丹参素异丙酯的药效学研究
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摘要
自由基(Free Radical,FR)通常指能独立存在的带有未成对电子的原子、原子团、分子或离子,包括超氧阴离子自由基(O_2~-·),羟自由基(·OH)、脂氧自由基(LO·)等。在生理情况下,自由基在体内不断产生,也不断被消除,不但对机体无害,而且还有有益作用;但是在病理情况下,机体由于自由基产生增加、清除自由基能力减弱等原因导致体内自由基过剩,这些过剩的自由基将会导致不良的生物学效应,从而对机体构成危害。大量的研究表明:有超过100种疾病与自由基代谢紊乱有关,包括各种癌症、心脑血管疾病、胃肠道疾病等。因此,深入研究自由基对机体的生理、病理作用及其与疾病的关系对与其有关疾病的防治具有重要意义。
复方丹参滴丸(Compound danshen dropping prill,CDDP)是一种中成药,主要成分有丹参、三七、冰片,具有活血化瘀、理气止痛等作用。我们的课题协作组在对复方丹参滴丸体内代谢产物的研究中发现丹参素异丙酯(Danshensuisopropyl ister,DIE)是复方丹参滴丸在体内的一种活性代谢物,其常温下为棕黄色液体,易溶于水、甲醇、乙醇、乙酸乙酯等溶剂。在对其研究中发现丹参素异丙酯具有扩张大鼠肠系膜动脉,增加大鼠心室肌收缩力等作用,而关于其抗自由基损伤及其他药理作用及其作用机制尚未研究。本课题主要从以下三个方面来研究丹参素异丙酯的药理作用:(1)建立D-半乳糖(D-galactose,D-gal)致衰老模型,观察衰老大鼠小脑组织SOD活力、MDA含量和浦肯野细胞凋亡指数的改变及丹参素异丙酯对它们的影响,并用维生素E(Vitamine E,VE)作为阳性对照;(2)利用Langendroff离体灌流技术,建立大鼠心肌缺血再灌注损伤模型,观察丹参素异丙酯对离体大鼠模拟缺血再灌注损伤心肌线粒体结构改变及能量物质代谢的影响,并用复方丹参滴丸作为阳性对照;(3)利用Langendroff灌流技术,建立外源性自由基致离体大鼠心律失常模型,观察丹参素异丙酯对外源性自由基所致心律失常(Cardiac arrhythmias,CA)的影响,并用维拉帕米(Verapamil,VPL)作为阳性对照。
研究方法
第一部分:取健康6月龄Wistar大鼠80只,雌雄各半,随机分为空白对照组、衰老模型组、阳性对照药(维生素E 100.0 mg/kg)组、丹参素异丙酯21.0 mg/kg和7.0 mg/kg两个剂量组,共5组,每组16只。空白对照组:每天皮下注射4 mL/kg的0.85%生理盐水;衰老模型组:每天皮下注射2.5%D-半乳糖2mL/kg的同时皮下注射0.85%生理盐水2.0 mL/kg;各给药组:每天皮下注射2.5%D-半乳糖2.0 mL/kg的同时,皮下注射相应浓度的药物2.0 mL/kg。每天一次,连续8周。末次给药1小时后对各组动物进行处理,各组一半动物小脑用于测定大鼠小脑组织中的超氧化物歧化酶(Superoxide dismutase,SOD)活性及丙二醛(Malondialdehyde,MDA)含量;另一半动物小脑采用甲基绿—哌洛宁染色法计数各组大鼠小脑皮质浦肯野细胞的凋亡指数(Apoptotic index,AI)。
第二部分:取健康Wistar大鼠80只,随机分为空白对照组、单纯缺血再灌注模型组、阳性对照药(复方丹参滴丸50.00 mg/L)前保护及后保护组、丹参素异丙酯4.00 mg/L和0.04 mg/L两个剂量的前保护及后保护组。共8组,每组10只。利用Langendroff灌流技术,制备心肌缺血再灌注损伤模型,分别在缺氧前及缺氧后,在灌流液中加入复方丹参滴丸或丹参素异丙酯对心肌给予保护。空白对照组:用Krebs-Hanseleit磷酸缓冲液持续灌流75 min;单纯缺血再灌注模型组:用Krebs-Hanseleit磷酸缓冲液预灌15 min,然后停灌40 min,再恢复灌流20min;各用药组:分别在预灌或再灌时用加入相应浓度药物的灌流液灌流,其他处理同单纯缺血再灌注模型组。各组所用灌流液的药物浓度分别是50.00 mg/L、50.00 mg/L、4.00 mg/L、4.00 mg/L、0.04 mg/L、0.04 mg/L。各组大鼠心脏灌流结束后,7只动物心脏采用高效液相色谱仪测定其组织中的高能磷酸化合物的含量变化;剩余3只动物利用H-7500透射电镜观察心肌细胞超微结构。
第三部分:取健康Wistar大鼠50只,随机分为空白对照组、心律失常模型组、阳性对照药(维拉帕米1.00 mg/L)组、丹参素异丙酯4.00 mg/L、0.04 mg/L两个剂量组,共5组,每组10只。采用Langendorf灌流装置对大鼠离体心脏灌注硫酸亚铁(0.025 mmol/L)/抗坏血酸(0.100 mmol/L)的方法,复制自由基致心律失常的模型,并观察丹参素异丙酯对其影响。空白对照组:离体心脏用Krebs-Hanseleit磷酸缓冲液灌注45 min;心律失常模型组:离体心脏用Krebs-Hanseleit磷酸缓冲液预灌15 min后,灌流液中加入硫酸亚铁(0.025mmol/L)/抗坏血酸(0.100 mmol/L)持续灌注30min;阳性对照药(维拉帕米1.00 mg/L)组、丹参素异丙酯4.00 mg/L、0.04 mg/L两个剂量组用加入相应浓度药物的Krebs-Hanseleit磷酸缓冲液预灌15 min,其余处理同心律失常模型组。每只心脏在灌流的同时,将心电图机上的导联线通过电极分别放在右心房和左心室尖部并记录心电图。
结果
第一部分:与空白对照组比较,衰老模型组大鼠小脑组织SOD活性降低、MDA含量增加、蒲肯野细胞的凋亡指数增加(P＜0.01);阳性对照药(维生素E100.0 mg/kg)组、丹参素异丙酯21.0 mg/kg和7.0 mg/kg两个剂量组与衰老模型组相比,小脑组织SOD活性均明显增加、MDA含量显著降低,蒲肯野细胞凋亡指数显著降低(P＜0.01);丹参素异丙酯21.0 mg/kg组与维生素E 100.0 mg/kg组的SOD活力、MDA含量、蒲肯野细胞的凋亡指数差异无显著性(P＞0.05);丹参素异丙酯7.0 mg/kg组与前两组相比大鼠小脑组织SOD活性降低、MDA含量增加、浦肯野细胞的凋亡指数增加(P＜0.05)。
第二部分:高能磷酸化合物(High energy phosphates,HEP)的含量:与空白对照组相比,模型组的三磷酸腺苷(Adenosine triphosphate,ATP)、二磷酸腺苷(Adenosine diphosphate,ADP)、一磷酸腺苷(Adenosine monophosphate,AMP)及腺嘌呤核苷酸总量(Total adenine nucleotides,TAN)显著降低(P＜0.01);复方丹参滴丸50.00 mg/L前保护和后保护组、丹参素异丙酯4.00 mg/L前保护和后保护组及丹参素异丙酯0.04 mg/L前保护和后保护组的ADP、AMP及AN与模型组相比显著升高(P＜0.01)(丹参素异丙酯0.04 mg/L前保护组的ADP除外);丹参素异丙酯0.04 mg/L前保护组的ADP除外与模型组相比有一定升高(P＜0.05);复方丹参滴丸50.00 mg/L前保护和后保护组、丹参素异丙酯4.00 mg/L前保护和后保护组的ATP含量与模型组相比升高(P＜0.05)。电镜显示:空白对照组心肌肌原纤维排列整齐,明暗带清晰,心肌细胞核膜和线粒体膜完整,染色质均匀、细密,线粒体基质致密,嵴排列紧密整齐,线粒体和糖原颗粒丰富;单纯缺氧/复氧模型组心肌细胞超微结构损伤最严重,心肌细胞有少量轻度坏死区,肌节排列紊乱或消失,核的一端基质明显水肿,线粒体大部分或全部的脊和膜融合或消失,线粒体和糖原数量明显减少;复方丹参滴丸50.00 mg/L前保护、后保护组及丹参素异丙酯4.00 mg/L前保护、后保护组超微结构接近空白对照组,损伤较轻;丹参素异丙酯0.04 mg/L前保护、后保护组心肌细胞超微结构的损伤与模型组相比有所改善,但损伤仍较严重。
第三部分:空白对照组离体大鼠心脏无一例发生心律失常;模型组离体大鼠心脏总心律失常发生率100%,主要发生传导阻滞和室性早搏两种类型的心律失常;阳性对照药(维拉帕米1.00 mg/L)组、丹参素异丙酯4.00 mg/L、0.04 mg/L两个剂量组与模型组相比,心律失常发生率明显降低。
结论
1.丹参素异丙酯具有抗氧化作用及抗凋亡作用,与阳性对照药维生素E相似。
2.丹参素异丙酯具有保护缺血再灌注损伤心肌的作用,与阳性对照药复方丹参滴丸相似。
3.丹参素异丙酯具有抗外源性自由基生成系统所致心律失常的作用,与阳性对照药维拉帕米相似。
Free radicals are atomic or molecular species with unpaired electrons on an otherwise open shell configuration, such as superoxide anion radical (O_2~-·), hydroxyl radical (OH), Lipid alkoxyl radical (LO·). Under normal conditions, free radicals are continuously produced and eliminated at good balance and benefit the body; while, under pathological conditions, excessive free radicals can be accumulated in the body due to either increase in generation or decrease in elimination, which will cause damages to the body. Many studies showed that over 100 kinds of diseases are related to metabolic disturbance of free radical. For example, cancers, cardio-cerebrovascular diseases, gastrointestinal diseases and so on. Therefore, it is important to illuminate the physiological and pathologic effects of free radical in order to prevent and treat the diseases caused by free radicals.
