能够使胞内钙水平在DOX加入后保持不变，5min时其荧光强度与基础值的比值分别为1．09±0．04、1．06±0．02。所有THSG组与DOX单独处理组比较均有统计学差异(P＜0．01)。
结论：
①THSG对DOX诱导的小鼠急性心脏毒性有保护作用，且作用与抑制DOX引起的氧化损伤有关。
②THSG能够减少DOX诱导的细胞凋亡，而且作用与减少细胞线粒体膜电位下降，抑制caspase-3激活，增加Bcl-2及减少Bax的蛋白表达有关。
③THSG抑制DOX引起的活性氧自由基增多。
④THSG能够抑制DOX诱导的心肌细胞胞内钙浓度增高。
第二部分体外培育牛黄镇痛作用的电生理机制研究
牛黄是名贵中药，《本草纲目》记载：其“清心解毒、凉肝息风”，主治惊厥抽搐、癫痫发狂、咽喉肿痛等症。以牛黄为主要成分的牛黄千金散、牛黄解毒丸等经典中成药主要用于解热、镇痛，具有良好的临床疗效。已证实，牛黄及培植牛黄均能显著降低醋酸所致的小鼠疼痛反应。关于牛黄镇痛的作用机制，除与抗炎止痛有关外，推测也可能涉及对痛觉信息传入的直接干预作用。天然牛黄药源匮乏、价格昂贵。国家药品监督管理部门自1972年陆续批准了3个牛黄代用品：人工牛黄、培植牛黄、体外培育牛黄。体外培育牛黄(calculus bovis sativus，CBS；曾译名in vitro cultivated calculusbovis，ICCB)是模拟体内胆结石形成的原理和生化过程，在体外牛胆汁内培育的牛胆红素钙结石，其性状、结构、成分、含量、药效及疗效等与天然牛黄一致。批准文号国药准字20010075，已收载入2005年药典(一部)。
实验以CBS为对象，以蟾蜍坐骨神经干和三叉神经节细胞为标本，观察CBS对复合动作电位及电压依赖性总钠通道电流(I_(Na)-T)、TTX不敏感型钠通道电流(I_(Na)-TTX-r)及电压依赖性钙通道电流(I_(Ca))的影响，探讨牛黄镇痛作用的电生理机制，以证实其阻滞痛觉信息传导的推测。
1．CBS对蟾蜍坐骨神经干复合动作电位的影响
方法：复合动作电位记录法
结果：0．4 mg·mL~(-1) CBS能轻度抑制蟾蜍坐骨神经干复合动作电位的幅度，30 min时使动作电位由给药前的2．68±0．26 mV下降为给药后的2．55±0．29 mV，抑制率为5．0±2．4％(n=7，P＞0．05)。4 mg·mL~(-1) CBS可时间依赖性的显著抑制动作电位，给药后10 min、20 min和30 min时，对动作电位的抑制率分别为21．3±10．3％、36．8±14．2％、42．5±17．6％(n=8，P＜0．01)，用任氏液冲洗10～20 min可使动作电位部分恢复。同样条件下，给予0．02％利多卡因10 min、20 min和30 min时，动作电位分别下降26．8±14．9％、48．0±19．1％和60．7±14．2％(n=6，P＜0．05)。溶剂对照组对动作电位无明显影响。
2．CBS对大鼠三叉神经元总钠通道电流(I_(Na)-T)的影响
方法：全细胞膜片钳记录
结果：溶剂对照对于TRG细胞I_(Na)-T无明显影响。CBS能够剂量依赖性的抑制TRG细胞的总钠电流幅值，0．2、2、20μg·mL~(-1) CBS可使神经元钠电流幅值分别减少25．9±5．7％、45．0±7．6％和55．8±7．8％(n=6，P＜0．01)。20μg·mL~(-1) CBS使I_(Na)由给药前的12．2±0．7 nA减少至给药后的5．4±0．9 nA，但冲洗后电流未见明显恢复(5．6±1．0 nA，P＞0．05)，提示药物作用难以逆转。以I_(Na)幅值对应各测试电位作图，得I_(Na) I-V曲线。各测试电压下，CBS使I_(Na)-T幅值减小，给药前后最大激活电位及激活阈值电位无改变。根据所记录到的I_(Na)，用Boltzmann函数拟合比较激活曲线发现：给药前半数激活电压V_(1/2)=-19．0±10．6 mV，k=4．3±1．7，给予20μg·mL~(-1) CBS后V_(1/2)=-24．5±10．3 mV，k=4．9±2．5(n=6，P＞0．05)，表明20μg·mL~(-1) CBS对I_(Na)-T的激活动力学无明显影响。
3．CBS对大鼠三叉神经元TTX不敏感型钠通道电流(I_(Na)-TTX-r)的影响
方法：全细胞膜片钳记录
结果：给药前I_(Na)-TTX-r幅值为10．2±0．2 nA，给予溶剂对照后为9．9±0．4 nA(n=5，P＞0．05)，表明溶剂对TRG细胞I_(Na)-TTX-r无明显影响。CBS可剂量依赖性的抑制TRG细胞I_(Na)-TTX-r幅值，0．2、2、20μg·mL~(-1) CBS可使TRG细胞I_(Na)-TTX-r幅值分别减少8．1±2．0％、28±5．2％和39．3±5．7％(n=6，P＜0．01)。20μg·mL~(-1) CBS使I_(Na)-TTX-r幅值由给药前的11．4±1．4 nA减少至给药后的6．9±0．6 nA，冲洗后电流同样未见明显恢复(6．8±0．8 nA，P＞0．05)。I_(Na)-TTX-r给药前后的I-V曲线显示，在各测试电压下CBS可使I_(Na)-TTX-r幅值减小，对I-V曲线的形状无明显影响。比较给药前后电流的激活曲线发现：给药前半数激活电压V_(1/2)=-24．1±0．2 mV，k=1．6±0．1，给予20μg·mL~(-1) CBS后V_(1/2)=-23．0±1．4 mV，k=1．6±0．2(n=6，P＞0．05)，表明20μg·mL~(-1) CBS对TRG细胞I_(Na)-TTX-r的激活时间过程无明显影响。
4．CBS对大鼠三叉神经元细胞电压依赖性钙通道电流(I_(Ca))的影响
方法：全细胞膜片钳记录
结果：0．05％溶剂对照对于TRG细胞I_(Ca)无明显影响。CBS能够剂量依赖性的抑制TRG细胞电压依赖性钙电流幅值，0．2、2、20μg·mL~(-1) CBS可使神经元钙电流幅值分别减少303±4．7％、41．9±3．6％和56．7±6．8％(n=6，P＜0．01)。20μg·mL~(-1) CBS使I_(Ca)由给药前的5．7±0．9 nA减少至给药后的2．5±0．4 nA，但冲洗后电流未见明显恢复(2．4±0．6 nA，P＞0．05)，提示药物作用难以逆转。I_(Ca) I-V曲线显示，各测试电压下，CBS使I_(Ca)幅值减小，给药前后最大激活电位及激活阈值电位无改变。比较给药前后电流的激活曲线发现：给药前半数激活电压V_(1/2)=-15．5±3．2m V，k=3．4±0．8，给予20μg·mL~(-1) CBS后V_(1/2)=-12．3±4．6 mV，k=4．5±0．6(n=6，P＞0．05)，表明20μg·mL~(-1) CBS对I_(Ca)的激活动力学无明显影响。比较给药前后I_(Ca)的稳态失活曲线发现：给药前半数失活电压V_(1/2)=-36．3±4．5 m V，k=21．4±5．4，给予20μg·mL~(-1) CBS后V_(1/2)=-33．5±5．8mV，k=18．5±4．3(n=6，P＞0．05)，表明20μg·mL~(-1) CBS对I_(Ca)的失活动力学无明显影响。
结论：
CBS对电压依赖性钠电流和钙电流的阻滞作用可能是其镇痛作用的机制之一。
Part 1Protective effect of THSG on cardiotoxicity induced bydoxorubicin in vitro and in vivo
Doxorubicin (also named adriamycin) is an anthracycline antibiotic that has beenused for more than 30 years for the treatment of a wide variety of cancers. However, severecardiomyopathy and heart failure have been observed in DOX-treated cancer patient, whichlimits the clinical chemotheraphy. It has been proposed that doxorubicin-inducedcardiomyopathy is at least partially caused by increased oxidant production in the heart, andthere is a great deal of supportive evidence for this hypothesis.Tetrahydroxystilbene-glucoside(THSG) was the antioxidative component of Polygonummultiflorum Thunb and it showed strong antioxidant activity in vitro. Previous researcheselucidated THSG could diminish peroxidation level of brain in Alzheimer's Disease modelmice and ischemia-reperfusion model mice. Structurally, THSG belongs to hydroxystilbene,and its structure is similar to that of resveratrol extracted from red wine which hassignificantly protective effects on cardiomyocyte injury. Based on the previous reportsthat THSG protects normal tissues against oxidative stress, we envisioned a possibility thatTHSG might protect heart from DOX injury.
In the present study we used neonate rattus cardiomyocytes in vitro and acutemouse model of DOX-induced cardiotoxicity to examine the protective effect of THSG.
§1. Protective effects and mechamism of THSG on DOX induced actue cardiotoxicityin mice
1. Protective effects of THSG on DOX-induced cardiotoxicity in mice
Method: A single DOX (15 mg/kg) was intraperitoneally injected to the animals to inducedthe actue cardiotoxicity, THSG (0.1g/kg·d) were administered with intragastric injection(p.o.) for 7 days continuously. Then it was observed whether THSG have protective effectson DOX-induced cardiotoxity in mice.
Results:①Animal body weight and survival: There was no difference on body weightamong 3 groups at day 1. On the 7th day after DOX administration, animals' body weightswere reduced(21.8±3.6 g) by DOX treatment compared with control(25.6±2.6 g,P＜0.01). Treatment with THSG did not increase body weights of animals (22.5±2.8 g,P＞0.05 compared with DOX alone). There was no difference in the heart weight/bodyweight ratio among the 3 groups(4.5±0.6mg/g in control group, 4.3±0.7mg/g in DOXgroup and 4.3±0.4 mg/g in THSG group). The survival rate of animals in the control groupwas 100%. DOX treated animals had a compromised survival rate (70%), and there was atrend of increased survival in THSG treated animals (85%) compared with DOX treatedgroup (P＞0.05, log rank test).②Histological findings : Normal heart histologicalfindings were seen in the control group.On the other hand, there were histological changesin DOX group by the presence of marked interstitial oedema, focal subendocardial bleed,cardiac muscle fibre destruction or loss, which was qualitatively recognized as DOX-inducecardiac damage. The lesions were significantly reduced in the groups treated with THSGcompared to DOX group.
2. Effects of THSG on serum LDH and CK in DOX administrated mice
Method: Serum LDH and CK were investigated with biochemical method.
Results: On the 7th day after DOX adminstration, serum LDH increased from 3.87±0.40U/ml of normal control group to 6.00±0.27 U/ml of the DOX group (P＜0.01). However,THSG (0.1g/kg) could prevent the increase of serum LDH induced by DOX, with the value4.86±0.20U/ml (P＜0.05, compared with DOX alone). Serum CK increased from 1.55±0.49 U/ml of control group to 3.02±0.28U/ml of the DOX alone administrationgroup (P＜0.01). THSG administration could inhibit the increase of serum CK induced byDOX, with the value of 1.94±0.4U/ml (P＜0.05, compared with DOX alone)。
3. Effects of THSG on MDA and GSH in DOX administrated mice
Method: MDA and GSH were investigated with biochemical method.
Results: On the 7th day after DOX adminstration, MDA content increased from 1.15±0.30nmol/mg protein of normal control group to 4.17±0.91 nmol/mg protein of the DOX group(P＜0.01). However, THSG (0.1g/kg) could significantly prevent the increase of MDAinduced by DOX, with the value 2.32±0.76 nmol/mg protein (P＜0.01, compared withDOX alone). GSH content decreased from 24.43±6.57mg/g protein of control group to13.65±3.46 mg/g protein of the DOX alone administration group(P＜0.01). THSGadministration could inhibit the decrease of GSH induced by DOX, with the value of18.31±3.27 mg/g protein (P＜0.01, compared with DOX alone).
§2 Protective effects of THSG on DOX induced cardiomyocyte lesion
1. Effects of THSG on viability of DOX treated cells
Method: Cell viability was determinated by MTT assay.
Results: Cell viability of control group was 100%. DOX markedly affected cell survival asthe rate of cell survival after exposure to 1μM DOX for 24h was 72.68±8.52%(P＜0.01),while in the presence of THSG(3,10,30,100,300μM), cell viability was significantlyenhanced in a concentration-dependent manner with the values of 76.09±10.24 %, 81.37±9.84 %, 83.43±11.47 %, 91.21±7.54 %, 95.24±10.87 % respectively. Except 3μMTHSG group, 10-300μM THSG groups had significant difference compared with the DOXalone group (P＜0.05, P＜0.01).
2. Effects of THSG on DOX-induced apoptosis
Method: Cell apoptosis was investigated by TUNEL assay.
Results: The rate of apoptotic cells of control group was 4.57±0.52%. DOX markedlyincreased the rate of positive cells after exposure to 1μM DOX for 24h with the value was32.55±3.47% (P＜0.01), while in the presence of THSG(10,30,100,300μM), the rate ofapoptotic cells was significantly decreased in a concentration-dependent manner with thevalues 26.53±3.27%,20.12±2.64 %,13.34±8.64 %,10.48±1.57 % respectively. Allgroups had significant difference compared with the DOX alone group (P＜0.05, P＜0.01).
3. Effects of THSG on DOX-induced loss of mitochondrial membrane potential
Method: Mitochondrial membrane potential was investigated by fluorescence probe.
Results: Nearly all cells were well spread and exhibited red or orange fluorescence in anuntreated culture, Quantitative analysis showed ratio of JC-1 aggregates/monomericwas14.52±3.86 %. In contrast, a culture treated with DOX for 24h demonstrated multiplerounded cells, a majority of which fluoresce green exclusively with the value of 0.61±0.16% (P＜0.01, compared with control), indicating loss of mitochondrial membrane potential.A culture treated with DOX in the presence of THSG(10,30,100,300μM) appeared similarto untreated control, with spread cells exhibiting red or orange fluorescence with thevaluesl.48±0.34%, 2.32±0.46 %,3.70±0.87%,5.35±1.25% respectively. Except 10μMTHSG group, 30-300μM THSG groups had significant difference compared with DOXgroup (P＜0.01).
4. Effects of THSG on DOX-mediated ROS generation
Method: ROS were investigated by fluorescence probe.
Results: Quantitative results (green fluorescence area) are expressed as % of values foundin cells untreated. The value was increased from 100% of control group to 337.56±45.56 % after exposure of 1μM DOX for 24h (P＜0.01), indicated that DOX stimulatedsignificant increase of ROS level. A culture treated with DOX in the presence of THSG(30,100,300μM) could inhibited DOX-mediated ROS generation with the values 265.65±23.46 %, 227.70±26.57%, 184.17±34.52%. All groups had significant differencecompared with the DOX alone group (P＜0.05, P＜0.01).
5. Effects of THSG on apoptosis-related proteins expression
Method: Protein expressions were investigated by Western blot.
Results:①Treatment of the cells with 1μM DOX for 24h induced the cleavage ofprocaspase-3(32 kDa) to its 17 kDa subunits. Caspase-3(17 kDa)/β-actin density ratio wassignificantly increased from normal control group 0.08±0.01 to DOX group 1.31±0.27(P＜0.01). THSG (30,100,300μM) could inhibit caspase-3 protein activation indose-dependent manner, and density ratio were 0.80±0.14,0.42±0.06,0.12±0.13. Allthese THSG groups had significant difference from DOX alone group (P＜0.05, P＜0.01) .②DOX treated for 24h markly increased Bcl-2 protein expression. Bcl-2/β-actin densityratio was significantly increased from normal control group 0.42±0.04 to DOX group0.88±0.09 (P＜0.05). THSG (30,100,300μM) could dose-dependently make this potentiallyadaptive response more outstanding, and density ratio were 0.99±0.10, 1.37±0.08,1.64±0.12. Except 30μM THSG group, 100 and 300μM THSG groups had significantdifference from DOX alone group (P＜0.05, P＜0.01).③DOX treated for 24h marklyincreased Bax protein expression. Bax/β-actin density ratio was increased from normalcontrol group 0.64±0.07 to DOX group 1.09±0.12 (P＜0.05). THSG (30,100,300μM) coulddose-dependently inhibit the increase of Bax protein expression, and density ratio were0.95±0.08, 0.75±0.06, 0.66±0.05. Except 30μM THSG group, 100 and 300μM THSGgroups had significant difference from DOX alone group (P＜0.05, P＜0.01).
6. Effects of THSG on DOX-induced increase of [Ca~(2+)]_i
Method: [Ca~(2+)]_i was investigated by fluorescence probe.
Results: Treatment of cardiomyocytes with DOX greatly increased level of [Ca~(2+)]_i. TheCa~(2+) increase was very quick and did not reach plateau at 5 min. Quantitative analysisshowed fluorescence intensity after treatment of DOX /basal value was 2.33±0.43.Pretreatment of THSG inhibited DOX-induced increase of [Ca~(2+)]_i. It reached plateau atapproximately 2 min with pretreatment of 30μM THSG and the ratio was 1.33±0.07.Treatment of 100 and 300μM THSG nearly maintained basal [Ca~(2+)]_i after DOX was addedwith the value 1.09±0.04 and 1.06±0.02. All these THSG groups had siginificant differencefrom DOX group (P＜0.01).
Conclusions:
1. THSG had protective effects on acute cardiotoxicity induced by DOX in mice, and itseffects may be associated with inhibiting oxidative damage by DOX.
2. THSG could inhibit DOX-induced apoptosis and it may be involved that it preventedDOX-mediated loss of mitochondrial membrane potential, caspase-3 activation andchanges of Bax and Bcl-2 protein expression.
3. THSG could inhibit DOX-mediated ROS generation.
4. THSG could inhibit the increase of [Ca~(2+)]_i induced by DOX.
Part 2
The electrophysiological mechanism of calculus bovissativus on analgesic effect
Calculus bovis has been used for thousand years as the Chinese traditional medicine.The classical Chinese patent medicine NiuHuang JieDu Wan and NiuHuang QianJing Shanwhose major ingredient were calculus bovis had antipyretic and analgesic effect andapproved for clinical use. Previous reports had verified that calculus bovis and culturalcalculus bovis could relieve pain induced by acid in mice. We presumed that themechanism of calculus bovis on analgesic effect was not only involved itsanti-inflammatory effect but also the directly intervention of afferent pain sense. In order toresolve the shortage raw material of calulus bovis, the national management of drugsurveilance had validated in succession three substitutes since 1972: artificial bezoar,cultural calulus boris and calulus bovis stativus (CBS).CBS is prepared in ox bile bysimulating the biochemical process of bilirubin calcium formation and by employingbioengineering techniques. It has same quality as that of natural calculus bovis in terms ofproperties, structure, component and content.
In the present study we used sciatic nerve trunk of toads and cultured adult rattrigeminal ganglion neurons to examine the effect of CBS on action potential, currents ofvoltage-dependent sodium channel and voltage-dependent calcium channel.
1. The effect of CBS on action potential of sciatic nerve trunk of toads
Method: The action potential of sciatic nerve trunk of toads was recorded by RM6240BDpolygraph.
Results: 0.4 mg·mL~(-1) CBS could solfly decrease the action potential of satatic nerve trunkof toads. The amplitude of action potential was decreased from 2.68±0.26 mV to 2.55±0.29 mV of CBS treatment for 30min. The inhibitory rate was 5.0±2.4% (n=7, P＞0.05). 4mg·mL~(-1) CBS could significantly decrease the action potential of satatic nerve trunk oftoads in time-dependent manner. The inhibitory rates were 21.3±10.3%, 36.8±14.2%,42.5±17.6% respectively when CBS treated for 10, 20 and 30min (n=8, P＜0.01), washed byRinger solution for 30 min could recovery the action potential partly. Under the samecondition, treated satatic nerve trunk of toads with 0.02% lidocaine for10, 20 and 30min,the inhibitory rates of action potential were 26.8±14.9 %, 48.0±19.1%, 60.7±14.2 %respectively (n=6, P＜0.05). Dissolvant control had no effect on action potential.
2. The effect of CBS on total currents of voltage-dependent sodium channel (I_(Na)-T).
Method: whole cell patch clamp record.
Results: The amplitude of total currents before treatment was 12.1±0.5 nA and the valuewas 12.1±0.6 nA (n=5, P＞0.05) after 0.05% dissolvent treatment, indicated that thedissolvent had no effect on I_(Na)-T. CBS could dose-dependently inhibit the total currents ofvoltage-dependent sodium channel on trigeminal ganglion neurons (TRG). The inhibitoryrates of 0.2, 2 and 20μg·mL~(-1) CBS on I_(Na)-T were 25.9±5.7%, 45.0±7.6%, 55.8±7.8%respectively (n=6, P＜0.01). Treatment with 20μg·mL~(-1) CBS decreased the currents from12.2±0.7 nA to 5.4±0.9 nA and washing could not make it recovery(5.6±1.0 nA,P＞0.05), indicated that the drug action was inreversible. Current-voltage (I-V) relation ofI_(Na)-T under different concentration showed CBS had no effect on maximum activationpotential and threshold of activation potential. Conductance-voltage (G-V) relation showedno significant change after 20μg·mL~(-1) CBS treatment (control: V_(1/2)=-19.0±10.6 mV,k=4.3±1.7; 20μg·mL~(-1) CBS:V_(1/2)=-24.5±10.3 mV, k=4.9±2.5 (n=6, P＞0.05)), indicatedthat CBS had no effect on activation kinetics of I_(Na)-T.
3. The effect of CBS on TTX-resistant currents of voltage-dependent sodium channel(I_(Na)-TTX-r).
Method: whole cell patch clamp record.
Results: The dissolvent had no effect on I_(Na)-TTX-r. CBS could dose-dependently inhibitthe TTX-resistant currents of voltage-dependent sodium channel on TRG. The inhibitoryrate of 0.2, 2 and 20μg·mL~(-1) CBS on I_(Na)-TTX-r were 8.1±2.0%, 28±5.2%, 39.3±5.7%respectively(n=6, P＜0.01). Treatment with 20μg·mL~(-1) CBS decreased the currents from11.4±1.4 nA to 6.9±0.6 nA and washing could not make it recovery(6.8±0.8 nA,P＞0.05), indicated that the drug action was inreversible. Current-voltage (I-V) relation ofI_(Na)-TTX-r under different concentration showed CBS had no effect on maximum activationpotential and threshold of activation potential. Conductance-voltage (G-V) relation showedno significant change after 20μg·mL~(-1) CBS treatment.( control: V_(1/2)= -24.1±0.2 mV,k=1.6±0.1; 20μg·mL~(-1) CBS: V_(1/2)= -23.0±1.4 mV, k=1.6±0.2 (n=6, P＞0.05)), indicatedthat CBS had no effect on activation kinetics of I_(Na)-TTX-r.
4. The effect of CBS on the currents of voltage-dependent calcium channel (I_(Ca)).
Method: whole cell patch clamp record.
Results: The amplitude of the currents before treatment was 5.3±0.8 nA and the value was5.1±0.6 nA (n=6, P＞0.05) after treated with 0.05% dissolvent, indicated that the dissolventhad no effect on I_(Ca). CBS could dose-dependently inhibit the currents of voltage-dependentcalcium channel on TRG. The inhibitory rate of 0.2, 2, 20μg·mL~(-1) CBS on I_(Ca) were30.3±4.7%, 41.9±3.6% and 56.7±6.8% respectively (n=6, P＜0.01). Treatment with 20μg·mL~(-1) CBS decreased the currents from 5.7±0.9 nA to 2.5±0.4 nA and washing couldnot make it recovery(2.4±0.6 nA, P＞0.05). Current-voltage (I-V) relation of I_(Ca) underdifferent concentration showed CBS had no effect on maximum activation potential andthreshold of activation potential. Conductance-voltage (G-V) relation showed no significantchange after 20μg·mL~(-1) CBS treatment( control:V_(1/2)=-15.5±3.2 mV, k=3.4±0.8; 20 μg·mL~(-1) CBS:V_(1/2)= -12.3±4.6 mV, k=4.5±0.6 (n=6, P＞0.05)), indicated that CBS had noeffect on activation kinetics of I_(Ca). Steady-state inactivation curves showed no significantchange after 20μg·mL~(-1) CBS treatment( control:V_(1/2)= -36.3±4.5 mV, k=21.4±5.4; 20μg·mL~(-1) CBS: V_(1/2)= -33.5±5.8 mV, k=18.5±4.3 (n=6, P＞0.05)), indicated that CBS hadno effect on inactivation kinetics of I_(Ca).
Conclusion:
The inhibitory effect of CBS on the voltage-dependent sodium and calcium currents maycontribute to its relieving pain.
引文
[1]Bagchi D, Bagchi M, Hassoun EA, et al. Adriamycin-induced hepatic and myocardial lipid peroxidation and DNA damage, and enhanced excretion of urianary lipid metabolites in rats. Toxicology, 1995, 95:1-9.
[2]Giantris A, Abdurrahman L, Hinkle A, et al. Anthracyline-induced cardiotoxicity in children and young adults. Crit Rev Oncol Hematol,1998,27:53-6S.
[3]Kotamraju S, Konorev EA, Joseph J, Kalyanaraman B. Doxorubicin-induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone spin traps and ebselen. Role of reactive oxygen and nitrogen species. J Biol Chem, 2000, 275:33585-33592.
[4]Kang YJ, Chen Y, Epstein PN. Suppression of doxorubicin cardiotoxicity by overexpression of catalase in the heart of transgenic mice. J Biol Chem, 1996, 271:12610-12616.
[5]Kang YJ, Chen Y, Yu A, Voss-McCowan M, Epstein PN. Overexpresssion of metallothionein in the heart of transgenic mice suppresses doxorubicin cardiotoxicity. J Clin Investig, 1997,100:1501-1506.
[6]Doroshow JH, Locker GY, Baldinge RJ, Myers CE. The effect of doxorubicin on hepatic and cardiac glutathione Res Che Pathol Pharmacol, 1979, 26:285-295.
[7]Doroshow JH. Effect of anthracycline antibiotics on oxygen redical formation in rat heart. Cancer Res, 1983, 43:460-472.
[8]Yen HC, Oberley TD, Vichitbandha S, Ho YS, St Clair DK. The protective role of manganese superoxide dismutase against adriamycin-induced acute cardiac toxicity in transgenic mice. J Clin Invest, 1996,98:1253-1260.
[9] Gutierrez PL. The role of NAD(P)H oxidoreductase(DT-dia-phorase) in the bioactivation of quinine-containing antitumor agents. Free Radi Biol Med, 2000, 29:263-275.
[10]Shadle SE, Bammel BP, Cusack BJ, et al. Daunorubicin cardiotoxicity: evidence for the importance of the quinine moiety in a free-radical-independent mechanism. Biochem Pharmacol, 2000, 60:1435-1444.