Compound danshen dropping prill (CDDP), a combination of Radix Salviae Miltiorrhizae, Radix Notoginseng and Borneolum Syntheticum, is a Chinese remedy, which has been reported has the effects of activating the blood and reducing phlegm. In the study of metabolins which are from CDDP in vivo, our cooperative team find that Danshensu isopropyl ister (DIE), a kind of active metabolin which is from CDDP in vivo, is a yellowish-brown liquid which dissolves easy in some dissolvent such as water, colonial spirit, alcohol and acetic ether. In the study to DIE, it was found that DIE can expand mesentery artery in rats and enhance ventricular contraction force. But nobody study the good positive effect, or other pharmacologic action and mechanism against free radical of DIE. In this project, we studied mainly the pharmacological action of DIE along the following three aspects. First, Model of D-galactose induced aged rats was established to observe how DIE effect SOD vigour, MDA density and the apoptotic index of purkinje fibers in cerebellum of D-galactose induced aged rat , and Vitamine E (VE) was used as positive control. Second, the model of myocardial ischemia/reperfusion injury was established in isolated rat heart perfused with Langendorff thechnique to observe how DIE effect the content of high energy phosphates and myocardial ultrastructure, and CDDP was used as positive control. Third, the model of cardiac arrhythmia was established to observe how DIE effect the arrhythmias induced by exogenous free radical, and verapamil (VPL) was used as positive control.
Methods:
In the first part, 80 healthy Wistar rats which were 6 months, were applied in this study and divided into 5 groups at random: normal group, aging model group, positive control (VE 100.0 mg/Kg) group, two DIE groups with the dose of 21.0 mg/Kg and 7.0 mg/Kg, and each group had 16 rats. The rats of normal group was treated with daily subcutaneous injection of 4.0 mL/kg saline (0.85%), The rats of model group was treated with daily subcutaneous injection of 2.0 mL/kg saline (0.85%) and 2.0 mL/kg D-gal (2.5%), The rats of VE group and two DIE groups with the dose of 21.0 mg/Kg and 7.0 mg/Kg was treated with daily subcutaneous injection of 2.0 mL/kg drug of corresponding concentration and 2.0 mL/kg D-gal (2.5%), for successive 8 weeks. In each group, the half of animal were used to detect SOD vigour and the content of MDA; the other half to detect the apoptotic index of purkinje fibers of cerebellum cortex of rats in each group by methy/green pyronin stain.
In the second part, 80 healthy Wistar rats were applied in this study and divided into 8 groups at random: normal group, ischemia/reperfusion injury model group, CDDP prevention group, CDDP protection group, two DIE prevention groups with the dose of 4.00 mg/L and 0.04 mg/L, two DIE protection groups with the dose of 4.00 mg/L and 0.04 mg/L, and each group had 10 rats. A myocardial ischemical/ reperfusion injury model was established with the Langendroff method. Normal group: the hearts of rats were mouted on a Langendorff apparatus and perfused with modified Krebs-Hanseleit phosphate buffer for 75 min; I/R group: the hearts were perfused with modified Krebs-Hanseleit phosphate buffer for 15 min followed by global ischemia for 40 min, and then reperfused for 20 min; Each drug groups: the hearts were perfused or reperfused with modified Krebs-Hanseleit phosphate buffer which has drug of corresponding concentration, the concentration of drug is by turns 50.00 mg/L, 50.00 mg/L, 4.00 mg/L, 4.00 mg/L, 0.04 mg/L or 0.04 mg/L. In each group, High energy phosphates concentrations were determined by HPLC in myocardium of seven rats and the ultrastructure morphological changes of the other three rats'myocardial cell were observed by transmission electron microscope.
In the third part, 50 healthy Wistar rats were applied in this study and divided into 5 groups at random: normal group, model group, Verapamil 1.00 mg/L group, two DIE groups with the dose of 4 mg/L and 0.04 mg/L, and each group had 10 rats. The arrhythmias in the isolated rat heart were induced by FeSO_4 perfusing (0.025 mmol/L)/Vitamine C (VC)(0.100 mmol/L) with the Langendroff method. normal group: the hearts of rats were mouted on a Langendorff apparatus and perfused with modified Krebs-Hanseleit phosphate buffer for 45 min; Model group: the hearts were perfused with modified Krebs-Hanseleit phosphate buffer which has FeSO_4 perfusing (0.025 mmol/L)/VC (0.100 mmol/L) for 15 min, and then with modified Krebs-Hanseleit phosphate buffer for 30 min; Each drug groups: the hearts were perfused with modified Krebs-Hanseleit phosphate buffer which has FeSO_4 perfusing (0.025 mmol/L)/VC (0.100 mmol/L) and drug of corresponding concentration for 15 min, and then with modified Krebs-Hanseleit phosphate buffer for 30 min. The ECG was recorded when the hearts were perfused.
Results:
In the first part, comparing the aging group with the normal group, there is significant decrease in SOD vigour, significant increase in the content of MDA, significant decrease in apoptotic index of purkinje fibers (P<0.01). comparing the effects between two DIE groups with the dose of 21.0 mg/Kg and 7.0 mg/Kg and VE 100.0 mg/Kg group with the aging group, there is significant increase in SOD vigour, significant decrease in the content of MDA, significant decrease in apoptotic index of purkinje fibers (P<0.01). The difference of the effects between DIE group with the dose of 21.0 mg/Kg and VE 100.0 mg/Kg group is insignificant (P>0.05). comparing DIE group with dose of 7.0 mg/Kg with the former two groups, there is significant decrease in SOD vigour, increase in the content of MDA, significant decrease in apoptotic index of purkinje fibers(P<0.05).
In the second part, Energy-rich phosphate concentrations: the study showed that as compared with the normal control group, ATP, ADP, AMP and TAN levels were significantly decreased in the ischemia/reperfusion injury model group (P<0.01), as compared with the model group, ADP, AMP and TAN levels were significantly increased in the CDDP prevention group, the CDDP protection group, the DIE prevention groups with the dose of 4.00 mg/L, the DIE protection groups with the dose of 4.00 mg/L, the DIE prevention groups with the dose of 0.04 mg/L and the DIE protection groups with the dose of 0.04 mg/L (P<0.01) (except ADP levels in the DIE prevention groups with the dose of 0.04 mg/L); ADP levels was increased in the DIE prevention groups with the dose of 0.04 mg/L (P<0.05); ATP levels were significantly increased in the CDDP prevention group, the CDDP protection group, the DIE prevention groups with the dose of 4.00 mg/L and the DIE protection groups with the dose of 4.00 mg/L (P<0.05). The ultrastructure damages in myocardium tissue. In the normal control group, myoneme of myocardium cell was integral, sarcomere was regulation, light band and dark band was clear, Karyotheca of myocardium cell and mitochondrial membrane was integrity. In nucleus density of chromatin was proport, distribution of chromatin was uniformity, mitochondrial matrix was pycnotic. It was rich in the number of mitochondria and glycogen particle. The ultrastructure damages of myocardium tissue was very severe in the ischemia/reperfusion injury model group. sarcomere was arranged disorderly or disappeared, the dropsy of base material was very obvious in nucleus, mitochondria was swollen, mitochondrial cristae was fused or disappeared, Mitochondria and glycogen particle significantly diminished. The ultrastructure is damaged quite a little in the CDDP prevention group, the CDDP protection group, the DIE prevention groups with the dose of 4.00 mg/L, the DIE protection groups with the dose of 4.00 mg/L. In the DIE prevention groups with the dose of 0.04 mg/L and the DIE protection groups with the dose of 0.04 mg/L, the ultrastructure damages of myocardium tissue was lessened, as compared with the ischemia/reperfusion injury model group, but it was still severe.
In the third part, normal group has no the arrhythmias; the incidence rate of arrhythmias induced by the exogenous free radical was 100%, it has chiefly conduction blockade and premature ventricualr contraction; the incidence rate of arrhythmias reduces significantly in VPL 1.00 mg/L group and two DIE groups with the dose of 4.00 mg/L and 0.04 mg/L, as compared with the model group.
Conclusions:
1. DIE has the anti-oxidative and anti-apoptotic effects, The anti-oxidative and anti-apoptotic effects of DIE is similar to VE.
2. DIE can protect cardiomyocytes against ischemia/reperfusion injury, The protection of DIE is similar to CDDP.
3. DIE has the preventing effect on the arrhythmias induced by the exogenous free radical, the effect of DIE is similar to VPL.