[11]Wang L, Ma W, Markovich R, Chen JW, Wang PH. Regulation of cardiomyocyte apoptotic signaling by insulin-like growth factor Ⅰ. Circ Res, 1998, 83: 516-512.
[12]Arola OJ, Saraste A, Pulkki K, et al. Acute doxorubicin cardiotoxicity involves cardiomocyte apoptosis. Cancer Res, 2000, 60: 1789-1792.
[13]Orrenius S. Mitochondrial regulation of apoptotic cell death. Toxico Lett, 2004, 149: 19-23.
[14]Wu S, Ko YS, Teng MS, et al. Adriamycin-induced cardiomyocyte and endothelial cell apoptosis: in vitro and in vivo studies. J Mol Cell Cardiol, 2002, 34: 1595-1607.
[15]Green PS and Leeuwenburgh C. Mitochondrial dysfunction is an early indicator of doxorubicin-induced apoptosis. Biochim Biophys Acta, 2002, 1588: 94-101.
[16]Swain SM, Whaley FS, Gerber MC, et al. Cardioprotection with dexrazoxane for doxorubicin-containing therapy in advanced breast cancer. J Clin Oncol, 1997, 15: 1318-1332.
[17]Sehested M, Jensen PB, Sorensen BS, et al. Antagonistic effect of the cardioprotector (+)-1, 2-bis(3,5-dioxopiperazinyl-1-yl)propane(ICRF-187) on DNA breaks and cytotoxicity induced by the topoisomerase Ⅱ directed drugs daunorubicin and etoposide(VP-16). Biochem Pharmacol, 1993, 46: 389-393.
[18]Bian XJ, Bao GR. Research on capability of Emodin and Stilbene Glucoside of scavenging superoxide onions. Fuzhou Zhong Yi Xue Yuan, 2002, 4: 12-13.
[19]Liu HC, Chen WS. Research on antioxidative effects of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucoside in vitro. J Pharm. Pract., 2000, 18: 232-234.
[20]Chu J, Ye CF, Li L. Effects of stilbene-glycoside on learning and memory function and free radicals metabolism in dementia model mice. Chin J Rehabil Theory Practice, 2003, 9: 643-645.
[21]Liu L, Zhao L, Li YL. Zhang L, Ye CF, Li L. Protective effect of 2, 3, 5, 4'-Tetrahydroxystilbene-2-β-O-D-glucoside on hippocampal neurons in dementia rats induced by chronic cerebral ischemia. Chin J Pharm, 2006, 41: 354-357.
[1]班翊，刘其礼，金悠等．二苯乙烯苷的含量测定及其稳定性研究．中草药，2004，35：1235-1237．
[2]Wang X, Zhao L, ttan T, et al. Protective effects of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-d-glucoside, an active component of Polygonum multiflorum Thunb, on experimental colitis in mice. Eur J Pharmacol, 2008, 578: 339-348.
[3]Kotamraju S, Konorev EA, Joseph J, Kalyanaraman B. Doxorubicin-induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone spin traps and ebselen. Role of reactive oxygen and nitrogen species. J Biol Chem, 2000, 275: 33585-33592.
[4]Kang YJ, Chen Y, Epstein PN. Suppression of doxorubicin cardiotoxicity by overexpression of catalase in the heart of transgenic mice. J Biol Chem, 1996, 271: 12610-12616.
[5]Kang YJ, Chen Y, Yu A, Voss-McCowan M, Epstein PN. Overexpresssion of metallothionein in the heart of transgenic mice suppresses doxorubicin cardiotoxicity. J Clin Investig, 1997, 100: 1501-1506.
[6]Doroshow JH, Locker GY, Baldinge RJ, Myers CE. The effect of doxorubicin on hepatic and cardiac glutathione. Res Che Pathol Pharmacol, 1979, 26: 285-295.
[7]Doroshow JH. Effect of anthracycline antibiotics on oxygen redical formation in rat heart. Cancer Res, 1983, 43: 460-472.
[8]Yen HC, Oberley TD, Vichitbandha S, Ho YS, St Clair DK. The protective role of manganese superoxide dismutase against adriamycin-induced acute cardiac toxicity in transgenic mice. J Clin Invest, 1996, 98: 1253-1260.
[9]Goormaghtigh E, Chatelain P, Caspers J, Ruysschaert JM. Evidence of a complex between adriamycin derivatives and cardiolipin: possible role in cardiotoxicity. Biochem Pharmacol, 1980, 29: 3003-3010.
[10]Anderson AB, Arriaga EA. Subcellular metabolite profiles of the parent CCRF-CEM and the derived CEM/C2 cell lines after treatment with doxorubicin. J Chromatogr B Analyt Technol Biomed Life Sci, 2004, 808: 295-302
[11]Tokarska-Schlattner M, Zaugg M, da Silva R, Lucchinetti E, Schaub MC, Wallimann T, et al. Acute toxicity of doxorubicin on isolated perfused heart: response of kinases regulating energy supply. Am J Physiol Heart Circ Physiol, 2005, 289: 37-47.
[12]Doroshow JH, Locker GY, Myers CE. Enzymatic defenses of the mouse heart against reactive oxygen metabolites: alterations produced by doxorubicin. J Clin Invest, 1980, 65: 128-135.
[13]Berlin V, Haseltine WA. Reduction of adriamycin to a semiquinone-free radical by NADPH cytochrome P-450 reductase produces DNA cleavage in a reaction mediated by molecular oxygen. J Biol Chem, 1981, 256: 4747-4756.
[14]Bachur NR, Gordon SL, Gee MV, Kon H. NADPH cytochrome P-450 reductase activation of quinone anticancer agents to free radicals. Proc Natl Acad Sci USA, 1979, 76: 954-957.
[15]De Beer EL, Bottone AE, Voest EE. Doxorubicin and mechanical performance of cardiac trabeculae after acute and chronic treatment: a review. Eur J Pharmacol, 2001, 415: 1-11.
[16]Gianni L, Zweier JL, Levy A, Myers CE. Characterization of the cycle of iron-mediated electron transfer from Adriamycin to molecular oxygen. J Biol Chem, 1985, 260: 6820-6826.
[17]Doroshow JH, Locker GY, Baldinger J,. Myers CE. The effect of doxorubicin on hepatic and cardiac glutathione. Res Commun Chem Pathol Pharmacol, 1979, 2: 85-295.
[18]吕丽爽．何首乌中二苯乙烯苷的体外抗氧化研究．食品科学，2007，28：313-317．
[19]Liu HC, Chen WS. Research on antioxidative effects of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucoside in vitro. J Pharm Pract, 2000, 18: 232-234.
[20]Hu J, Ye CF, Li L. Effects of stilbene-glycoside on learning and memory function and free radicals metabolism in dementia model mice. Chin J Rehabil Theory Practice, 2003, 9: 643-645.
[21]Liu L, Zhao L, Li YL. Zhang L, Ye CF, Li L. Protective effect of 2, 3, 5, 4'-Tetrahydroxystilbene-2-β-O-D-glucoside on hippocampal neurons in dementia rats induced by chronic cerebral ischemia. Chin J Pharm, 2006, 41: 354-357.
[1]Kalyanaraman B, Joseph J, Kalivendi S, et al. Doxorubicin-induced apoptosis: implication in cardiotoxicity. Mol Cell Biochem, 2002, 234-235: 119-124.
[2]Oliverira PG, Santos MS, Wallace KB. Doxorubicin-induccd thiol-dependent alteration of cardiac mitochondrial permeability transition and respiration. Biochemistry, 2006, 71: 194-199.
[3]Green PS, Leeuwenburgh C. Mitochondrial dysfunction is an early indicator doxorubicin-induced apoptosis. Biochim Biophys Acta, 2002, 1588: 94-101.
[4]Fisher PW, Salloum F, Das A, et al. Phosphodiesterase-5 inhibition with sildenafil attenuates cardiomyocyte apoptosis and left ventricular dysfunction in a chronic model of doxorubicin cardiotoxicity. Circulation, 2005, 111: 1601-1610.
[5]Chae HJ, Kim HR, Lee WG, et al. Radiation protects adriamycin-induced apoptosis. Immunopharmocology and immunotoxicology, 2005, 27: 211-232.
[6]Wang L, Ma W, Markovich R, Chen JW, Wang PH. Regulation of cardiomyocyte apoptotic signaling by insulin-like growth factor Ⅰ. Circ Res, 1998, 83: 516-512.
[7]Wang L, Ma W, Markovich R, et al. Insulin-like growth factor Ⅰ mosulates induction of apoptotic signaling in H9C2 cardiac muscle cells. Endocrinology, 1998, 139: 1354-1360.
[8]Kim Y, Ma AG, Kitta K, et al. Anthracycline-induced suppression of GATA-4 transcription factor: implication in the regulation of cardiac myocyte apoptosis. Mol Pharmacol, 2003, 63: 368-377.
[9]Kitta K, Day RM, Kim Y, et al. Hepatocyte growth factor induces GATA-4 phosphorylation and cell survival in cardiac muscle cells. J Biol Chem, 2003, 278: 4705-4712.
[10]Sugawara T, Chan PH. Reactive oxygen radicals and pathogenesis of neuronal death after cerebral ischemia. Antioxid Redox Signal, 2003, 5: 597-607.
[11] Yamada J, Yoshimura S, Yamakawa H, Sawada M, Nakagawa M, Hara S, et al. Cell permeable ROS scavengers, Tiron and Tempol, rescue PC12 cell death caused by pyrogallol or hypoxia/reoxygenation. Neurosci Res, 2003, 45:1-8.
[12]Zhuang S, Lynch MC, Kochecar IE. Caspase-8 mediated caspase-3 activation and cytochrome c release during singlet oxygen-induced apoptosis of HL-60 cells. Exp Cell Res, 1999, 250:203-212.
[13]Clementi ME, Giardina B, Di Stasio E, et al. Doxorubicin-derived metabolites induce release of cytochrome c and inhibition of respiration on cardiac isolated mitochondria. Anticancer Res, 2003, 23: 2445-2450.
[14]Gille L, Nohl H. Analyses of the molecular mechanism of adriamycin-induced cardiotoxicity. Free Radic Biol Med, 1997, 23: 775-782.
[15]Zorzato F, Sarviati G, Facchinetti T, Volpe P. Doxorubicin induces calcium release from terminal cisternae of skeletal muscle. J Cell Biol, 1985, 99:875-85.
[16]Kim DH, Landry AB, Lee YS, Katz AM. Doxorubicin-induced calcium release from sarcoplasmic reticulum vesicles. J Moll Cell Cardiol, 1989, 21:433-436.
[17]Caroni P, Villani F, Carafoli, E. The cardiotoxic antibiotic doxorubicin inhibits the Na~+/Ca~(2+) exchange of dog heart sarcolemmal vesicles. FEBS Lett, 1981, 30:184-186.
[18]Keung EC, Toll L, Ellis M, Jensen RA. L-type cardiac calcium channels in doxorubicin cardiomyopathy in rats morphological, biochemical, and functional correlations. J Clin Invest, 1991, 87:2108-2113.
[19]Przygodzki T, Sokal A, Bryszewska M. Calcium ionophore A 23187 action on cardiomyocytes is accompanied by enhanced production of reactive oxygen species. Biochim Bioph Acta, 2005, 1740:481-488.
[20]Petrosillo G, Ruggiero F, Pistolese M, Paradies, G. Ca~(2+)-induced reactive oxygen species production promotes cytochrome c release from rat liver mitochondria via mitochondirial permeability transition (MPT)-dependent and MPT-independent mechanisms. J Biol Chem, 2004, 279:53103-53108.
[21]Waring P. Redox active calcium ion channels, cell death. Arc Biochem Biophys, 2005, 434:33-42.
[22]Kim SY, Kim SJ, Kim BJ, et al. Doxorubicin-induced reactive oxygen species generation and intracellular Ca~(2+) increase are reciprocally modulated in rat cardiomyocytes. Exp Mol Med, 2006, 38: 535-545.
[23]Kimura Y, Okusa H. Effects of naturally occurring stilbene glucosides from medicinal plants and wine, on tumor growth and lung metastasis in Lewis lung caraionma-bearing mice. J Pharm Pharmacol, 2000, 52: 1287-1292.
[1]李培锋，哈斯苏红，关红。胆红素含量不同培植牛黄的镇静、镇痛及抗惊厥作用研究。内蒙古农牧学院学报，1998，19：22-27。
[2]袁惠南。培植牛黄药物作用和研究。 中国中药杂志，1991，16：105-108
[3]贺春阳，关红，李培锋。人工培育牛黄的药理作用。中草药，1988，19：21-24
[4]蔡红娇，裘法祖，刘仁则。体外培育牛黄的药学研究。中国天然药物，2004，2：335-338．
[5]Waxman SG. The molecular pathophysiology of pain: abnormal expression of sodium channel genes and its contributions to hyperexcitability of primary sensory neurons. Pain, 1999, 6: 132-140.
[6]Porreca F, Lai J, Bian D, et al. A comparison of the potential role of the tetrodotoxin-insensitive sodium channels PN3/SNS and NaN/SNS2 in rat models of chronic pain. Proc Natl Acad Sci USA, 1999, 96: 7640-7644.
[7]Gold MS. Tetrodotoxin-resistant Na~+ currents and inflammatory hyperalgesia. ProcNatlAcad Sci USA, 1999, 96: 7645-7649.
[8]Coward K, Plumpton C, Plumpton C, Facer P, et al. Immunolocalization of SNS/PN3 and NaN/SNS2 sodium channels in human pain states. Pain, 2000, 85: 41-50.