复方丹参滴丸(Compound danshen dropping prill,CDDP)是一种中成药,主要成分有丹参、三七、冰片,具有活血化瘀、理气止痛等作用。我们的课题协作组在对复方丹参滴丸体内代谢产物的研究中发现丹参素异丙酯(Danshensuisopropyl ister,DIE)是复方丹参滴丸在体内的一种活性代谢物,其常温下为棕黄色液体,易溶于水、甲醇、乙醇、乙酸乙酯等溶剂。在对其研究中发现丹参素异丙酯具有扩张大鼠肠系膜动脉,增加大鼠心室肌收缩力等作用,而关于其抗自由基损伤及其他药理作用及其作用机制尚未研究。本课题主要从以下三个方面来研究丹参素异丙酯的药理作用:(1)建立D-半乳糖(D-galactose,D-gal)致衰老模型,观察衰老大鼠小脑组织SOD活力、MDA含量和浦肯野细胞凋亡指数的改变及丹参素异丙酯对它们的影响,并用维生素E(Vitamine E,VE)作为阳性对照;(2)利用Langendroff离体灌流技术,建立大鼠心肌缺血再灌注损伤模型,观察丹参素异丙酯对离体大鼠模拟缺血再灌注损伤心肌线粒体结构改变及能量物质代谢的影响,并用复方丹参滴丸作为阳性对照;(3)利用Langendroff灌流技术,建立外源性自由基致离体大鼠心律失常模型,观察丹参素异丙酯对外源性自由基所致心律失常(Cardiac arrhythmias,CA)的影响,并用维拉帕米(Verapamil,VPL)作为阳性对照。
研究方法
第一部分:取健康6月龄Wistar大鼠80只,雌雄各半,随机分为空白对照组、衰老模型组、阳性对照药(维生素E 100.0 mg/kg)组、丹参素异丙酯21.0 mg/kg和7.0 mg/kg两个剂量组,共5组,每组16只。空白对照组:每天皮下注射4 mL/kg的0.85%生理盐水;衰老模型组:每天皮下注射2.5%D-半乳糖2mL/kg的同时皮下注射0.85%生理盐水2.0 mL/kg;各给药组:每天皮下注射2.5%D-半乳糖2.0 mL/kg的同时,皮下注射相应浓度的药物2.0 mL/kg。每天一次,连续8周。末次给药1小时后对各组动物进行处理,各组一半动物小脑用于测定大鼠小脑组织中的超氧化物歧化酶(Superoxide dismutase,SOD)活性及丙二醛(Malondialdehyde,MDA)含量;另一半动物小脑采用甲基绿—哌洛宁染色法计数各组大鼠小脑皮质浦肯野细胞的凋亡指数(Apoptotic index,AI)。
第二部分:取健康Wistar大鼠80只,随机分为空白对照组、单纯缺血再灌注模型组、阳性对照药(复方丹参滴丸50.00 mg/L)前保护及后保护组、丹参素异丙酯4.00 mg/L和0.04 mg/L两个剂量的前保护及后保护组。共8组,每组10只。利用Langendroff灌流技术,制备心肌缺血再灌注损伤模型,分别在缺氧前及缺氧后,在灌流液中加入复方丹参滴丸或丹参素异丙酯对心肌给予保护。空白对照组:用Krebs-Hanseleit磷酸缓冲液持续灌流75 min;单纯缺血再灌注模型组:用Krebs-Hanseleit磷酸缓冲液预灌15 min,然后停灌40 min,再恢复灌流20min;各用药组:分别在预灌或再灌时用加入相应浓度药物的灌流液灌流,其他处理同单纯缺血再灌注模型组。各组所用灌流液的药物浓度分别是50.00 mg/L、50.00 mg/L、4.00 mg/L、4.00 mg/L、0.04 mg/L、0.04 mg/L。各组大鼠心脏灌流结束后,7只动物心脏采用高效液相色谱仪测定其组织中的高能磷酸化合物的含量变化;剩余3只动物利用H-7500透射电镜观察心肌细胞超微结构。
第三部分:取健康Wistar大鼠50只,随机分为空白对照组、心律失常模型组、阳性对照药(维拉帕米1.00 mg/L)组、丹参素异丙酯4.00 mg/L、0.04 mg/L两个剂量组,共5组,每组10只。采用Langendorf灌流装置对大鼠离体心脏灌注硫酸亚铁(0.025 mmol/L)/抗坏血酸(0.100 mmol/L)的方法,复制自由基致心律失常的模型,并观察丹参素异丙酯对其影响。空白对照组:离体心脏用Krebs-Hanseleit磷酸缓冲液灌注45 min;心律失常模型组:离体心脏用Krebs-Hanseleit磷酸缓冲液预灌15 min后,灌流液中加入硫酸亚铁(0.025mmol/L)/抗坏血酸(0.100 mmol/L)持续灌注30min;阳性对照药(维拉帕米1.00 mg/L)组、丹参素异丙酯4.00 mg/L、0.04 mg/L两个剂量组用加入相应浓度药物的Krebs-Hanseleit磷酸缓冲液预灌15 min,其余处理同心律失常模型组。每只心脏在灌流的同时,将心电图机上的导联线通过电极分别放在右心房和左心室尖部并记录心电图。
结果
第一部分:与空白对照组比较,衰老模型组大鼠小脑组织SOD活性降低、MDA含量增加、蒲肯野细胞的凋亡指数增加(P＜0.01);阳性对照药(维生素E100.0 mg/kg)组、丹参素异丙酯21.0 mg/kg和7.0 mg/kg两个剂量组与衰老模型组相比,小脑组织SOD活性均明显增加、MDA含量显著降低,蒲肯野细胞凋亡指数显著降低(P＜0.01);丹参素异丙酯21.0 mg/kg组与维生素E 100.0 mg/kg组的SOD活力、MDA含量、蒲肯野细胞的凋亡指数差异无显著性(P＞0.05);丹参素异丙酯7.0 mg/kg组与前两组相比大鼠小脑组织SOD活性降低、MDA含量增加、浦肯野细胞的凋亡指数增加(P＜0.05)。
第二部分:高能磷酸化合物(High energy phosphates,HEP)的含量:与空白对照组相比,模型组的三磷酸腺苷(Adenosine triphosphate,ATP)、二磷酸腺苷(Adenosine diphosphate,ADP)、一磷酸腺苷(Adenosine monophosphate,AMP)及腺嘌呤核苷酸总量(Total adenine nucleotides,TAN)显著降低(P＜0.01);复方丹参滴丸50.00 mg/L前保护和后保护组、丹参素异丙酯4.00 mg/L前保护和后保护组及丹参素异丙酯0.04 mg/L前保护和后保护组的ADP、AMP及AN与模型组相比显著升高(P＜0.01)(丹参素异丙酯0.04 mg/L前保护组的ADP除外);丹参素异丙酯0.04 mg/L前保护组的ADP除外与模型组相比有一定升高(P＜0.05);复方丹参滴丸50.00 mg/L前保护和后保护组、丹参素异丙酯4.00 mg/L前保护和后保护组的ATP含量与模型组相比升高(P＜0.05)。电镜显示:空白对照组心肌肌原纤维排列整齐,明暗带清晰,心肌细胞核膜和线粒体膜完整,染色质均匀、细密,线粒体基质致密,嵴排列紧密整齐,线粒体和糖原颗粒丰富;单纯缺氧/复氧模型组心肌细胞超微结构损伤最严重,心肌细胞有少量轻度坏死区,肌节排列紊乱或消失,核的一端基质明显水肿,线粒体大部分或全部的脊和膜融合或消失,线粒体和糖原数量明显减少;复方丹参滴丸50.00 mg/L前保护、后保护组及丹参素异丙酯4.00 mg/L前保护、后保护组超微结构接近空白对照组,损伤较轻;丹参素异丙酯0.04 mg/L前保护、后保护组心肌细胞超微结构的损伤与模型组相比有所改善,但损伤仍较严重。
第三部分:空白对照组离体大鼠心脏无一例发生心律失常;模型组离体大鼠心脏总心律失常发生率100%,主要发生传导阻滞和室性早搏两种类型的心律失常;阳性对照药(维拉帕米1.00 mg/L)组、丹参素异丙酯4.00 mg/L、0.04 mg/L两个剂量组与模型组相比,心律失常发生率明显降低。
结论
1.丹参素异丙酯具有抗氧化作用及抗凋亡作用,与阳性对照药维生素E相似。
2.丹参素异丙酯具有保护缺血再灌注损伤心肌的作用,与阳性对照药复方丹参滴丸相似。
3.丹参素异丙酯具有抗外源性自由基生成系统所致心律失常的作用,与阳性对照药维拉帕米相似。
Free radicals are atomic or molecular species with unpaired electrons on an otherwise open shell configuration, such as superoxide anion radical (O_2~-·), hydroxyl radical (OH), Lipid alkoxyl radical (LO·). Under normal conditions, free radicals are continuously produced and eliminated at good balance and benefit the body; while, under pathological conditions, excessive free radicals can be accumulated in the body due to either increase in generation or decrease in elimination, which will cause damages to the body. Many studies showed that over 100 kinds of diseases are related to metabolic disturbance of free radical. For example, cancers, cardio-cerebrovascular diseases, gastrointestinal diseases and so on. Therefore, it is important to illuminate the physiological and pathologic effects of free radical in order to prevent and treat the diseases caused by free radicals.
Compound danshen dropping prill (CDDP), a combination of Radix Salviae Miltiorrhizae, Radix Notoginseng and Borneolum Syntheticum, is a Chinese remedy, which has been reported has the effects of activating the blood and reducing phlegm. In the study of metabolins which are from CDDP in vivo, our cooperative team find that Danshensu isopropyl ister (DIE), a kind of active metabolin which is from CDDP in vivo, is a yellowish-brown liquid which dissolves easy in some dissolvent such as water, colonial spirit, alcohol and acetic ether. In the study to DIE, it was found that DIE can expand mesentery artery in rats and enhance ventricular contraction force. But nobody study the good positive effect, or other pharmacologic action and mechanism against free radical of DIE. In this project, we studied mainly the pharmacological action of DIE along the following three aspects. First, Model of D-galactose induced aged rats was established to observe how DIE effect SOD vigour, MDA density and the apoptotic index of purkinje fibers in cerebellum of D-galactose induced aged rat , and Vitamine E (VE) was used as positive control. Second, the model of myocardial ischemia/reperfusion injury was established in isolated rat heart perfused with Langendorff thechnique to observe how DIE effect the content of high energy phosphates and myocardial ultrastructure, and CDDP was used as positive control. Third, the model of cardiac arrhythmia was established to observe how DIE effect the arrhythmias induced by exogenous free radical, and verapamil (VPL) was used as positive control.