[9]Yiangou Y, Birch R, Sangmeswaran L, et al. SNS/PN3 and SNS2/NaN sodium channel-like immunoreactivity in human adult and neonate injured sensory nerves. FEBS Let, 2000, 467: 249-252.
[10]Lai J, Gold MS, Kim CS, et al. Inhibition of neuropathic pain by decreased expression of the tetrodotoxin-resistant sodium channel NaV1.8. Pain, 2002, 95: 143-152.
[11]Tanaka M, Cummins TR, Ishikawa K, et al. SNS Na~+ channel expression increases in dorsal root ganglion neurons in the carrageenan inflammatory pain model. Neuroreport, 1998, 9: 967-972.
[12]Lyu YS, Park SK, Chung K. Low dose of tetrodotoxin reduces neuropathic pain behaviors in an animal model. Brain Res, 2000, 871: 98-103.
[13]Leigh AS, Emmaunel B. Voltage-gated calcium channels in chronic pain. Eur J Physiol, 2008, 456:459-466.
[14]Zamponi GW, Lewis RJ, Todorovic SM, et al. Role of voltage-gated calcium chnnels in ascending pain pathways. Brain Res Rev, 2009, 60:84-89.
[15]Vanegas H, Schaible H. Efeects of antagonists to high-threshold calcium channels upon spinal mechanisms of pain, hyperalgesia and allodynia. Pain, 2000, 85:9-18.
[16]Hara K, Saito Y, Kirihara Y, et al. Antinociceptive effects of intrathecal L-type calcium channel blocker on visceral an somatic stimuli in the rat.Anesth Analg, 1998, 87:382-389.
[17]Bowersox SS, Gadbois T, Singh T, et al. Selective N-type neuraonal voltage-sensitive calcium channel blocker, SNX-111, produces spinal antinociception in rat models of acute persistent and neuropathic pain. J Pharmacol Exp Ther, 1996, 279:1243-1249.
[18]Elizabeth AM, Anthony H. Effects of ethosuximide, a T-type Ca~(2+) channel blocker, on dorsal horn neuronal responses in rats. Europ J Pharm, 2001, 415:141-149.
[19]Kim HC, Chung MK. Voltage-dependent sodium and calcium currents in acutely isolated adult rat trigeminal root ganglion neurons. J Neurophysiol, 1999, 81:1123-1134.
[1]Signal PK, Iliskovic N. Doxorubicin-induced cardiomyopathy. N Engl J Med, 1998, 339:900-905.
[2]Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev, 2004, 56:185-229.
[3]Goormaghtigh E, Chatelain P, Caspers J, Ruysschaert JM. Evidence of a complex between adriamycin derivatives and cardiolipin: possible role in cardiotoxicity. Biochem Pharmacol, 1980, 29:3003-3010.
[4]Anderson AB, Arriaga EA. Subcellular metabolite profiles of the parent CCRF-CEM and the derived CEM/C2 cell lines after treatment with doxorubicin. J Chromatogr B Analyt Technol Biomed Life Sci, 2004, 808:295-302
[5]Tokarska-Schlattner M, Zaugg M,da Silva R, Lucchinetti E, Schaub MC, Wallimann T,et al. Acute toxicity of doxorubicin on isolated perfused heart: response of kinases regulating energy supply. Am J Physiol Heart Circ Physiol, 2005, 289:37-47.
[6]Doroshow JH, Locker GY, Myers CE. Enzymatic defenses of the mouse heart against reactive oxygen metabolites: alterations produced by doxorubicin. J Clin Invest, 1980, 65:128-135.
[7]Berlin V, Haseltine WA. Reduction of adriamycin to a semiquinone-free radical by NADPH cytochrome P-450 reductase produces DNA cleavage in a reaction mediated by molecular oxygen. J Biol Chem, 1981, 256:4747-4756.
[8]Bachur NR, Gordon SL, Gee MV, Kon H. NADPH cytochrome P-450 reductase activation of quinone anticancer agents to free radicals. Proc Natl Acad Sci USA, 1979,76:954-957.
[9]De Beer EL, Bottone AE, Voest EE. Doxorubicin and mechanical performance of cardiac trabeculae after acute and chronic treatment: a review. Eur J Pharmacol, 2001, 415:1-11.
[10]Gianni L, Zweier JL, Levy A, Myers CE. Characterization of the cycle of iron-mediated electron transfer from Adriamycin to molecular oxygen. J Biol Chem, 1985, 260:6820-6826.
[11]Solern LE, Heller LJ, Wallace KB. Dose-dependent increase in sensitivity to calcium-induced mitochondrial dysfunction and cardiomyocyte cell injury by doxorubicin. J Mol Cell Cardiol, 1996, 28:1023-1032.
[12]Green PS, Leeuwenburgh C. Mitochondrial dysfunction is an early indicator of doxorubicin-induced apoptosis. Biochim Biophys Acta, 2002,1588:94-101.
[13]Palmeira CM, Serrano J, Kuehl DW, Wallace KB. Preferential oxidation of cardiac mitochondrial DNA following acute intoxication with doxorubicin. Biochim Biophys Acta, 1997,1321:101-106.
[14]Zhou S, Palmeira CM, Wallace KB. Doxorubicin-induced persistent oxidative stress to cardiac myocytes. Toxicol Lett, 2001,121:151-157.
[15]Kotamraju S, Chitambar CR, Kalivendi SV, Joseph J, Kalyanaraman B. Transferrin receptor-dependent iron uptake is responsible for doxorubicin-mediated apoptosis in endothelial cells: role of oxidant-induced iron signaling in apoptosis. J Biol Chem, 2002,277:17179-17187.
[16]Minotti G. Sources and role of iron in lipid peroxidation. Chem Res Toxicol, 1993, 6: 134-146.
[17]Cairo G, Recalcati S, Pietrangelo A, and Minotti G. The iron regulatory proteins: targets and modulators of free radical reactions and oxidative damage. Free Radic Biol Med, 2002,32: 1237-1243.
[18]Minotti G, Cairo G, and Monti E. Role of iron in anthracycline cardiotoxicity: new tunes for an old song? FASEB J, 1999,13:199-212.
[19]Kwok JC and Richardson DR. Anthracyclines induce accumulation of iron in ferritin in myocardial and neoplastic cells: inhibition of the ferritin iron mobilization pathway. Mol Pharmacol, 2003, 63: 849-861.
[20]Kwok JC and Richardson DR. Examination of the mechanism(s) involved in doxorubicin-mediated iron accumulation in ferritin: studies using metabolic inhibitors, protein synthesis inhibitors and lysosornotropic agents. Mol Pharmacol, 2004, 65: 181-195.
[21]Hasinoff BB, Schnabl KL, Marusak RA, Patel D, and Huebner E. Dexrazoxane (ICRF-187) protects cardiac myocytes against doxorubicin by preventing damage to mitochondria. Cardiovasc Toxicol, 2003, 3: 89-99.
[22]Sawyer DB, Fukazawa R, Arstall MA, and Kelly RA. Daunorubicin-induced apoptosis in rat cardiac myocytes is inhibited by dexrazoxane. Circ Res, 1999, 84: 257-265.
[23]Kaiserov(?) H, den Hartog GJ, Simunek T, Schr(?)terov(?) L, Kvasnickov(?) E, Bast A. Iron is not involved in oxidative stress-mediated cytotoxicity of doxorubicin and bleomycin. Br J Pharmacol, 2006,149: 920-930.
[24]van Acker SA, van Balen GP, van den Berg DJ, Bast A, van der Vijgh WJ. Influence of iron chelation on the antioxidant activity of flavonoids. Biochem Pharmacol, 1998, 56: 935-943.
[25]Wencker D, Chandra M, Nguyen K, Miao W, Garantziotis S, Factor SM, Shirani J,Armstrong RC, and Kitsis RN. A mechanistic role for cardiac myocyte apoptosis in heart failure. J Clin Investig, 2003, 111: 1497-1504.
[26]Narula J, Haider N, Virmani R, Disalvo TG, Kilodgie FD, Hajjar RJ, et al. Apoptosis in myocytes in end-stage heart failure. N Engl J Med, 1996, 335:1182-1189.
[27]Feuerstein GZ. Apoptosis-new opportunities for novel therapeutics for heart diseases. Cardiovusc Drugs Ther, 2001, 15:547-551.
[28]Wang L, Ma W, Markovich R, Chen JW, Wang PH. Regulation of cardiomyocyte apoptotic signaling by insulin-like growth factor I. Circ Res, 1998, 83:516-522.
[29]Kotamraju S, Konorev EA, Joseph J, Kalyanaraman B. Doxorubicin-induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone spin traps and ebselen. Role of reactive oxygen and nitrogen species. J Biol Chem, 2000, 275:33585-33592.
[30]Arola OJ, Saraste A, Pulkki K, Kallajoki M, Parvinen M, Voipio-Pulkki LM. Acute doxorubicin cardiotoxicity involves cardiomyocyte apoptosis. Cancer Res, 2000, 60: 1789-1792.
[31]Kotamraju S, Konorev EA, Joseph J, Kalyanaraman B. Doxorubicin induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone sp in traps and ebselen. Role of reactive oxygen and nitrogen species. J Biol Chem, 2000, 275:33585 - 33592.
[32]ChildsAC, Phaneuf SL, DirksAJ, Phillip s T, Leeuwenburgh C.Doxorubicin treatment in vivo causes cytochrome C release and cardiomyocyte apoptosis, as well as increased mitochondrial efficiency, superoxide dismutase activity, and Bcl2 : Bax ratio. Cancer Res, 2002, 62:4592 - 4598.
[33] Wang GW, Zhou ZX, Kang YJ. Effect of metallothionein on doxorubicin induced apoptosis in cardiomyocytes: role of cytochrome C activated caspase-3. Toxicologist, 2000,54:114-121.
[34]Wang GW, Klein JB, Kang YJ. Metallothionein inhibits doxorubicin induced mitochondrial cytochrome C release and caspase-3 activation in cardiomyocytes. J Pharm acol Exp Ther, 2001, 298:461 - 468.
[35]Sugawara T, Chan PH. Reactive oxygen radicals and pathogenesis of neuronal death after cerebral ischemia.Antioxid Redox Signal, 2003, 5:597-607
[36]Yamada J, Yoshimura S, Yamakawa H, Sawada M, Nakagawa M, Hara S, et al. Cell permeable ROS scavengers, Tiron and Tempol, rescue PC12 cell death caused by pyrogallol or hypoxia/reoxygenation. Neurosci Res, 2003, 45:1-8.
[37]Zhuang S, Lynch MC, Kochecar IE. Caspase-8 mediated caspase-3 activation and cytochrome c release during singlet oxygen-induced apoptosis of HL-60 cells. Exp Cell Res, 1999, 250:203-212.
[38]Sugioka R, Shimizu S, Funatsu T, Tamagawa H, Sawa Y, Kawakaml T, et al. BH-4 domain peptide from Bcl-xL exerts anti-apoptotic activity in vivo. Oncogene, 2003, 22:8432-8440.
[39]Kunisada K, Tone E, Negoro S, Nakaoka Y, Oshima Y, Osugi T, et al. Bcl-xL reduces doxorubicin-induced myocardial damage but fails to control cardiac gene downregulation. Cardiovasc Res, 2002, 53:936-943.
[40]Chen Z, Chua CC, Ho YS, Harndy RC, Chua BH. Gverexpression of Bcl-2 attenuates apoptosis and protects against myocardial I/R injury in transgenie mice. Am J Physiol Heart Circ Physiol, 2001, 280:2313-2320.
[41]Wang L, Ma W, Markovich R, Chen JW, Wang PH. Regulation of cardiomyocyte apoptotic signaling by insulin-like growth factor Ⅰ. Circ Res, 1998, 83:516-512.
[42]Wang L, Ma W, Markovich R, et al. Insulin-like growth factor Ⅰ mosulates induction of apoptotic signaling in H9C2 cardiac muscle cells. Endocrinology, 1998,139:1354-1360.
[43]Kim Y, Ma AG, Kitta K, et al. Anthracycline-induced suppression of GATA-4 transcription factor: implication in the regulation of cardiac myocyte apoptosis. Mol Pharmacol, 2003, 63:368-377.
[44]Kitta K, Day RM, Kim Y, et al. Hepatocyte growth factor induces GATA-4 phosphorylation and cell survival in cardiac muscle cells. J Biol Chem, 2003, 278:4705-4712.
[45]Nakamura T, Ueda Y, Juan Y, et al. Fas-mediated apoptosis in adriamycin-induced cardiomyopathy in rats in vivo study. Circulation, 2002,102:572-578.
[46]Yamaoka M, Yamaguchi S, Suzuki T, et al. Apoptosis in rat cardiacmyocytes induced by Fas ligand:priming for Fas-mediated apoptosis with doxorubicin. J Mol Cell Cardol, 2002, 32:881-889.
[47]Kalivendi SV, Konorev EA, Cunningham S, et al. Doxorubicin activates nuclear factor of activated T-lymphocytes and Fas ligand transcription: role of mitochondrial reactive oxygen species and calcium. Biochem J, 2005, 389:527-539.
[48]Janice LR, Eva S, Susan EL, Triona NC, Timothy O, Thomas R, et al. Distinct mechanisms of cardiomyocyte apoptosis induced by doxorubicin and hypoxia converge on mitochondria and are inhibited by Bcl-xL. J Cell Mol Med, 2007,11:509-520.
[49]Tanaka M, Ito H, Adachi S, Akimoto H, Nishikawa T, Kasajima T, et al. Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes. Circ Res, 1994, 75:426-433.
[50]Yaniv G, Shilkrut M, Lotan R, Berke G,Larisch S, Binah O. Hypoxia predisposes neonatal rat ventricular myocytes to apoptosis induced by activation of the Fas receptor:Fas activation and apoptosis in hypoxic myocytes. Cardiovasc Res, 2002, 54:611-623.
[51]Nitobe J, Yamaguchi S, Okuyama M, Nozaki N, Sata M, Miyamoto T, et al. Reactive oxygen species regulate FLICE inhibitory protein(FLIP) and susceptibility to Fas-mediated apoptosis in cardiac myocytes. Cardiovasc Res, 2003, 57:119-128.
[52]Gang L.Regulation of the p53 transcriptional activity. J Cell Biochem, 2006,97:448-458.
[53]Shizukuda Y, Matoba S, Mian OY. Targeted disruption of p53 attenuates doxorubicin-induced cardiac toxicity in mice. Mol Cell Biochem, 2005, 273:25-28.
[54]Youn HJ, Kim HS, Jeon MH, et al. Induction of caspase-independent apoptosis in H9C2 cardiomyocytes by adriamycin treatment. Mol Cell Biochem, 2005, 270:13-19.
[55]Liu X, Chua CC, Gao J. Pifithrin-alpha protects against doxorubicin-induced apoptosis and acute cardiotoxicity in mice.Am J Physiol Heart Physiol, 2004, 286:933-939.
[56]Wang S, Leonard SS, Ye J, Ding M, Shi X. The role of hydroxyl radical as a messenger in Cr(Ⅵ)-induced p53 activation. Am J Physiol Cell Physiol, 2000, 279: C868-875.
[57]Huang C, Zhang Z, Ding M, Li J, Ye J, Leonard SS, et al. Vanadate induces p53 transactivation through hydrogen peroxide and causes apoptosis. J Biol Chem, 2000,275:32516-32522.
[58]Zhao Y, Chaiswing L, Velez JM, Batinic I, Colbum NH, Oberley TD, et al. p53 translocation to mitochondria preccedes its nuclear translotion and targets mitichondrial oxidative defense protein-manganese superoxide dismutase. Cancer Res, 2005, 65:3745-3750.
[59]Sayan BS, Sayan AE, Knight RA, Melino G, Cohen GM. P53 is cleaved by caspases generating fragments localizing to mitochondria. J Biol Chem, 2006, 281:13566-13573.
[60]Chen Y, Jungsuwadee P, Vore M, Butterfield DA. Collateral damage in cancer chemotherapy: oxidative stress in nontargeted tissues. Mol Intervent, 2007, 7:147-156.
[61]Hwang PM, Bunz F, Yu J, Rago C, Chan TA, Murphy MP, et al. Ferredoxin reductase affects p53-dependent, 5-fluorouracil-induced apoptosis in colorectal cancer cells. Nat Med, 2001,7:1111-1117.
[62]Ueno M, Masutani H, Arai RJ, Yamaguchi A, Hirota K, Sakai T, et al. Thioredoxin-dependent redox regulation of p53-mediated p21 activation. J Biol Chem,1999, 274: 35809-35815.
[63]Zhou LJ, Zhu XZ. Reactive oxygen species-induced apoptosis in pcl2 cells and protective effect of bilobalide. J Pharmacol Exp Ther, 2000, 293:982-988.
[64]Birjoccio A, Benassi B, Amodei S, Gabellini C, Del D, Zupi G. c-Myc down-regulation increases susceptibility to cisplatin through reactive oxygen species-mediated apoptosis in m14 human melanoma cells. Mol Pharmacol, 2001, 60:174-182.
[65]Jang Y, Chen C, Li Z, Guo W, Geger JA, Lin S, et al. Characterization of the structure and function of a new mitogen-activated protein kinase. J Biol Chem, 1996,271:17920-17926.
[66]Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature, 1994,372:739-746.
[67]Young PR, Mclaughlin MM, Kumar S, Kassis S, Doyle ML, McNulty D, et al. Pyridinyl imidazole ihibitors of p38 mitogen-activated protein kinase bind in the ATP site. J Biol Chem, 1997,272:12116-12121.
[68]Chen QM, Tu VC, Purdon S, Wood J, Dilley T. Molecular mechanisms of cardiac hypertrophy induced by toxicants. Cardiovasc Toxicol, 2001, 1:267-283.
[69]Xia Z, Dickens M, Raingeaud J, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science, 1995, 270: 1326-1331.
[70]Ichijo H, Nishida E, Irie K, ten Dijke P, Saitoh M, Moriguchi T, et al. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science, 1997, 275: 90-94.
[71]Tsuruta F, Sunayama J, Mori Y, Hattori S, Shimizu S, Tsujimoto Y, et al. JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3 proteins.EMBO J, 2004, 23:1889-1899.
[72]Wang XZ, Ron D. Stress-induced phosphorylation and activation of the transcription factor CHOP by p38MAPK. Science, 1996, 272:1347-1349.
[73]Tan Y, Rouse J, Zhang A, Cariati S, Cohen P, Comb MJ. FGF and stress regulate CREB and ATF-1 via a pathway involving p38MAP kinase and MAPKAP kinase-2. EMBO J, 1996,15:4629-4642.
[74]Yin T, Sandhu G, Wolggang CD, Burrier A, Webb RL, Rigel DF. Tissue-specific pattern of stress kinase activation in ischemic/reperfused heart and kidney. J Biol Chem, 1997, 272:19943-19950.
[75]Kumar D, Lou H, Signal PK. Oxidative stress and apoptosis in heart dysfunction. Herz, 2002, 27:662-668.
[76]Kang YJ, Zhou ZX, Wang GW, Burid A, Klein JB. Suppression by metallothionein of doxorubicin-induced cardiomyocyte apoptosis through inhibition of p38MAPK. J Biol Chem, 2000, 275: 13690-13698.
[77]Wang Y, Huang S, Sah VP, Ross J, Brown JH, Han J, et al. Cardiac muscle cell hypertropy and apoptosis induced by distinct members of the p38 mitogen-activated protein kinase family. J Biol Chem, 1998, 273:2161-2168.
[78]Pennica D, Shaw KH, Swanson TA, Moore MW, Shelton DL, Zioncheck KA, et al.Cardiotrophin-1:biological activities and binding to the leukemia inhibitory factor receptor/gp130 signaling complex. J Biol Chem, 1995, 270:10915-10922.
[79]Fujio Y, Kunisada K, Hirota H, Yamauchi-Takihara K, Kishimoto T. Signals through gp130 upregulate bcl-x gene expression via STAT1-binding cis-element in cardiac myocytes. J Clin Invest, 1997, 99:2898-2905.
[80]Sheng Z, Pennica D, Wood WI, Chien KR. Cardiotrophin-1 displays early expression in the murine heart tube and promotes cardiac myocyte survival. Development, 1996, 122:419-428.
[81]Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, et al.Regulation of cell death protease caspase-9 by phosphorylation. Science, 1998,282:1318-1321.
[82]Peso L, Gonzalez-Gacia M, Page C, Herrera R, Nuiez G. Interleukin-3-induced phosphorylation of Bad through the protein kinase Akt. Science, 1997, 278:687-689.
[83]Suhara T, Kim HS, Kirshenbaum LA, Walsh K. Suppression of Akt signaling induces Fas ligand expression: involvment of caspase and Jun kinase activation in Akt-mediated Fas ligand regulation. Mol Cell Biol, 2002, 22:680-691.
[84]Suhara T, Mano T, Oliveira BE, Walsh K. Phosphatidylinositol 3-kinase/Akt signaling controls endothelial cells sensitivity to Fas-mediated apoptosis via regulation of FLICE-inhibitory protein. Circ Res, 2001, 89:13-19.
[85]Panka DJ, Mano T, Suhara T, Walsh K, Mier JW. Phosphatidylinositol 3-kinase/Akt activity regulates c-FLIP expression in tumor cells. J Biol Chem, 2001, 276:6893-6896.
[86]Ahmed NN, Grimes HL, Bellacosa A, Chan TO, Tsichlis PN. Transduction of interleukin-2 antiapoptotic and proliferative signals via Akt protein kinase. Proc Natl Acad Sci USA, 1997, 94:3627-3632.
[87]Yao R, Cooper GM. Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science, 1995, 267:2003-2006.
[88]Negoro S, Oh H, Tone E, Kunisada K, Fujio Y, Walsh K, et al. Glycoprotein 130 regulates cardiac myocyte survival in doxorubicin-induced apoptosis through phosphatidylinositol 3-kinase/Akt phosphorylation and Bcl-xL/caspase-3 interaction. Circulation, 2001,103:555-561.
[89]Wu W, Lee WL, Wu YY, Chen D, Liu TJ, Jiang A, et al. Expression of constitutively active phosphatidylinositol 3-kinase inhibits activation of caspase 3 and apoptosis of cardiac muscle cells. J Biol Chem, 2000, 275:40113-40119.
[90]Lai HC, Liu TJ, Ting CT, Sharma PM, Wang PH. Insulin-like growth factor-1 prevents loss of electrochemical gradient in cardiac muscle mitochondria via actibation of PI3 kinase/Akt pathway. Mol Cell Endocrinol, 2003, 205:99-106.
[91]Zhang J, Clark JR, Herman EH, Ferrans VJ. Doxorubicin-induced apoptosis in spontaneously hypertensive rats: differential effects in heart, kidney and intestine and inhibition by ICRF-187. J Mol Cell Cardiol, 1996, 28:1931-1943.
[92]Saraste A, Pulkki K, Kallajoki M, Heikkila P, Laine P, Mattila S, et al. Cardiomyocyte apoptosis and progression of heart failure to transplantation. Eur J Clin Investig, 1999, 29:380-386.
[93]Zorzato F, Salviati G, Facchinetti T, Volpe P. Doxorubicin induces calcium release from terminal cisternae of skeletal muscle. A study on isolated sarcoplasmic reticulum and chemically skinned fibers. J Biol Chem, 1985, 260:7349-7355.
[94]Kim DH, Landry AB 3rd, Lee YS, Katz AM.Doxorubicin-induced calcium release from cardiac sarcoplasmic reticulum vesicles. J Mol Cell Cardiol, 1989, 21:433-436.
[95]Caroni P, Villani F, Carafoli E. The cardiotoxic antibiotic doxorubicin inhibits the Na+/Ca2+ exchange of dog heart sarcolemmal vesicles. FEBS Lett, 1981,130: 184-186.
[96]Keung EC, Toll L, Ellis M, Jensen RA. L-type cardiac calcium channels in doxorubicin cardiomyopathy in rats morphological, biochemical, and functional correlations. J Clin Invest, 1991, 87:2108-2013.
[97]Kalivendi SV, Konorev EA, Cunningham S, Vanamala SK, Kaji EH, Joseph J, et al. Doxorubicin activates nuclear factor of activated T-lymphocytes and Fas ligand transcription: role of mitochondrial reactive oxygen species and calcium. Biochem J,2005, 389: 527-539.
[98]Kim SY, Kim SJ, Kim BJ, Rah SY, Chung SM, Im MJ, et al. Doxorubicin-induced reactive oxygen species generation and intracellular Ca~(2+) increase are reciprocally modulated in rat cardiomyocytes. Exp Mol Med, 2006, 38: 535-545.
[99]Park KH, Kim SY, Gul R, Kim B J, Jang KY, Chung HT, et al. Fatty acids ameliorate doxorubicin-induced intracellular Ca~(2+) increase and apoptosis in rat cardiomyocytes. Biol Pharm Bull, 2008, 31: 809-815.
[100]#12
[101]Olson RD, Mushlin PS, Brenner DE, Fleischer S, Cusack BJ, Chang BK, et al. Doxorubicin cardiotoxicity may be caused by its metabolite, doxorubicinol. Proc Natl Acad Sci USA, 1988, 85: 3585-3589.
[102]Takemura G, Fujiwara H. Doxorubicin-induced cardiomyopathy from the cardiotoxic mechanisms to managemen. Prog Cardiovasc Dis, 2007, 49: 330-352.
[103]Kim Y, Ma AG, Kitta K, Fitch SN, Ikeda T, Ihara Y, et al. Anthracycline-induced suppression of GATA-4 transcription factor: implication in the regulation of cardiac myocyte apoptosis. Mol Pharmacol, 2003, 63: 368-377.
[104]Aries A, Paradis P, Lefebvre C, Schwartz RJ, Nemer M. Essential role of GATA-4 in cell survival and drug-induced cardiotoxicity. Proc Natl Acad Sci USA, 2004, 101: 6975-6980.
[105]Lou H, Danelisen I, Singal PK. Involvement of mitogen-activated protein kinases in adriamycin-induced cardiomyopathy. Am J Physiol Heart Circ Physiol, 2005, 288: H1925-30.
[2]Giantris A, Abdurrahman L, Hinkle A, et al. Anthracyline-induced cardiotoxicity in children and young adults. Crit Rev Oncol Hematol,1998,27:53-6S.
[3]Kotamraju S, Konorev EA, Joseph J, Kalyanaraman B. Doxorubicin-induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone spin traps and ebselen. Role of reactive oxygen and nitrogen species. J Biol Chem, 2000, 275:33585-33592.
[4]Kang YJ, Chen Y, Epstein PN. Suppression of doxorubicin cardiotoxicity by overexpression of catalase in the heart of transgenic mice. J Biol Chem, 1996, 271:12610-12616.
[5]Kang YJ, Chen Y, Yu A, Voss-McCowan M, Epstein PN. Overexpresssion of metallothionein in the heart of transgenic mice suppresses doxorubicin cardiotoxicity. J Clin Investig, 1997,100:1501-1506.