Methods:
In the first part, 80 healthy Wistar rats which were 6 months, were applied in this study and divided into 5 groups at random: normal group, aging model group, positive control (VE 100.0 mg/Kg) group, two DIE groups with the dose of 21.0 mg/Kg and 7.0 mg/Kg, and each group had 16 rats. The rats of normal group was treated with daily subcutaneous injection of 4.0 mL/kg saline (0.85%), The rats of model group was treated with daily subcutaneous injection of 2.0 mL/kg saline (0.85%) and 2.0 mL/kg D-gal (2.5%), The rats of VE group and two DIE groups with the dose of 21.0 mg/Kg and 7.0 mg/Kg was treated with daily subcutaneous injection of 2.0 mL/kg drug of corresponding concentration and 2.0 mL/kg D-gal (2.5%), for successive 8 weeks. In each group, the half of animal were used to detect SOD vigour and the content of MDA; the other half to detect the apoptotic index of purkinje fibers of cerebellum cortex of rats in each group by methy/green pyronin stain.
In the second part, 80 healthy Wistar rats were applied in this study and divided into 8 groups at random: normal group, ischemia/reperfusion injury model group, CDDP prevention group, CDDP protection group, two DIE prevention groups with the dose of 4.00 mg/L and 0.04 mg/L, two DIE protection groups with the dose of 4.00 mg/L and 0.04 mg/L, and each group had 10 rats. A myocardial ischemical/ reperfusion injury model was established with the Langendroff method. Normal group: the hearts of rats were mouted on a Langendorff apparatus and perfused with modified Krebs-Hanseleit phosphate buffer for 75 min; I/R group: the hearts were perfused with modified Krebs-Hanseleit phosphate buffer for 15 min followed by global ischemia for 40 min, and then reperfused for 20 min; Each drug groups: the hearts were perfused or reperfused with modified Krebs-Hanseleit phosphate buffer which has drug of corresponding concentration, the concentration of drug is by turns 50.00 mg/L, 50.00 mg/L, 4.00 mg/L, 4.00 mg/L, 0.04 mg/L or 0.04 mg/L. In each group, High energy phosphates concentrations were determined by HPLC in myocardium of seven rats and the ultrastructure morphological changes of the other three rats'myocardial cell were observed by transmission electron microscope.
In the third part, 50 healthy Wistar rats were applied in this study and divided into 5 groups at random: normal group, model group, Verapamil 1.00 mg/L group, two DIE groups with the dose of 4 mg/L and 0.04 mg/L, and each group had 10 rats. The arrhythmias in the isolated rat heart were induced by FeSO_4 perfusing (0.025 mmol/L)/Vitamine C (VC)(0.100 mmol/L) with the Langendroff method. normal group: the hearts of rats were mouted on a Langendorff apparatus and perfused with modified Krebs-Hanseleit phosphate buffer for 45 min; Model group: the hearts were perfused with modified Krebs-Hanseleit phosphate buffer which has FeSO_4 perfusing (0.025 mmol/L)/VC (0.100 mmol/L) for 15 min, and then with modified Krebs-Hanseleit phosphate buffer for 30 min; Each drug groups: the hearts were perfused with modified Krebs-Hanseleit phosphate buffer which has FeSO_4 perfusing (0.025 mmol/L)/VC (0.100 mmol/L) and drug of corresponding concentration for 15 min, and then with modified Krebs-Hanseleit phosphate buffer for 30 min. The ECG was recorded when the hearts were perfused.
Results:
In the first part, comparing the aging group with the normal group, there is significant decrease in SOD vigour, significant increase in the content of MDA, significant decrease in apoptotic index of purkinje fibers (P<0.01). comparing the effects between two DIE groups with the dose of 21.0 mg/Kg and 7.0 mg/Kg and VE 100.0 mg/Kg group with the aging group, there is significant increase in SOD vigour, significant decrease in the content of MDA, significant decrease in apoptotic index of purkinje fibers (P<0.01). The difference of the effects between DIE group with the dose of 21.0 mg/Kg and VE 100.0 mg/Kg group is insignificant (P>0.05). comparing DIE group with dose of 7.0 mg/Kg with the former two groups, there is significant decrease in SOD vigour, increase in the content of MDA, significant decrease in apoptotic index of purkinje fibers(P<0.05).
In the second part, Energy-rich phosphate concentrations: the study showed that as compared with the normal control group, ATP, ADP, AMP and TAN levels were significantly decreased in the ischemia/reperfusion injury model group (P<0.01), as compared with the model group, ADP, AMP and TAN levels were significantly increased in the CDDP prevention group, the CDDP protection group, the DIE prevention groups with the dose of 4.00 mg/L, the DIE protection groups with the dose of 4.00 mg/L, the DIE prevention groups with the dose of 0.04 mg/L and the DIE protection groups with the dose of 0.04 mg/L (P<0.01) (except ADP levels in the DIE prevention groups with the dose of 0.04 mg/L); ADP levels was increased in the DIE prevention groups with the dose of 0.04 mg/L (P<0.05); ATP levels were significantly increased in the CDDP prevention group, the CDDP protection group, the DIE prevention groups with the dose of 4.00 mg/L and the DIE protection groups with the dose of 4.00 mg/L (P<0.05). The ultrastructure damages in myocardium tissue. In the normal control group, myoneme of myocardium cell was integral, sarcomere was regulation, light band and dark band was clear, Karyotheca of myocardium cell and mitochondrial membrane was integrity. In nucleus density of chromatin was proport, distribution of chromatin was uniformity, mitochondrial matrix was pycnotic. It was rich in the number of mitochondria and glycogen particle. The ultrastructure damages of myocardium tissue was very severe in the ischemia/reperfusion injury model group. sarcomere was arranged disorderly or disappeared, the dropsy of base material was very obvious in nucleus, mitochondria was swollen, mitochondrial cristae was fused or disappeared, Mitochondria and glycogen particle significantly diminished. The ultrastructure is damaged quite a little in the CDDP prevention group, the CDDP protection group, the DIE prevention groups with the dose of 4.00 mg/L, the DIE protection groups with the dose of 4.00 mg/L. In the DIE prevention groups with the dose of 0.04 mg/L and the DIE protection groups with the dose of 0.04 mg/L, the ultrastructure damages of myocardium tissue was lessened, as compared with the ischemia/reperfusion injury model group, but it was still severe.
In the third part, normal group has no the arrhythmias; the incidence rate of arrhythmias induced by the exogenous free radical was 100%, it has chiefly conduction blockade and premature ventricualr contraction; the incidence rate of arrhythmias reduces significantly in VPL 1.00 mg/L group and two DIE groups with the dose of 4.00 mg/L and 0.04 mg/L, as compared with the model group.
Conclusions:
1. DIE has the anti-oxidative and anti-apoptotic effects, The anti-oxidative and anti-apoptotic effects of DIE is similar to VE.
2. DIE can protect cardiomyocytes against ischemia/reperfusion injury, The protection of DIE is similar to CDDP.
3. DIE has the preventing effect on the arrhythmias induced by the exogenous free radical, the effect of DIE is similar to VPL.
引文
[1]李鸿香.对衰老机理及抗衰老问题的探讨[J].天津医学,1996,8(4):3-7.
[2]Butterfield DA,Howard BJ,Yatin S,et al.Free radical oxidation of brain proteins in accelerated senescence and its modulation by N-tert-butyl-alpha-phenylnitrone[J].Proc Natl Acad Sci U S A.1997.94(2):674-678.
[3]Gutierrez AM,Reboredo GR,Catala A.Fatty acid profiles and lipid peroxidation of microsomes and mitochondria from liver,heart and brain of Cairina moschata.Int J Biochem Cell Biol.2002,34(6):605-612.
[4]田晓华,顾景范,孙存普,等.氧化应激与细胞凋亡[J].军事医学,1996,15(4):109-111.
[5]杨东丽,张宗玉,童坦君,等.细胞凋亡与衰老[J].生理科学进展,1996,27(1):64-66.
[6]崔旭.D-半乳糖衰老模型及其氧化损伤机理与α-硫辛酸的干预作用研究.[博士论文].北京:中国协和医科大学,2001.53.
[7]张熙,张葆樽,杨新平,等.D-半乳糖诱导拟衰老动物模型的记忆行为改变[J].中国老年杂志,1996,16(4):230-232.
[8]王中林,顾一煌,焦瑞请,等.D-半乳糖致小鼠衰老模型海马突触蛋白的改变[J].南京中医药大学学报,1997,13(4):216-217.
[9]杨志文,王孝铭.复方丹参滴丸对D-半乳糖致衰大鼠小脑的作用[J].哈尔滨医科大学学报,2002,36(2):115-117.
[10]姜泊.细胞凋亡基础与临床[M].北京:人民军医出版社,1999.9.
[11]朱曰林,赵崇德.甲基绿-哌洛宁染色法研究进展[J].中国组织化学与细胞化学杂志,1996,5(2):227-230.
[12]张均田,张庆柱.神经药理学研究技术与方法[M].北京:人民卫生出版社,2001.01.
[13]Harman DJ.Role of free radicals in aging and disease aging[M].New York:Raven Press,1998.113.
[14]Harman D.The free radical theory of aging:Effect of age on serum copper levels[J].J Gerontol.1965,20:151-153.
[15]Saltman P.Oxidative Stress:A radical view[J].Seminars in Hematology,1989,26 (4):249-256
[16]Harman D.Free radical theory of aging:an update:increasing the functional life span[J].Ann N Y Acad Sci.2006,1067:10-21.
[17]Miquel J.Mitochondrial role in cell aging[J].Exp Gerontol,1980,15(6):575-579.
[18]崔旭,李文彬,张炳烈,等.D-半乳糖脑老化模型的脂质过氧化机理[J].中国老年学杂志,1998,18(1):38-40.
[19]方允中,李文杰.自由基与酶[M].北京:科学出版社,1989:153-155.
[20]Liu K,Wang C,Li G,et al.A comparison study between D-galactose treated mice and natural aging mice[J].Wei Sheng Yah Jiu.2007,36(6):685-688.
[21]李文彬,韦丰,范明,等.D-半乳糖在大鼠上诱导的拟脑老化效应[J].中国药理学与毒理学杂志,1995,9(2):93-95.
[22]张利娟,潘秀华,王玉红,等.脑活素改善血管性痴呆患者智能状况的临床研究[J].中国临床康复,2003,7(28):3850-3851.
[23]柯开富,周鸣鸣,强亮,等.神经生长液对衰老模型小鼠学习记能力的影响[J].中国临床康复,2003,7(25):3444-3445.
[24]Lantos J,Temes G,G(o|¨)b(o|¨)l(o|¨)s L,et al.Is peripheral blood a reliable indicator of acute oxidative stress following heart ischemia and reperfusion?[J].Med Sci Monit,2001,7(6):1166-1170.
[25]Dmitriev LF.Shortage of lipid-radical cycles in membranes as a possible prime cause of energetic failure in aging and Alzheimer disease[J].Neurochem Res.2007,32(8):1278-1291.
[26]李英哲,黄连珍,周丽玲.维生素A缺乏对大鼠脂质过氧化和抗氧化系统的影响[J].营养学报,2001,23(1):1-4.
[27]Rice-Evans C,Burdon R.Free radical-lipid interactions and their pathological consequences.Prog Lipid Res.1993,32(1):71-110.
[28]de Zwart LL,Meeman JH,Commandeur JN,et al.Biomarkers of free radical damage applications in experimental animals and in humans[J].Free Radic Biol Meal,1999,26(1-2):202-226.
[29]Kerr JF,Wyllie AH,Currie AR.Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics[J].Br J Cancer.1972,26(4):239-257.
[30]Schulze CJ,Wang W,Kumari R,et al.Imbalance between tissue inhibitor of metalloproteinase-4 and matrix metalloproteinases during acute myocardial correction of myocardial ischemia-reperfusion injury[J].Circulation,2003,107(19):2487-2492.
[31]Forman MB,Virmani R,Puett DW.Mechanisms and therapy of myocardial reperfusion injury[J].Circulation.1990,81(3 Suppl):Ⅳ69-78.
[32]Menger MD,Vollmar B.Role of microcirculation in transplantation[J].Microcirculation.2000,7(5):291-306.
[33]Yellon DM,Baxter GF.Protecting the ischaemic and reperfused myocardium in acute myocardial infarction:distant dream or near reality?[J].Heart.2000,83(4):381-387.
[34]赵雅君,史从宁,王孝铭,等.复方丹参滴丸对离体大鼠缺氧/复氧心肌的保护作用[J].中国病理生理杂志.2002,18(10):1271-1275.
[35]徐叔云,卞如濂,陈修.药理学实验方法[M].第三版,北京:人民卫生出版社,2001.1054-1056.
[36]Reimer KA,Jennings RB,Tatum AH.Pathobiology of acute myocardial ischemia:metabolic,functional and ultrastructural studies[J].Am J Cardiol,1983,52(2):72A-81A.
[37]Classen JB,Mergner WJ,Costa M.ATP hydrolysis by ischemic mitochondria[J].J Cell Physiol,1989,141(1):53-59.
[38]Lopaschuk G.Regulation of carbohydrate metabolism in ischemia and reperfusion [J].Am Heart J,2000,139(2 pt 3),S115-S119.
[39]Sutherland FJ.Hearse DJ.The isolated blood and peffusion fluid perfused heart [J].Pharmacol Res,2000,41(6):613-627.
[40]Curtis MJ,Macleod BA,Tabrizchi R,et al.An improved perfusion apparatus for small animal hearts[J].J Pharmacol Methods,1986,15(1):87-94.
[41]李春杰,余柏林.心肌缺血再灌注损伤机制研究进展[J].人民军医,2004,47(1):47-49.
[42]Sadek HA,Nulton-Persson AC,Szweda PA,et al.Cardiac ischemia/reperfusion,aging,and redox-dependent alterations in mitochondrial function[J].Arch Biochem Biophys.2003,420(2):201-208.
[43]Oriaku G,Xiang B,Dai G,et al.Effects of retrograde cardioplegia on myocardial perfusion and energy metabolism in immature porcine myocardium[J].J Thorac Cardiovasc Surg,2000,119(6):1102-1109.
[44]Harman D.Free radicals in aging[J].Mol Cell Biochem,1988,84(2):155-161.
[45]Wallace KB,Eells JT,Madeira VM,et al.Mitochondria-mediated cell injury.Symposium overview[J].Fundam Appl Toxicol,1997,38(1):23-37.
[46]Woodward B,Zakaria MN.Effect of some free radical scavengers on reperfusion induced arrhythmias in the isolated rat heart[J].J Mol Cell Cardiol.1985,17(5):485-493.
[47]Curtis MJ,Pugsley MK,Walker MJ.Endogenous chemical mediators of ventricular arrhythmias in ischaemic heart disease[J].Cardiovasc Res.1993,27(5):703-719.
[48]时飒,徐长庆,王孝铭,等.复方丹参滴丸对外源性自由基所致心律失常的影响[J].哈尔滨医科大学学报,2001,35(5):335-336.
[49]钮伟真,刘萍,高燕丽,等.豚鼠及大鼠离体心脏希氏束电图的记录[J].中国药理学通报,2001,17(4):476-477.
[50]Niu WZ,Gao YL,Liu P,et al.A comparison of calcitonin gene-related peptide effects on coronary flow and cardiac conduction system in the guinea pig[J].Sheng Li Xue Bao,2000,52(3):259-262.
[51]Goldhaber JI,Ji S,Lamp ST,et al.Effects of exogenous free radicals on electromechanical function and metabolism in isolated rabbit and guinea pig ventricle.Implications for ischemia and reperfusion injury[J].J Clin Invest,1989,83(6):1800-1809.
[52]苏哲坦.再灌流心律失常.国外医学生理、病理科学与临床分册,1986,4:181.
[53]Brooks WW,Conrad CH,Morgan JP.Reperfusion induced arrhythmias following ischaemia in intact rat heart:role of intracellular calcium[J].Cardiovasc Res,1995,29(4):536-542.
[54]Cerbai E,Ambrosio G,Porciatti F,et al.Cellular electrophysiological basis for oxygen radical-induced arrhythmias.A patch-clamp study in guinea pig ventricular myocytes.Circulation,1991,84(4):1773-1782.
[55]宋文良,唐朝枢.PH反常与心肌缺血再灌注损伤.国外医学病理生理科学与临床分册,1991,12:9-11.
[56]Cerbai E,Ambrosio G,Porciatti F,et al.Cellular electrophysiological basis for oxygen radical-induced arrhythmias.A patch-clamp study in guinea pig ventricular myocytes[J].Circulation,1991,84(4):1773-1782.
[57]Markov AK.Hemodynamics and metabolic effects of fructose 1-6 diphosphate in ischemia and shock—experimental and clinical observations[J].Ann Emerg Med,1986,15(12):1470-1477.
[58]王新一,许宏远,徐长庆,等.不同浓度的外源性自由基生成系统致心律失常作用的比较[J].哈尔滨医科大学学报,1999,33(4):251-253.
[1]孙存普,张建中,段绍瑾.自由基生物学导论[M].安徽:中国科技大学出版社,1999.1.
[2]方允中,郑荣梁.自由基生物学的理论与应用[M].北京:科学出版社,2002.7.
[3]Palmer RM,Ferrige AG,Momcada S.Nitric oxide release accounts for the biological activity of endothelial-derived relaxing factor[J].Nature,1987,327(6122):524-526.
[4]詹晖,葛新发.自由基消除与中医药抗运动性疲劳[J].武汉体育学院学报.1998,2:61-65.
[5]周永平,王恬.运动与自由基代谢[J].浙江体育科学,996,18(1):12-15.
[6]吕国蔚主编.医学神经生物学fM].北京:高等教育出版社,2000.390-392.
[7]郑荣梁主编.自由基生命科学进展(第6集)[M].北京:原子能出版社,1998.36-37.
[8]吕国蔚主编.医学神经生物学[M].北京:高等教育出版社,2000.390-392.
[9]代毅,金文泉,高卫.运动对自由基代谢的影响[J].成都体育学院学报,1995,21(3):65-68.
[10]曹锡清.脂质过氧化对细胞与机体的作用[J].生物化学与生物物理进展,1986,2:17-23.
[11]方允中,李文杰.自由基与酶—基础理论及其在生物学和医学中的应用[M].北京:科学出版社,1998.147-162.
[12]Bhatnagar A.Biochemical mechanism of irreversible cell injury caused by free radical-initiated reactions[J].Mol Cell Biochem,1994,137(1):9-16.
[13]Benzie IF.Lipid peroxidation:a review of causes,consequences,measurement and dietary influences[J].Int J Food Sci Nutr,1996,47(3):233-261.
[14]方允中,郑荣梁,沈文梅.自由基生命科学进展(第3集)[M].北京:原子能出版社,1995.99-102.
[15]韩立强.肌肽清除自由基及抗氧化性研究[硕士论文].郑州:河南农业大学,2004.5.
[16]顾有方,陈会良,刘德义,等.自由基的生理和病理作用[J].动物医学进展,2005,26(1):94-97.
[17]程时,宋国兴.生物物理学[M].北京:北京医科大学中国协和医科大学联合出版社,1997.223-230.
[18]Halliwell B,Gutteridge JM.Free Radicols in Biology and Medicine.Oxford:Clarendon Press,1989.188-276.