[6]Doroshow JH, Locker GY, Baldinge RJ, Myers CE. The effect of doxorubicin on hepatic and cardiac glutathione Res Che Pathol Pharmacol, 1979, 26:285-295.
[7]Doroshow JH. Effect of anthracycline antibiotics on oxygen redical formation in rat heart. Cancer Res, 1983, 43:460-472.
[8]Yen HC, Oberley TD, Vichitbandha S, Ho YS, St Clair DK. The protective role of manganese superoxide dismutase against adriamycin-induced acute cardiac toxicity in transgenic mice. J Clin Invest, 1996,98:1253-1260.
[9] Gutierrez PL. The role of NAD(P)H oxidoreductase(DT-dia-phorase) in the bioactivation of quinine-containing antitumor agents. Free Radi Biol Med, 2000, 29:263-275.
[10]Shadle SE, Bammel BP, Cusack BJ, et al. Daunorubicin cardiotoxicity: evidence for the importance of the quinine moiety in a free-radical-independent mechanism. Biochem Pharmacol, 2000, 60:1435-1444.
[11]Wang L, Ma W, Markovich R, Chen JW, Wang PH. Regulation of cardiomyocyte apoptotic signaling by insulin-like growth factor Ⅰ. Circ Res, 1998, 83: 516-512.
[12]Arola OJ, Saraste A, Pulkki K, et al. Acute doxorubicin cardiotoxicity involves cardiomocyte apoptosis. Cancer Res, 2000, 60: 1789-1792.
[13]Orrenius S. Mitochondrial regulation of apoptotic cell death. Toxico Lett, 2004, 149: 19-23.
[14]Wu S, Ko YS, Teng MS, et al. Adriamycin-induced cardiomyocyte and endothelial cell apoptosis: in vitro and in vivo studies. J Mol Cell Cardiol, 2002, 34: 1595-1607.
[15]Green PS and Leeuwenburgh C. Mitochondrial dysfunction is an early indicator of doxorubicin-induced apoptosis. Biochim Biophys Acta, 2002, 1588: 94-101.
[16]Swain SM, Whaley FS, Gerber MC, et al. Cardioprotection with dexrazoxane for doxorubicin-containing therapy in advanced breast cancer. J Clin Oncol, 1997, 15: 1318-1332.
[17]Sehested M, Jensen PB, Sorensen BS, et al. Antagonistic effect of the cardioprotector (+)-1, 2-bis(3,5-dioxopiperazinyl-1-yl)propane(ICRF-187) on DNA breaks and cytotoxicity induced by the topoisomerase Ⅱ directed drugs daunorubicin and etoposide(VP-16). Biochem Pharmacol, 1993, 46: 389-393.
[18]Bian XJ, Bao GR. Research on capability of Emodin and Stilbene Glucoside of scavenging superoxide onions. Fuzhou Zhong Yi Xue Yuan, 2002, 4: 12-13.
[19]Liu HC, Chen WS. Research on antioxidative effects of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucoside in vitro. J Pharm. Pract., 2000, 18: 232-234.
[20]Chu J, Ye CF, Li L. Effects of stilbene-glycoside on learning and memory function and free radicals metabolism in dementia model mice. Chin J Rehabil Theory Practice, 2003, 9: 643-645.
[21]Liu L, Zhao L, Li YL. Zhang L, Ye CF, Li L. Protective effect of 2, 3, 5, 4'-Tetrahydroxystilbene-2-β-O-D-glucoside on hippocampal neurons in dementia rats induced by chronic cerebral ischemia. Chin J Pharm, 2006, 41: 354-357.
[1]班翊，刘其礼，金悠等．二苯乙烯苷的含量测定及其稳定性研究．中草药，2004，35：1235-1237．
[2]Wang X, Zhao L, ttan T, et al. Protective effects of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-d-glucoside, an active component of Polygonum multiflorum Thunb, on experimental colitis in mice. Eur J Pharmacol, 2008, 578: 339-348.
[3]Kotamraju S, Konorev EA, Joseph J, Kalyanaraman B. Doxorubicin-induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone spin traps and ebselen. Role of reactive oxygen and nitrogen species. J Biol Chem, 2000, 275: 33585-33592.
[4]Kang YJ, Chen Y, Epstein PN. Suppression of doxorubicin cardiotoxicity by overexpression of catalase in the heart of transgenic mice. J Biol Chem, 1996, 271: 12610-12616.
[5]Kang YJ, Chen Y, Yu A, Voss-McCowan M, Epstein PN. Overexpresssion of metallothionein in the heart of transgenic mice suppresses doxorubicin cardiotoxicity. J Clin Investig, 1997, 100: 1501-1506.
[6]Doroshow JH, Locker GY, Baldinge RJ, Myers CE. The effect of doxorubicin on hepatic and cardiac glutathione. Res Che Pathol Pharmacol, 1979, 26: 285-295.
[7]Doroshow JH. Effect of anthracycline antibiotics on oxygen redical formation in rat heart. Cancer Res, 1983, 43: 460-472.
[8]Yen HC, Oberley TD, Vichitbandha S, Ho YS, St Clair DK. The protective role of manganese superoxide dismutase against adriamycin-induced acute cardiac toxicity in transgenic mice. J Clin Invest, 1996, 98: 1253-1260.
[9]Goormaghtigh E, Chatelain P, Caspers J, Ruysschaert JM. Evidence of a complex between adriamycin derivatives and cardiolipin: possible role in cardiotoxicity. Biochem Pharmacol, 1980, 29: 3003-3010.
[10]Anderson AB, Arriaga EA. Subcellular metabolite profiles of the parent CCRF-CEM and the derived CEM/C2 cell lines after treatment with doxorubicin. J Chromatogr B Analyt Technol Biomed Life Sci, 2004, 808: 295-302
[11]Tokarska-Schlattner M, Zaugg M, da Silva R, Lucchinetti E, Schaub MC, Wallimann T, et al. Acute toxicity of doxorubicin on isolated perfused heart: response of kinases regulating energy supply. Am J Physiol Heart Circ Physiol, 2005, 289: 37-47.
[12]Doroshow JH, Locker GY, Myers CE. Enzymatic defenses of the mouse heart against reactive oxygen metabolites: alterations produced by doxorubicin. J Clin Invest, 1980, 65: 128-135.
[13]Berlin V, Haseltine WA. Reduction of adriamycin to a semiquinone-free radical by NADPH cytochrome P-450 reductase produces DNA cleavage in a reaction mediated by molecular oxygen. J Biol Chem, 1981, 256: 4747-4756.
[14]Bachur NR, Gordon SL, Gee MV, Kon H. NADPH cytochrome P-450 reductase activation of quinone anticancer agents to free radicals. Proc Natl Acad Sci USA, 1979, 76: 954-957.
[15]De Beer EL, Bottone AE, Voest EE. Doxorubicin and mechanical performance of cardiac trabeculae after acute and chronic treatment: a review. Eur J Pharmacol, 2001, 415: 1-11.
[16]Gianni L, Zweier JL, Levy A, Myers CE. Characterization of the cycle of iron-mediated electron transfer from Adriamycin to molecular oxygen. J Biol Chem, 1985, 260: 6820-6826.
[17]Doroshow JH, Locker GY, Baldinger J,. Myers CE. The effect of doxorubicin on hepatic and cardiac glutathione. Res Commun Chem Pathol Pharmacol, 1979, 2: 85-295.
[18]吕丽爽．何首乌中二苯乙烯苷的体外抗氧化研究．食品科学，2007，28：313-317．
[19]Liu HC, Chen WS. Research on antioxidative effects of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucoside in vitro. J Pharm Pract, 2000, 18: 232-234.
[20]Hu J, Ye CF, Li L. Effects of stilbene-glycoside on learning and memory function and free radicals metabolism in dementia model mice. Chin J Rehabil Theory Practice, 2003, 9: 643-645.
[21]Liu L, Zhao L, Li YL. Zhang L, Ye CF, Li L. Protective effect of 2, 3, 5, 4'-Tetrahydroxystilbene-2-β-O-D-glucoside on hippocampal neurons in dementia rats induced by chronic cerebral ischemia. Chin J Pharm, 2006, 41: 354-357.
[1]Kalyanaraman B, Joseph J, Kalivendi S, et al. Doxorubicin-induced apoptosis: implication in cardiotoxicity. Mol Cell Biochem, 2002, 234-235: 119-124.
[2]Oliverira PG, Santos MS, Wallace KB. Doxorubicin-induccd thiol-dependent alteration of cardiac mitochondrial permeability transition and respiration. Biochemistry, 2006, 71: 194-199.
[3]Green PS, Leeuwenburgh C. Mitochondrial dysfunction is an early indicator doxorubicin-induced apoptosis. Biochim Biophys Acta, 2002, 1588: 94-101.
[4]Fisher PW, Salloum F, Das A, et al. Phosphodiesterase-5 inhibition with sildenafil attenuates cardiomyocyte apoptosis and left ventricular dysfunction in a chronic model of doxorubicin cardiotoxicity. Circulation, 2005, 111: 1601-1610.
[5]Chae HJ, Kim HR, Lee WG, et al. Radiation protects adriamycin-induced apoptosis. Immunopharmocology and immunotoxicology, 2005, 27: 211-232.
[6]Wang L, Ma W, Markovich R, Chen JW, Wang PH. Regulation of cardiomyocyte apoptotic signaling by insulin-like growth factor Ⅰ. Circ Res, 1998, 83: 516-512.
[7]Wang L, Ma W, Markovich R, et al. Insulin-like growth factor Ⅰ mosulates induction of apoptotic signaling in H9C2 cardiac muscle cells. Endocrinology, 1998, 139: 1354-1360.
[8]Kim Y, Ma AG, Kitta K, et al. Anthracycline-induced suppression of GATA-4 transcription factor: implication in the regulation of cardiac myocyte apoptosis. Mol Pharmacol, 2003, 63: 368-377.
[9]Kitta K, Day RM, Kim Y, et al. Hepatocyte growth factor induces GATA-4 phosphorylation and cell survival in cardiac muscle cells. J Biol Chem, 2003, 278: 4705-4712.
[10]Sugawara T, Chan PH. Reactive oxygen radicals and pathogenesis of neuronal death after cerebral ischemia. Antioxid Redox Signal, 2003, 5: 597-607.
[11] Yamada J, Yoshimura S, Yamakawa H, Sawada M, Nakagawa M, Hara S, et al. Cell permeable ROS scavengers, Tiron and Tempol, rescue PC12 cell death caused by pyrogallol or hypoxia/reoxygenation. Neurosci Res, 2003, 45:1-8.
[12]Zhuang S, Lynch MC, Kochecar IE. Caspase-8 mediated caspase-3 activation and cytochrome c release during singlet oxygen-induced apoptosis of HL-60 cells. Exp Cell Res, 1999, 250:203-212.
[13]Clementi ME, Giardina B, Di Stasio E, et al. Doxorubicin-derived metabolites induce release of cytochrome c and inhibition of respiration on cardiac isolated mitochondria. Anticancer Res, 2003, 23: 2445-2450.
[14]Gille L, Nohl H. Analyses of the molecular mechanism of adriamycin-induced cardiotoxicity. Free Radic Biol Med, 1997, 23: 775-782.
[15]Zorzato F, Sarviati G, Facchinetti T, Volpe P. Doxorubicin induces calcium release from terminal cisternae of skeletal muscle. J Cell Biol, 1985, 99:875-85.
[16]Kim DH, Landry AB, Lee YS, Katz AM. Doxorubicin-induced calcium release from sarcoplasmic reticulum vesicles. J Moll Cell Cardiol, 1989, 21:433-436.
[17]Caroni P, Villani F, Carafoli, E. The cardiotoxic antibiotic doxorubicin inhibits the Na~+/Ca~(2+) exchange of dog heart sarcolemmal vesicles. FEBS Lett, 1981, 30:184-186.
[18]Keung EC, Toll L, Ellis M, Jensen RA. L-type cardiac calcium channels in doxorubicin cardiomyopathy in rats morphological, biochemical, and functional correlations. J Clin Invest, 1991, 87:2108-2113.
[19]Przygodzki T, Sokal A, Bryszewska M. Calcium ionophore A 23187 action on cardiomyocytes is accompanied by enhanced production of reactive oxygen species. Biochim Bioph Acta, 2005, 1740:481-488.
[20]Petrosillo G, Ruggiero F, Pistolese M, Paradies, G. Ca~(2+)-induced reactive oxygen species production promotes cytochrome c release from rat liver mitochondria via mitochondirial permeability transition (MPT)-dependent and MPT-independent mechanisms. J Biol Chem, 2004, 279:53103-53108.
[21]Waring P. Redox active calcium ion channels, cell death. Arc Biochem Biophys, 2005, 434:33-42.
[22]Kim SY, Kim SJ, Kim BJ, et al. Doxorubicin-induced reactive oxygen species generation and intracellular Ca~(2+) increase are reciprocally modulated in rat cardiomyocytes. Exp Mol Med, 2006, 38: 535-545.
[23]Kimura Y, Okusa H. Effects of naturally occurring stilbene glucosides from medicinal plants and wine, on tumor growth and lung metastasis in Lewis lung caraionma-bearing mice. J Pharm Pharmacol, 2000, 52: 1287-1292.
[1]李培锋，哈斯苏红，关红。胆红素含量不同培植牛黄的镇静、镇痛及抗惊厥作用研究。内蒙古农牧学院学报，1998，19：22-27。
[2]袁惠南。培植牛黄药物作用和研究。 中国中药杂志，1991，16：105-108
[3]贺春阳，关红，李培锋。人工培育牛黄的药理作用。中草药，1988，19：21-24
[4]蔡红娇，裘法祖，刘仁则。体外培育牛黄的药学研究。中国天然药物，2004，2：335-338．
[5]Waxman SG. The molecular pathophysiology of pain: abnormal expression of sodium channel genes and its contributions to hyperexcitability of primary sensory neurons. Pain, 1999, 6: 132-140.