[19]Hallliwell B,Chirico S.Lipid peroxidation:its mechanism measurement and significance[J].Am J Clin Nutr,1993,57(5 Suppl):715S-725S.
[20]Radi R,Beckman JS,Bush KM,et al.Peroxynitrite-induced membrane lipid peroxidation:the cytotoxic potential of superoxide and nitric oxide[J].Arch Biochem Biophys,1991,288(2):481-487.
[21]Frank H,Hintze T,Bimboes D,et al.Monitoring lipid peroxidation by breath analysis:endogenous hydrocarbons and their metabolic elimination[J].Toxicol Appl Pharmacol,1980,56(3):337-344.
[22]Van Gossum A,Decuyper J,Breath alkanes as an index of lipid peroxidation[J],Eur Respir J,1989,2(8):787-791.
[23]Fang YZ,Zheng RL.Theory and application of free radical biology[M].Beijing:The Science Publishing House,2002.23-48.
[24]Zheng RL,Huang ZY.Basics of free radical medicine and agriculture[M].Beijing:China Higher Education Press,2001.105-143.
[25]高庆,张克英.自由基与动物营养[J].中国饲料,2004,(4):13-14.
[26]刘斌.小鼠早期胚胎发育过程中活性自由基生成机制初探[硕士论文].湖南:湖南农业大学,2002.8.
[27]赵学纲.针灸对亚急性衰老大鼠睾丸自由基代谢和Bcl-2表达的影响[硕士论文].山东:山东中医药大学,2005.21.
[28]孙婷婷,徐建雄.微生物源性抗氧化剂对母猪繁殖性能和自由基代谢的影响[J].上海交通大学学报(农业科学版),2007,25(4):342-346.
[29]金婧.LPS致新生大鼠急性肺损伤促炎性细胞因子、抗炎性细胞因子和NF-κB变化及N-乙酰半胱氨酸干预作用的研究[博士论文].沈阳:中国医科大学.2004.46.
[30]王桔红,谢宗平,陈文.衰老机理及抗衰老研究进展[J].河西学院学报,2003,(2):94-98.
[31]Melov S.Animal models of oxidative stress,aging,and therapeutic antioxidant interventions[J].Int J Biochem Cell Biol,2002,34(11):1395-1400.
[32]Linton S,Davies MJ,Dean RT.Protein oxidation and ageing[J].Exp Gerontol,2001,36(9):1503-1518.
[33]陈瑾,李荣享.衰老的自由基机制[J].中国老年学杂志,2004,24(7):677-679.
[34]Squier TC.Oxidative stress and protein aggregation during biological aging[J].Exp Gerontol,2001,36(9):1539-1550.
[35]王锋青,陈秉初.生物衰老机理研究进展[J].生物学教学,2004,29(2):4-7.
[36]周玫,张宝,陈瑗.机体抗氧化防御系统,自由基医学基础与病理生理[M].北京:人民卫生出版社,2002.121.
[37]Human D.Aging:overview[J].Ann N Y Acad Sci,2001,928:1-21.
[38][美]简.卡著.邱巍,张敏译.延缓衰老[M].北京:新华出版社,1998.
[39]谢文海,甘耀坤.自由基超氧化物歧化酶(SOD)及抗衰老[J].玉林师专学报(自然科学),1999,20(3):70-72.
[40]李大庆.氧自由基与当代医学[J].论文选刊,1999,9:18-19.
[41]敖拉哈.衰老与自由基[J].老年医学与保健,1999,5(2):51-52.
[42]张冬齐,抗氧化剂对帕金森病的保护作用及机制的进展[J].医学综述,2006,12(2):113-115.
[43]Richie JP Jr.The role of glutathione in aging and cancer[J].Exp Gerontol,1992,27(5-6):615-626.
[44]Fernandez-checa JC,Colell A,Garcia-Ruiz C.S-AdenosyI-L-methionine and mitochondrial reduced glutathione depletion in alcoholic liver disease[J].Alcohol,2002,27(3):179-183.
[45]车祺,蒋涛,林伊凤,等.醛糖还原酶基因转染对体外培养大鼠肾脏系膜细胞增殖的影响[J].中华病理学杂志,2005,34(7):417-420.
[46]张洪泉,余文新.中华抗衰老医药学[M].北京:科学出版社,2000.82-109.
[47]张宗玉,范新青.衰老分子机制研究新进展[J].实用老年医学杂志,1994,8(3):97-99.
[48]Trounce I,Byme E,Marzuki S.Decline in skeletal muscle mitochondrial respiratory chain function:possible factor in ageing[J].Lancet,1989,1(8639): 637-639.
[49]Cooper JM,Mann VM,Schapira AH.Analysis of mitochondrial respiratory chain function and mitochondrial DNA deletion in human skeletal muscle:effect of aging[J].J Neurol Sci,1992,113(1):91-98.
[50]Bowling AC,Mutisya EM,Walker LC,et al.Age-dependent inpaiment of mitochondrial function in primate brain[J].J Neurochem,1993,60(5):1964-1967.
[51]Benzi G,Pastoris O,Marzatico F,et al.The mitochondrial electron transfer alteration as a factor involved in the brain aging[J].J Neurobiol Aging,1992,13(3):361-368.
[52]Von Zglinicki T.A mitochondrial membrane hypothesis of aging[J].J Theor Biol,1987,127(2):127-132.
[53]Harman D.Free radical theory of aging:Nutritionnal implications[J].Age,1978,1(4):145-153.
[54]方芳,陈晓春,朱元贵.抗氧化作用可能是人参皂甙Rg1抗细胞凋亡的机制[J].中国临床药理学与治疗学,2002,7(5):412-416.
[55]方雪铃,王锡田,黄淑如,等.杭白菊对小牛血管平滑肌细胞凋亡及其抗氧化性研究[J].浙江大学学报(医学版),2002,31(5):347-350.
[56]刘汴生.衰老过程中的病理生理[J].实用老年医学,2002,16(2):64-68.
[57]陈奇.中药药理研究方法学[M].北京:人民卫生出版社,1993.931-941.
[58]Stadman ER.Protein oxidation and aging[J].Science,1992,257(5074):1220-1224.
[59]Rice-Evans C,Burdon R.Free radical-lipid interactions and their pathological consequences[J].Prog Lipid Res,1993,32(1):71-110.
[60]Yu BP.Aging and oxidative stress:modulation by dietary restriction[J].Free Radic Biol Med,1996,21(5):651-658.
[61]Halliwell B.Oxidation of low-density lipoproteins:questions of initiation,propagation,and the effect of antioxidants[J].Am J Clin Nutr,1995,61(3Suppl):670S-677S.
[62]Paradies G,Petrosillo G,Pistolese M,et al.Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia and reperfusion.Free Radic Biol Med,1999,27(1-2):42-50.
[63]Chen Q,Moghaddas S,Hoppel CL,et al.Reversible blockade of electron transport during ischemia protects mitochondria and decreases myocardial injury following reperfusion.J Pharmacol Exp Ther,2006,319(3):1405-1412.
[64]Petrosillo G,Ruggiero FM,Di Venosa N,et al.Decreased complex Ⅲ activity in mitochondria isolated from rat heart subjected to ischemia and reperfusion:role of reactive oxygen species and cardiolipin[J].FASEB J,2003,17(6):714-716.
[65]李丹彤,冯雁,高妹娟,等.异丙基肾上腺素损伤的大鼠心肌线粒体中细胞色素氧化酶的复活研究[J].中国地方病防治杂志,1997,12(3):138-141.
[66]Chen J,Mehta JL,Haider N,et al.Role of caspases in Ox-LDL-induced apoptotic cascade in human coronary artery endothelial cells.Circ Res,2004,94(3):370-376.
[67]程贵蓉,施娣,陶圣祥,等.安氟醚对离体鼠心的心肌保护作用[J].广州医药,2000,31(5):13-15.
[68]伍静,姚尚龙,黄方敏,等.异丙酚预处理对大鼠离体心脏缺血-再灌注损伤的影响[J].中华麻醉学杂志,2004,24(12):905-908.
[69]徐万红,曹春梅,夏强,等.杭白菊提取液对抗缺血再灌注引起的离体大鼠心肌收缩功能下降[J].中国病理生理杂志,2004,20(5):822-826.
[70]程桂荣,易胜中.黄芪预处理大鼠心肌保护效应的实验研究[J].高血压杂志,2001,9(4):316-318.
[71]景桂霞,温健,王伟.高氧液预处理对兔心肌缺血再灌注损伤的保护作用[J].西安交通大学学报(医学版),2005,26(4):336-339.
[2]Butterfield DA,Howard BJ,Yatin S,et al.Free radical oxidation of brain proteins in accelerated senescence and its modulation by N-tert-butyl-alpha-phenylnitrone[J].Proc Natl Acad Sci U S A.1997.94(2):674-678.
[3]Gutierrez AM,Reboredo GR,Catala A.Fatty acid profiles and lipid peroxidation of microsomes and mitochondria from liver,heart and brain of Cairina moschata.Int J Biochem Cell Biol.2002,34(6):605-612.
[4]田晓华,顾景范,孙存普,等.氧化应激与细胞凋亡[J].军事医学,1996,15(4):109-111.
[5]杨东丽,张宗玉,童坦君,等.细胞凋亡与衰老[J].生理科学进展,1996,27(1):64-66.
[6]崔旭.D-半乳糖衰老模型及其氧化损伤机理与α-硫辛酸的干预作用研究.[博士论文].北京:中国协和医科大学,2001.53.
[7]张熙,张葆樽,杨新平,等.D-半乳糖诱导拟衰老动物模型的记忆行为改变[J].中国老年杂志,1996,16(4):230-232.
[8]王中林,顾一煌,焦瑞请,等.D-半乳糖致小鼠衰老模型海马突触蛋白的改变[J].南京中医药大学学报,1997,13(4):216-217.