[6]Porreca F, Lai J, Bian D, et al. A comparison of the potential role of the tetrodotoxin-insensitive sodium channels PN3/SNS and NaN/SNS2 in rat models of chronic pain. Proc Natl Acad Sci USA, 1999, 96: 7640-7644.
[7]Gold MS. Tetrodotoxin-resistant Na~+ currents and inflammatory hyperalgesia. ProcNatlAcad Sci USA, 1999, 96: 7645-7649.
[8]Coward K, Plumpton C, Plumpton C, Facer P, et al. Immunolocalization of SNS/PN3 and NaN/SNS2 sodium channels in human pain states. Pain, 2000, 85: 41-50.
[9]Yiangou Y, Birch R, Sangmeswaran L, et al. SNS/PN3 and SNS2/NaN sodium channel-like immunoreactivity in human adult and neonate injured sensory nerves. FEBS Let, 2000, 467: 249-252.
[10]Lai J, Gold MS, Kim CS, et al. Inhibition of neuropathic pain by decreased expression of the tetrodotoxin-resistant sodium channel NaV1.8. Pain, 2002, 95: 143-152.
[11]Tanaka M, Cummins TR, Ishikawa K, et al. SNS Na~+ channel expression increases in dorsal root ganglion neurons in the carrageenan inflammatory pain model. Neuroreport, 1998, 9: 967-972.
[12]Lyu YS, Park SK, Chung K. Low dose of tetrodotoxin reduces neuropathic pain behaviors in an animal model. Brain Res, 2000, 871: 98-103.
[13]Leigh AS, Emmaunel B. Voltage-gated calcium channels in chronic pain. Eur J Physiol, 2008, 456:459-466.
[14]Zamponi GW, Lewis RJ, Todorovic SM, et al. Role of voltage-gated calcium chnnels in ascending pain pathways. Brain Res Rev, 2009, 60:84-89.
[15]Vanegas H, Schaible H. Efeects of antagonists to high-threshold calcium channels upon spinal mechanisms of pain, hyperalgesia and allodynia. Pain, 2000, 85:9-18.
[16]Hara K, Saito Y, Kirihara Y, et al. Antinociceptive effects of intrathecal L-type calcium channel blocker on visceral an somatic stimuli in the rat.Anesth Analg, 1998, 87:382-389.
[17]Bowersox SS, Gadbois T, Singh T, et al. Selective N-type neuraonal voltage-sensitive calcium channel blocker, SNX-111, produces spinal antinociception in rat models of acute persistent and neuropathic pain. J Pharmacol Exp Ther, 1996, 279:1243-1249.
[18]Elizabeth AM, Anthony H. Effects of ethosuximide, a T-type Ca~(2+) channel blocker, on dorsal horn neuronal responses in rats. Europ J Pharm, 2001, 415:141-149.
[19]Kim HC, Chung MK. Voltage-dependent sodium and calcium currents in acutely isolated adult rat trigeminal root ganglion neurons. J Neurophysiol, 1999, 81:1123-1134.
[1]Signal PK, Iliskovic N. Doxorubicin-induced cardiomyopathy. N Engl J Med, 1998, 339:900-905.
[2]Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev, 2004, 56:185-229.
[3]Goormaghtigh E, Chatelain P, Caspers J, Ruysschaert JM. Evidence of a complex between adriamycin derivatives and cardiolipin: possible role in cardiotoxicity. Biochem Pharmacol, 1980, 29:3003-3010.
[4]Anderson AB, Arriaga EA. Subcellular metabolite profiles of the parent CCRF-CEM and the derived CEM/C2 cell lines after treatment with doxorubicin. J Chromatogr B Analyt Technol Biomed Life Sci, 2004, 808:295-302
[5]Tokarska-Schlattner M, Zaugg M,da Silva R, Lucchinetti E, Schaub MC, Wallimann T,et al. Acute toxicity of doxorubicin on isolated perfused heart: response of kinases regulating energy supply. Am J Physiol Heart Circ Physiol, 2005, 289:37-47.
[6]Doroshow JH, Locker GY, Myers CE. Enzymatic defenses of the mouse heart against reactive oxygen metabolites: alterations produced by doxorubicin. J Clin Invest, 1980, 65:128-135.
[7]Berlin V, Haseltine WA. Reduction of adriamycin to a semiquinone-free radical by NADPH cytochrome P-450 reductase produces DNA cleavage in a reaction mediated by molecular oxygen. J Biol Chem, 1981, 256:4747-4756.
[8]Bachur NR, Gordon SL, Gee MV, Kon H. NADPH cytochrome P-450 reductase activation of quinone anticancer agents to free radicals. Proc Natl Acad Sci USA, 1979,76:954-957.
[9]De Beer EL, Bottone AE, Voest EE. Doxorubicin and mechanical performance of cardiac trabeculae after acute and chronic treatment: a review. Eur J Pharmacol, 2001, 415:1-11.
[10]Gianni L, Zweier JL, Levy A, Myers CE. Characterization of the cycle of iron-mediated electron transfer from Adriamycin to molecular oxygen. J Biol Chem, 1985, 260:6820-6826.
[11]Solern LE, Heller LJ, Wallace KB. Dose-dependent increase in sensitivity to calcium-induced mitochondrial dysfunction and cardiomyocyte cell injury by doxorubicin. J Mol Cell Cardiol, 1996, 28:1023-1032.
[12]Green PS, Leeuwenburgh C. Mitochondrial dysfunction is an early indicator of doxorubicin-induced apoptosis. Biochim Biophys Acta, 2002,1588:94-101.
[13]Palmeira CM, Serrano J, Kuehl DW, Wallace KB. Preferential oxidation of cardiac mitochondrial DNA following acute intoxication with doxorubicin. Biochim Biophys Acta, 1997,1321:101-106.
[14]Zhou S, Palmeira CM, Wallace KB. Doxorubicin-induced persistent oxidative stress to cardiac myocytes. Toxicol Lett, 2001,121:151-157.
[15]Kotamraju S, Chitambar CR, Kalivendi SV, Joseph J, Kalyanaraman B. Transferrin receptor-dependent iron uptake is responsible for doxorubicin-mediated apoptosis in endothelial cells: role of oxidant-induced iron signaling in apoptosis. J Biol Chem, 2002,277:17179-17187.
[16]Minotti G. Sources and role of iron in lipid peroxidation. Chem Res Toxicol, 1993, 6: 134-146.
[17]Cairo G, Recalcati S, Pietrangelo A, and Minotti G. The iron regulatory proteins: targets and modulators of free radical reactions and oxidative damage. Free Radic Biol Med, 2002,32: 1237-1243.
[18]Minotti G, Cairo G, and Monti E. Role of iron in anthracycline cardiotoxicity: new tunes for an old song? FASEB J, 1999,13:199-212.
[19]Kwok JC and Richardson DR. Anthracyclines induce accumulation of iron in ferritin in myocardial and neoplastic cells: inhibition of the ferritin iron mobilization pathway. Mol Pharmacol, 2003, 63: 849-861.
[20]Kwok JC and Richardson DR. Examination of the mechanism(s) involved in doxorubicin-mediated iron accumulation in ferritin: studies using metabolic inhibitors, protein synthesis inhibitors and lysosornotropic agents. Mol Pharmacol, 2004, 65: 181-195.
[21]Hasinoff BB, Schnabl KL, Marusak RA, Patel D, and Huebner E. Dexrazoxane (ICRF-187) protects cardiac myocytes against doxorubicin by preventing damage to mitochondria. Cardiovasc Toxicol, 2003, 3: 89-99.
[22]Sawyer DB, Fukazawa R, Arstall MA, and Kelly RA. Daunorubicin-induced apoptosis in rat cardiac myocytes is inhibited by dexrazoxane. Circ Res, 1999, 84: 257-265.
[23]Kaiserov(?) H, den Hartog GJ, Simunek T, Schr(?)terov(?) L, Kvasnickov(?) E, Bast A. Iron is not involved in oxidative stress-mediated cytotoxicity of doxorubicin and bleomycin. Br J Pharmacol, 2006,149: 920-930.
[24]van Acker SA, van Balen GP, van den Berg DJ, Bast A, van der Vijgh WJ. Influence of iron chelation on the antioxidant activity of flavonoids. Biochem Pharmacol, 1998, 56: 935-943.
[25]Wencker D, Chandra M, Nguyen K, Miao W, Garantziotis S, Factor SM, Shirani J,Armstrong RC, and Kitsis RN. A mechanistic role for cardiac myocyte apoptosis in heart failure. J Clin Investig, 2003, 111: 1497-1504.
[26]Narula J, Haider N, Virmani R, Disalvo TG, Kilodgie FD, Hajjar RJ, et al. Apoptosis in myocytes in end-stage heart failure. N Engl J Med, 1996, 335:1182-1189.
[27]Feuerstein GZ. Apoptosis-new opportunities for novel therapeutics for heart diseases. Cardiovusc Drugs Ther, 2001, 15:547-551.
[28]Wang L, Ma W, Markovich R, Chen JW, Wang PH. Regulation of cardiomyocyte apoptotic signaling by insulin-like growth factor I. Circ Res, 1998, 83:516-522.
[29]Kotamraju S, Konorev EA, Joseph J, Kalyanaraman B. Doxorubicin-induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone spin traps and ebselen. Role of reactive oxygen and nitrogen species. J Biol Chem, 2000, 275:33585-33592.
[30]Arola OJ, Saraste A, Pulkki K, Kallajoki M, Parvinen M, Voipio-Pulkki LM. Acute doxorubicin cardiotoxicity involves cardiomyocyte apoptosis. Cancer Res, 2000, 60: 1789-1792.
[31]Kotamraju S, Konorev EA, Joseph J, Kalyanaraman B. Doxorubicin induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone sp in traps and ebselen. Role of reactive oxygen and nitrogen species. J Biol Chem, 2000, 275:33585 - 33592.
[32]ChildsAC, Phaneuf SL, DirksAJ, Phillip s T, Leeuwenburgh C.Doxorubicin treatment in vivo causes cytochrome C release and cardiomyocyte apoptosis, as well as increased mitochondrial efficiency, superoxide dismutase activity, and Bcl2 : Bax ratio. Cancer Res, 2002, 62:4592 - 4598.
[33] Wang GW, Zhou ZX, Kang YJ. Effect of metallothionein on doxorubicin induced apoptosis in cardiomyocytes: role of cytochrome C activated caspase-3. Toxicologist, 2000,54:114-121.
[34]Wang GW, Klein JB, Kang YJ. Metallothionein inhibits doxorubicin induced mitochondrial cytochrome C release and caspase-3 activation in cardiomyocytes. J Pharm acol Exp Ther, 2001, 298:461 - 468.
[35]Sugawara T, Chan PH. Reactive oxygen radicals and pathogenesis of neuronal death after cerebral ischemia.Antioxid Redox Signal, 2003, 5:597-607
[36]Yamada J, Yoshimura S, Yamakawa H, Sawada M, Nakagawa M, Hara S, et al. Cell permeable ROS scavengers, Tiron and Tempol, rescue PC12 cell death caused by pyrogallol or hypoxia/reoxygenation. Neurosci Res, 2003, 45:1-8.
[37]Zhuang S, Lynch MC, Kochecar IE. Caspase-8 mediated caspase-3 activation and cytochrome c release during singlet oxygen-induced apoptosis of HL-60 cells. Exp Cell Res, 1999, 250:203-212.
[38]Sugioka R, Shimizu S, Funatsu T, Tamagawa H, Sawa Y, Kawakaml T, et al. BH-4 domain peptide from Bcl-xL exerts anti-apoptotic activity in vivo. Oncogene, 2003, 22:8432-8440.
[39]Kunisada K, Tone E, Negoro S, Nakaoka Y, Oshima Y, Osugi T, et al. Bcl-xL reduces doxorubicin-induced myocardial damage but fails to control cardiac gene downregulation. Cardiovasc Res, 2002, 53:936-943.
[40]Chen Z, Chua CC, Ho YS, Harndy RC, Chua BH. Gverexpression of Bcl-2 attenuates apoptosis and protects against myocardial I/R injury in transgenie mice. Am J Physiol Heart Circ Physiol, 2001, 280:2313-2320.
[41]Wang L, Ma W, Markovich R, Chen JW, Wang PH. Regulation of cardiomyocyte apoptotic signaling by insulin-like growth factor Ⅰ. Circ Res, 1998, 83:516-512.
[42]Wang L, Ma W, Markovich R, et al. Insulin-like growth factor Ⅰ mosulates induction of apoptotic signaling in H9C2 cardiac muscle cells. Endocrinology, 1998,139:1354-1360.
[43]Kim Y, Ma AG, Kitta K, et al. Anthracycline-induced suppression of GATA-4 transcription factor: implication in the regulation of cardiac myocyte apoptosis. Mol Pharmacol, 2003, 63:368-377.
[44]Kitta K, Day RM, Kim Y, et al. Hepatocyte growth factor induces GATA-4 phosphorylation and cell survival in cardiac muscle cells. J Biol Chem, 2003, 278:4705-4712.
[45]Nakamura T, Ueda Y, Juan Y, et al. Fas-mediated apoptosis in adriamycin-induced cardiomyopathy in rats in vivo study. Circulation, 2002,102:572-578.