[9]杨志文,王孝铭.复方丹参滴丸对D-半乳糖致衰大鼠小脑的作用[J].哈尔滨医科大学学报,2002,36(2):115-117.
[10]姜泊.细胞凋亡基础与临床[M].北京:人民军医出版社,1999.9.
[11]朱曰林,赵崇德.甲基绿-哌洛宁染色法研究进展[J].中国组织化学与细胞化学杂志,1996,5(2):227-230.
[12]张均田,张庆柱.神经药理学研究技术与方法[M].北京:人民卫生出版社,2001.01.
[13]Harman DJ.Role of free radicals in aging and disease aging[M].New York:Raven Press,1998.113.
[14]Harman D.The free radical theory of aging:Effect of age on serum copper levels[J].J Gerontol.1965,20:151-153.
[15]Saltman P.Oxidative Stress:A radical view[J].Seminars in Hematology,1989,26 (4):249-256
[16]Harman D.Free radical theory of aging:an update:increasing the functional life span[J].Ann N Y Acad Sci.2006,1067:10-21.
[17]Miquel J.Mitochondrial role in cell aging[J].Exp Gerontol,1980,15(6):575-579.
[18]崔旭,李文彬,张炳烈,等.D-半乳糖脑老化模型的脂质过氧化机理[J].中国老年学杂志,1998,18(1):38-40.
[19]方允中,李文杰.自由基与酶[M].北京:科学出版社,1989:153-155.
[20]Liu K,Wang C,Li G,et al.A comparison study between D-galactose treated mice and natural aging mice[J].Wei Sheng Yah Jiu.2007,36(6):685-688.
[21]李文彬,韦丰,范明,等.D-半乳糖在大鼠上诱导的拟脑老化效应[J].中国药理学与毒理学杂志,1995,9(2):93-95.
[22]张利娟,潘秀华,王玉红,等.脑活素改善血管性痴呆患者智能状况的临床研究[J].中国临床康复,2003,7(28):3850-3851.
[23]柯开富,周鸣鸣,强亮,等.神经生长液对衰老模型小鼠学习记能力的影响[J].中国临床康复,2003,7(25):3444-3445.
[24]Lantos J,Temes G,G(o|¨)b(o|¨)l(o|¨)s L,et al.Is peripheral blood a reliable indicator of acute oxidative stress following heart ischemia and reperfusion?[J].Med Sci Monit,2001,7(6):1166-1170.
[25]Dmitriev LF.Shortage of lipid-radical cycles in membranes as a possible prime cause of energetic failure in aging and Alzheimer disease[J].Neurochem Res.2007,32(8):1278-1291.
[26]李英哲,黄连珍,周丽玲.维生素A缺乏对大鼠脂质过氧化和抗氧化系统的影响[J].营养学报,2001,23(1):1-4.
[27]Rice-Evans C,Burdon R.Free radical-lipid interactions and their pathological consequences.Prog Lipid Res.1993,32(1):71-110.
[28]de Zwart LL,Meeman JH,Commandeur JN,et al.Biomarkers of free radical damage applications in experimental animals and in humans[J].Free Radic Biol Meal,1999,26(1-2):202-226.
[29]Kerr JF,Wyllie AH,Currie AR.Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics[J].Br J Cancer.1972,26(4):239-257.
[30]Schulze CJ,Wang W,Kumari R,et al.Imbalance between tissue inhibitor of metalloproteinase-4 and matrix metalloproteinases during acute myocardial correction of myocardial ischemia-reperfusion injury[J].Circulation,2003,107(19):2487-2492.
[31]Forman MB,Virmani R,Puett DW.Mechanisms and therapy of myocardial reperfusion injury[J].Circulation.1990,81(3 Suppl):Ⅳ69-78.
[32]Menger MD,Vollmar B.Role of microcirculation in transplantation[J].Microcirculation.2000,7(5):291-306.
[33]Yellon DM,Baxter GF.Protecting the ischaemic and reperfused myocardium in acute myocardial infarction:distant dream or near reality?[J].Heart.2000,83(4):381-387.
[34]赵雅君,史从宁,王孝铭,等.复方丹参滴丸对离体大鼠缺氧/复氧心肌的保护作用[J].中国病理生理杂志.2002,18(10):1271-1275.
[35]徐叔云,卞如濂,陈修.药理学实验方法[M].第三版,北京:人民卫生出版社,2001.1054-1056.
[36]Reimer KA,Jennings RB,Tatum AH.Pathobiology of acute myocardial ischemia:metabolic,functional and ultrastructural studies[J].Am J Cardiol,1983,52(2):72A-81A.
[37]Classen JB,Mergner WJ,Costa M.ATP hydrolysis by ischemic mitochondria[J].J Cell Physiol,1989,141(1):53-59.
[38]Lopaschuk G.Regulation of carbohydrate metabolism in ischemia and reperfusion [J].Am Heart J,2000,139(2 pt 3),S115-S119.
[39]Sutherland FJ.Hearse DJ.The isolated blood and peffusion fluid perfused heart [J].Pharmacol Res,2000,41(6):613-627.
[40]Curtis MJ,Macleod BA,Tabrizchi R,et al.An improved perfusion apparatus for small animal hearts[J].J Pharmacol Methods,1986,15(1):87-94.
[41]李春杰,余柏林.心肌缺血再灌注损伤机制研究进展[J].人民军医,2004,47(1):47-49.
[42]Sadek HA,Nulton-Persson AC,Szweda PA,et al.Cardiac ischemia/reperfusion,aging,and redox-dependent alterations in mitochondrial function[J].Arch Biochem Biophys.2003,420(2):201-208.
[43]Oriaku G,Xiang B,Dai G,et al.Effects of retrograde cardioplegia on myocardial perfusion and energy metabolism in immature porcine myocardium[J].J Thorac Cardiovasc Surg,2000,119(6):1102-1109.
[44]Harman D.Free radicals in aging[J].Mol Cell Biochem,1988,84(2):155-161.
[45]Wallace KB,Eells JT,Madeira VM,et al.Mitochondria-mediated cell injury.Symposium overview[J].Fundam Appl Toxicol,1997,38(1):23-37.
[46]Woodward B,Zakaria MN.Effect of some free radical scavengers on reperfusion induced arrhythmias in the isolated rat heart[J].J Mol Cell Cardiol.1985,17(5):485-493.
[47]Curtis MJ,Pugsley MK,Walker MJ.Endogenous chemical mediators of ventricular arrhythmias in ischaemic heart disease[J].Cardiovasc Res.1993,27(5):703-719.
[48]时飒,徐长庆,王孝铭,等.复方丹参滴丸对外源性自由基所致心律失常的影响[J].哈尔滨医科大学学报,2001,35(5):335-336.
[49]钮伟真,刘萍,高燕丽,等.豚鼠及大鼠离体心脏希氏束电图的记录[J].中国药理学通报,2001,17(4):476-477.
[50]Niu WZ,Gao YL,Liu P,et al.A comparison of calcitonin gene-related peptide effects on coronary flow and cardiac conduction system in the guinea pig[J].Sheng Li Xue Bao,2000,52(3):259-262.
[51]Goldhaber JI,Ji S,Lamp ST,et al.Effects of exogenous free radicals on electromechanical function and metabolism in isolated rabbit and guinea pig ventricle.Implications for ischemia and reperfusion injury[J].J Clin Invest,1989,83(6):1800-1809.
[52]苏哲坦.再灌流心律失常.国外医学生理、病理科学与临床分册,1986,4:181.
[53]Brooks WW,Conrad CH,Morgan JP.Reperfusion induced arrhythmias following ischaemia in intact rat heart:role of intracellular calcium[J].Cardiovasc Res,1995,29(4):536-542.
[54]Cerbai E,Ambrosio G,Porciatti F,et al.Cellular electrophysiological basis for oxygen radical-induced arrhythmias.A patch-clamp study in guinea pig ventricular myocytes.Circulation,1991,84(4):1773-1782.
[55]宋文良,唐朝枢.PH反常与心肌缺血再灌注损伤.国外医学病理生理科学与临床分册,1991,12:9-11.
[56]Cerbai E,Ambrosio G,Porciatti F,et al.Cellular electrophysiological basis for oxygen radical-induced arrhythmias.A patch-clamp study in guinea pig ventricular myocytes[J].Circulation,1991,84(4):1773-1782.
[57]Markov AK.Hemodynamics and metabolic effects of fructose 1-6 diphosphate in ischemia and shock—experimental and clinical observations[J].Ann Emerg Med,1986,15(12):1470-1477.
[58]王新一,许宏远,徐长庆,等.不同浓度的外源性自由基生成系统致心律失常作用的比较[J].哈尔滨医科大学学报,1999,33(4):251-253.
[1]孙存普,张建中,段绍瑾.自由基生物学导论[M].安徽:中国科技大学出版社,1999.1.
[2]方允中,郑荣梁.自由基生物学的理论与应用[M].北京:科学出版社,2002.7.
[3]Palmer RM,Ferrige AG,Momcada S.Nitric oxide release accounts for the biological activity of endothelial-derived relaxing factor[J].Nature,1987,327(6122):524-526.
[4]詹晖,葛新发.自由基消除与中医药抗运动性疲劳[J].武汉体育学院学报.1998,2:61-65.
[5]周永平,王恬.运动与自由基代谢[J].浙江体育科学,996,18(1):12-15.
[6]吕国蔚主编.医学神经生物学fM].北京:高等教育出版社,2000.390-392.
[7]郑荣梁主编.自由基生命科学进展(第6集)[M].北京:原子能出版社,1998.36-37.
[8]吕国蔚主编.医学神经生物学[M].北京:高等教育出版社,2000.390-392.
[9]代毅,金文泉,高卫.运动对自由基代谢的影响[J].成都体育学院学报,1995,21(3):65-68.
[10]曹锡清.脂质过氧化对细胞与机体的作用[J].生物化学与生物物理进展,1986,2:17-23.
[11]方允中,李文杰.自由基与酶—基础理论及其在生物学和医学中的应用[M].北京:科学出版社,1998.147-162.
[12]Bhatnagar A.Biochemical mechanism of irreversible cell injury caused by free radical-initiated reactions[J].Mol Cell Biochem,1994,137(1):9-16.
[13]Benzie IF.Lipid peroxidation:a review of causes,consequences,measurement and dietary influences[J].Int J Food Sci Nutr,1996,47(3):233-261.
[14]方允中,郑荣梁,沈文梅.自由基生命科学进展(第3集)[M].北京:原子能出版社,1995.99-102.
[15]韩立强.肌肽清除自由基及抗氧化性研究[硕士论文].郑州:河南农业大学,2004.5.
[16]顾有方,陈会良,刘德义,等.自由基的生理和病理作用[J].动物医学进展,2005,26(1):94-97.
[17]程时,宋国兴.生物物理学[M].北京:北京医科大学中国协和医科大学联合出版社,1997.223-230.
[18]Halliwell B,Gutteridge JM.Free Radicols in Biology and Medicine.Oxford:Clarendon Press,1989.188-276.
[19]Hallliwell B,Chirico S.Lipid peroxidation:its mechanism measurement and significance[J].Am J Clin Nutr,1993,57(5 Suppl):715S-725S.
[20]Radi R,Beckman JS,Bush KM,et al.Peroxynitrite-induced membrane lipid peroxidation:the cytotoxic potential of superoxide and nitric oxide[J].Arch Biochem Biophys,1991,288(2):481-487.
[21]Frank H,Hintze T,Bimboes D,et al.Monitoring lipid peroxidation by breath analysis:endogenous hydrocarbons and their metabolic elimination[J].Toxicol Appl Pharmacol,1980,56(3):337-344.
[22]Van Gossum A,Decuyper J,Breath alkanes as an index of lipid peroxidation[J],Eur Respir J,1989,2(8):787-791.
[23]Fang YZ,Zheng RL.Theory and application of free radical biology[M].Beijing:The Science Publishing House,2002.23-48.
[24]Zheng RL,Huang ZY.Basics of free radical medicine and agriculture[M].Beijing:China Higher Education Press,2001.105-143.
[25]高庆,张克英.自由基与动物营养[J].中国饲料,2004,(4):13-14.
[26]刘斌.小鼠早期胚胎发育过程中活性自由基生成机制初探[硕士论文].湖南:湖南农业大学,2002.8.
[27]赵学纲.针灸对亚急性衰老大鼠睾丸自由基代谢和Bcl-2表达的影响[硕士论文].山东:山东中医药大学,2005.21.
[28]孙婷婷,徐建雄.微生物源性抗氧化剂对母猪繁殖性能和自由基代谢的影响[J].上海交通大学学报(农业科学版),2007,25(4):342-346.
[29]金婧.LPS致新生大鼠急性肺损伤促炎性细胞因子、抗炎性细胞因子和NF-κB变化及N-乙酰半胱氨酸干预作用的研究[博士论文].沈阳:中国医科大学.2004.46.
[30]王桔红,谢宗平,陈文.衰老机理及抗衰老研究进展[J].河西学院学报,2003,(2):94-98.
[31]Melov S.Animal models of oxidative stress,aging,and therapeutic antioxidant interventions[J].Int J Biochem Cell Biol,2002,34(11):1395-1400.
[32]Linton S,Davies MJ,Dean RT.Protein oxidation and ageing[J].Exp Gerontol,2001,36(9):1503-1518.
[33]陈瑾,李荣享.衰老的自由基机制[J].中国老年学杂志,2004,24(7):677-679.
[34]Squier TC.Oxidative stress and protein aggregation during biological aging[J].Exp Gerontol,2001,36(9):1539-1550.
[35]王锋青,陈秉初.生物衰老机理研究进展[J].生物学教学,2004,29(2):4-7.
[36]周玫,张宝,陈瑗.机体抗氧化防御系统,自由基医学基础与病理生理[M].北京:人民卫生出版社,2002.121.
[37]Human D.Aging:overview[J].Ann N Y Acad Sci,2001,928:1-21.
[38][美]简.卡著.邱巍,张敏译.延缓衰老[M].北京:新华出版社,1998.
[39]谢文海,甘耀坤.自由基超氧化物歧化酶(SOD)及抗衰老[J].玉林师专学报(自然科学),1999,20(3):70-72.
[40]李大庆.氧自由基与当代医学[J].论文选刊,1999,9:18-19.
[41]敖拉哈.衰老与自由基[J].老年医学与保健,1999,5(2):51-52.
[42]张冬齐,抗氧化剂对帕金森病的保护作用及机制的进展[J].医学综述,2006,12(2):113-115.
[43]Richie JP Jr.The role of glutathione in aging and cancer[J].Exp Gerontol,1992,27(5-6):615-626.
[44]Fernandez-checa JC,Colell A,Garcia-Ruiz C.S-AdenosyI-L-methionine and mitochondrial reduced glutathione depletion in alcoholic liver disease[J].Alcohol,2002,27(3):179-183.
[45]车祺,蒋涛,林伊凤,等.醛糖还原酶基因转染对体外培养大鼠肾脏系膜细胞增殖的影响[J].中华病理学杂志,2005,34(7):417-420.
[46]张洪泉,余文新.中华抗衰老医药学[M].北京:科学出版社,2000.82-109.
[47]张宗玉,范新青.衰老分子机制研究新进展[J].实用老年医学杂志,1994,8(3):97-99.
[48]Trounce I,Byme E,Marzuki S.Decline in skeletal muscle mitochondrial respiratory chain function:possible factor in ageing[J].Lancet,1989,1(8639): 637-639.
[49]Cooper JM,Mann VM,Schapira AH.Analysis of mitochondrial respiratory chain function and mitochondrial DNA deletion in human skeletal muscle:effect of aging[J].J Neurol Sci,1992,113(1):91-98.
[50]Bowling AC,Mutisya EM,Walker LC,et al.Age-dependent inpaiment of mitochondrial function in primate brain[J].J Neurochem,1993,60(5):1964-1967.
[51]Benzi G,Pastoris O,Marzatico F,et al.The mitochondrial electron transfer alteration as a factor involved in the brain aging[J].J Neurobiol Aging,1992,13(3):361-368.
[52]Von Zglinicki T.A mitochondrial membrane hypothesis of aging[J].J Theor Biol,1987,127(2):127-132.
[53]Harman D.Free radical theory of aging:Nutritionnal implications[J].Age,1978,1(4):145-153.
[54]方芳,陈晓春,朱元贵.抗氧化作用可能是人参皂甙Rg1抗细胞凋亡的机制[J].中国临床药理学与治疗学,2002,7(5):412-416.
[55]方雪铃,王锡田,黄淑如,等.杭白菊对小牛血管平滑肌细胞凋亡及其抗氧化性研究[J].浙江大学学报(医学版),2002,31(5):347-350.
[56]刘汴生.衰老过程中的病理生理[J].实用老年医学,2002,16(2):64-68.
[57]陈奇.中药药理研究方法学[M].北京:人民卫生出版社,1993.931-941.
[58]Stadman ER.Protein oxidation and aging[J].Science,1992,257(5074):1220-1224.
[59]Rice-Evans C,Burdon R.Free radical-lipid interactions and their pathological consequences[J].Prog Lipid Res,1993,32(1):71-110.
[60]Yu BP.Aging and oxidative stress:modulation by dietary restriction[J].Free Radic Biol Med,1996,21(5):651-658.
[61]Halliwell B.Oxidation of low-density lipoproteins:questions of initiation,propagation,and the effect of antioxidants[J].Am J Clin Nutr,1995,61(3Suppl):670S-677S.
[62]Paradies G,Petrosillo G,Pistolese M,et al.Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia and reperfusion.Free Radic Biol Med,1999,27(1-2):42-50.
[63]Chen Q,Moghaddas S,Hoppel CL,et al.Reversible blockade of electron transport during ischemia protects mitochondria and decreases myocardial injury following reperfusion.J Pharmacol Exp Ther,2006,319(3):1405-1412.
[64]Petrosillo G,Ruggiero FM,Di Venosa N,et al.Decreased complex Ⅲ activity in mitochondria isolated from rat heart subjected to ischemia and reperfusion:role of reactive oxygen species and cardiolipin[J].FASEB J,2003,17(6):714-716.
[65]李丹彤,冯雁,高妹娟,等.异丙基肾上腺素损伤的大鼠心肌线粒体中细胞色素氧化酶的复活研究[J].中国地方病防治杂志,1997,12(3):138-141.
[66]Chen J,Mehta JL,Haider N,et al.Role of caspases in Ox-LDL-induced apoptotic cascade in human coronary artery endothelial cells.Circ Res,2004,94(3):370-376.
[67]程贵蓉,施娣,陶圣祥,等.安氟醚对离体鼠心的心肌保护作用[J].广州医药,2000,31(5):13-15.
[68]伍静,姚尚龙,黄方敏,等.异丙酚预处理对大鼠离体心脏缺血-再灌注损伤的影响[J].中华麻醉学杂志,2004,24(12):905-908.
[69]徐万红,曹春梅,夏强,等.杭白菊提取液对抗缺血再灌注引起的离体大鼠心肌收缩功能下降[J].中国病理生理杂志,2004,20(5):822-826.
[70]程桂荣,易胜中.黄芪预处理大鼠心肌保护效应的实验研究[J].高血压杂志,2001,9(4):316-318.
[71]景桂霞,温健,王伟.高氧液预处理对兔心肌缺血再灌注损伤的保护作用[J].西安交通大学学报(医学版),2005,26(4):336-339.