[46]Yamaoka M, Yamaguchi S, Suzuki T, et al. Apoptosis in rat cardiacmyocytes induced by Fas ligand:priming for Fas-mediated apoptosis with doxorubicin. J Mol Cell Cardol, 2002, 32:881-889.
[47]Kalivendi SV, Konorev EA, Cunningham S, et al. Doxorubicin activates nuclear factor of activated T-lymphocytes and Fas ligand transcription: role of mitochondrial reactive oxygen species and calcium. Biochem J, 2005, 389:527-539.
[48]Janice LR, Eva S, Susan EL, Triona NC, Timothy O, Thomas R, et al. Distinct mechanisms of cardiomyocyte apoptosis induced by doxorubicin and hypoxia converge on mitochondria and are inhibited by Bcl-xL. J Cell Mol Med, 2007,11:509-520.
[49]Tanaka M, Ito H, Adachi S, Akimoto H, Nishikawa T, Kasajima T, et al. Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes. Circ Res, 1994, 75:426-433.
[50]Yaniv G, Shilkrut M, Lotan R, Berke G,Larisch S, Binah O. Hypoxia predisposes neonatal rat ventricular myocytes to apoptosis induced by activation of the Fas receptor:Fas activation and apoptosis in hypoxic myocytes. Cardiovasc Res, 2002, 54:611-623.
[51]Nitobe J, Yamaguchi S, Okuyama M, Nozaki N, Sata M, Miyamoto T, et al. Reactive oxygen species regulate FLICE inhibitory protein(FLIP) and susceptibility to Fas-mediated apoptosis in cardiac myocytes. Cardiovasc Res, 2003, 57:119-128.
[52]Gang L.Regulation of the p53 transcriptional activity. J Cell Biochem, 2006,97:448-458.
[53]Shizukuda Y, Matoba S, Mian OY. Targeted disruption of p53 attenuates doxorubicin-induced cardiac toxicity in mice. Mol Cell Biochem, 2005, 273:25-28.
[54]Youn HJ, Kim HS, Jeon MH, et al. Induction of caspase-independent apoptosis in H9C2 cardiomyocytes by adriamycin treatment. Mol Cell Biochem, 2005, 270:13-19.
[55]Liu X, Chua CC, Gao J. Pifithrin-alpha protects against doxorubicin-induced apoptosis and acute cardiotoxicity in mice.Am J Physiol Heart Physiol, 2004, 286:933-939.
[56]Wang S, Leonard SS, Ye J, Ding M, Shi X. The role of hydroxyl radical as a messenger in Cr(Ⅵ)-induced p53 activation. Am J Physiol Cell Physiol, 2000, 279: C868-875.
[57]Huang C, Zhang Z, Ding M, Li J, Ye J, Leonard SS, et al. Vanadate induces p53 transactivation through hydrogen peroxide and causes apoptosis. J Biol Chem, 2000,275:32516-32522.
[58]Zhao Y, Chaiswing L, Velez JM, Batinic I, Colbum NH, Oberley TD, et al. p53 translocation to mitochondria preccedes its nuclear translotion and targets mitichondrial oxidative defense protein-manganese superoxide dismutase. Cancer Res, 2005, 65:3745-3750.
[59]Sayan BS, Sayan AE, Knight RA, Melino G, Cohen GM. P53 is cleaved by caspases generating fragments localizing to mitochondria. J Biol Chem, 2006, 281:13566-13573.
[60]Chen Y, Jungsuwadee P, Vore M, Butterfield DA. Collateral damage in cancer chemotherapy: oxidative stress in nontargeted tissues. Mol Intervent, 2007, 7:147-156.
[61]Hwang PM, Bunz F, Yu J, Rago C, Chan TA, Murphy MP, et al. Ferredoxin reductase affects p53-dependent, 5-fluorouracil-induced apoptosis in colorectal cancer cells. Nat Med, 2001,7:1111-1117.
[62]Ueno M, Masutani H, Arai RJ, Yamaguchi A, Hirota K, Sakai T, et al. Thioredoxin-dependent redox regulation of p53-mediated p21 activation. J Biol Chem,1999, 274: 35809-35815.
[63]Zhou LJ, Zhu XZ. Reactive oxygen species-induced apoptosis in pcl2 cells and protective effect of bilobalide. J Pharmacol Exp Ther, 2000, 293:982-988.
[64]Birjoccio A, Benassi B, Amodei S, Gabellini C, Del D, Zupi G. c-Myc down-regulation increases susceptibility to cisplatin through reactive oxygen species-mediated apoptosis in m14 human melanoma cells. Mol Pharmacol, 2001, 60:174-182.
[65]Jang Y, Chen C, Li Z, Guo W, Geger JA, Lin S, et al. Characterization of the structure and function of a new mitogen-activated protein kinase. J Biol Chem, 1996,271:17920-17926.
[66]Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature, 1994,372:739-746.
[67]Young PR, Mclaughlin MM, Kumar S, Kassis S, Doyle ML, McNulty D, et al. Pyridinyl imidazole ihibitors of p38 mitogen-activated protein kinase bind in the ATP site. J Biol Chem, 1997,272:12116-12121.
[68]Chen QM, Tu VC, Purdon S, Wood J, Dilley T. Molecular mechanisms of cardiac hypertrophy induced by toxicants. Cardiovasc Toxicol, 2001, 1:267-283.
[69]Xia Z, Dickens M, Raingeaud J, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science, 1995, 270: 1326-1331.
[70]Ichijo H, Nishida E, Irie K, ten Dijke P, Saitoh M, Moriguchi T, et al. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science, 1997, 275: 90-94.
[71]Tsuruta F, Sunayama J, Mori Y, Hattori S, Shimizu S, Tsujimoto Y, et al. JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3 proteins.EMBO J, 2004, 23:1889-1899.
[72]Wang XZ, Ron D. Stress-induced phosphorylation and activation of the transcription factor CHOP by p38MAPK. Science, 1996, 272:1347-1349.
[73]Tan Y, Rouse J, Zhang A, Cariati S, Cohen P, Comb MJ. FGF and stress regulate CREB and ATF-1 via a pathway involving p38MAP kinase and MAPKAP kinase-2. EMBO J, 1996,15:4629-4642.
[74]Yin T, Sandhu G, Wolggang CD, Burrier A, Webb RL, Rigel DF. Tissue-specific pattern of stress kinase activation in ischemic/reperfused heart and kidney. J Biol Chem, 1997, 272:19943-19950.
[75]Kumar D, Lou H, Signal PK. Oxidative stress and apoptosis in heart dysfunction. Herz, 2002, 27:662-668.
[76]Kang YJ, Zhou ZX, Wang GW, Burid A, Klein JB. Suppression by metallothionein of doxorubicin-induced cardiomyocyte apoptosis through inhibition of p38MAPK. J Biol Chem, 2000, 275: 13690-13698.
[77]Wang Y, Huang S, Sah VP, Ross J, Brown JH, Han J, et al. Cardiac muscle cell hypertropy and apoptosis induced by distinct members of the p38 mitogen-activated protein kinase family. J Biol Chem, 1998, 273:2161-2168.
[78]Pennica D, Shaw KH, Swanson TA, Moore MW, Shelton DL, Zioncheck KA, et al.Cardiotrophin-1:biological activities and binding to the leukemia inhibitory factor receptor/gp130 signaling complex. J Biol Chem, 1995, 270:10915-10922.
[79]Fujio Y, Kunisada K, Hirota H, Yamauchi-Takihara K, Kishimoto T. Signals through gp130 upregulate bcl-x gene expression via STAT1-binding cis-element in cardiac myocytes. J Clin Invest, 1997, 99:2898-2905.
[80]Sheng Z, Pennica D, Wood WI, Chien KR. Cardiotrophin-1 displays early expression in the murine heart tube and promotes cardiac myocyte survival. Development, 1996, 122:419-428.
[81]Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, et al.Regulation of cell death protease caspase-9 by phosphorylation. Science, 1998,282:1318-1321.
[82]Peso L, Gonzalez-Gacia M, Page C, Herrera R, Nuiez G. Interleukin-3-induced phosphorylation of Bad through the protein kinase Akt. Science, 1997, 278:687-689.
[83]Suhara T, Kim HS, Kirshenbaum LA, Walsh K. Suppression of Akt signaling induces Fas ligand expression: involvment of caspase and Jun kinase activation in Akt-mediated Fas ligand regulation. Mol Cell Biol, 2002, 22:680-691.
[84]Suhara T, Mano T, Oliveira BE, Walsh K. Phosphatidylinositol 3-kinase/Akt signaling controls endothelial cells sensitivity to Fas-mediated apoptosis via regulation of FLICE-inhibitory protein. Circ Res, 2001, 89:13-19.
[85]Panka DJ, Mano T, Suhara T, Walsh K, Mier JW. Phosphatidylinositol 3-kinase/Akt activity regulates c-FLIP expression in tumor cells. J Biol Chem, 2001, 276:6893-6896.
[86]Ahmed NN, Grimes HL, Bellacosa A, Chan TO, Tsichlis PN. Transduction of interleukin-2 antiapoptotic and proliferative signals via Akt protein kinase. Proc Natl Acad Sci USA, 1997, 94:3627-3632.
[87]Yao R, Cooper GM. Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science, 1995, 267:2003-2006.
[88]Negoro S, Oh H, Tone E, Kunisada K, Fujio Y, Walsh K, et al. Glycoprotein 130 regulates cardiac myocyte survival in doxorubicin-induced apoptosis through phosphatidylinositol 3-kinase/Akt phosphorylation and Bcl-xL/caspase-3 interaction. Circulation, 2001,103:555-561.
[89]Wu W, Lee WL, Wu YY, Chen D, Liu TJ, Jiang A, et al. Expression of constitutively active phosphatidylinositol 3-kinase inhibits activation of caspase 3 and apoptosis of cardiac muscle cells. J Biol Chem, 2000, 275:40113-40119.
[90]Lai HC, Liu TJ, Ting CT, Sharma PM, Wang PH. Insulin-like growth factor-1 prevents loss of electrochemical gradient in cardiac muscle mitochondria via actibation of PI3 kinase/Akt pathway. Mol Cell Endocrinol, 2003, 205:99-106.
[91]Zhang J, Clark JR, Herman EH, Ferrans VJ. Doxorubicin-induced apoptosis in spontaneously hypertensive rats: differential effects in heart, kidney and intestine and inhibition by ICRF-187. J Mol Cell Cardiol, 1996, 28:1931-1943.
[92]Saraste A, Pulkki K, Kallajoki M, Heikkila P, Laine P, Mattila S, et al. Cardiomyocyte apoptosis and progression of heart failure to transplantation. Eur J Clin Investig, 1999, 29:380-386.
[93]Zorzato F, Salviati G, Facchinetti T, Volpe P. Doxorubicin induces calcium release from terminal cisternae of skeletal muscle. A study on isolated sarcoplasmic reticulum and chemically skinned fibers. J Biol Chem, 1985, 260:7349-7355.
[94]Kim DH, Landry AB 3rd, Lee YS, Katz AM.Doxorubicin-induced calcium release from cardiac sarcoplasmic reticulum vesicles. J Mol Cell Cardiol, 1989, 21:433-436.
[95]Caroni P, Villani F, Carafoli E. The cardiotoxic antibiotic doxorubicin inhibits the Na+/Ca2+ exchange of dog heart sarcolemmal vesicles. FEBS Lett, 1981,130: 184-186.
[96]Keung EC, Toll L, Ellis M, Jensen RA. L-type cardiac calcium channels in doxorubicin cardiomyopathy in rats morphological, biochemical, and functional correlations. J Clin Invest, 1991, 87:2108-2013.
[97]Kalivendi SV, Konorev EA, Cunningham S, Vanamala SK, Kaji EH, Joseph J, et al. Doxorubicin activates nuclear factor of activated T-lymphocytes and Fas ligand transcription: role of mitochondrial reactive oxygen species and calcium. Biochem J,2005, 389: 527-539.
[98]Kim SY, Kim SJ, Kim BJ, Rah SY, Chung SM, Im MJ, et al. Doxorubicin-induced reactive oxygen species generation and intracellular Ca~(2+) increase are reciprocally modulated in rat cardiomyocytes. Exp Mol Med, 2006, 38: 535-545.
[99]Park KH, Kim SY, Gul R, Kim B J, Jang KY, Chung HT, et al. Fatty acids ameliorate doxorubicin-induced intracellular Ca~(2+) increase and apoptosis in rat cardiomyocytes. Biol Pharm Bull, 2008, 31: 809-815.
[100]#12
[101]Olson RD, Mushlin PS, Brenner DE, Fleischer S, Cusack BJ, Chang BK, et al. Doxorubicin cardiotoxicity may be caused by its metabolite, doxorubicinol. Proc Natl Acad Sci USA, 1988, 85: 3585-3589.
[102]Takemura G, Fujiwara H. Doxorubicin-induced cardiomyopathy from the cardiotoxic mechanisms to managemen. Prog Cardiovasc Dis, 2007, 49: 330-352.
[103]Kim Y, Ma AG, Kitta K, Fitch SN, Ikeda T, Ihara Y, et al. Anthracycline-induced suppression of GATA-4 transcription factor: implication in the regulation of cardiac myocyte apoptosis. Mol Pharmacol, 2003, 63: 368-377.
[104]Aries A, Paradis P, Lefebvre C, Schwartz RJ, Nemer M. Essential role of GATA-4 in cell survival and drug-induced cardiotoxicity. Proc Natl Acad Sci USA, 2004, 101: 6975-6980.
[105]Lou H, Danelisen I, Singal PK. Involvement of mitogen-activated protein kinases in adriamycin-induced cardiomyopathy. Am J Physiol Heart Circ Physiol, 2005, 288: H1925-30.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |