线粒体损伤在重症休克中作用及虎杖苷治疗
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摘要
重症休克患者有很高的死亡率,在输血补液等一般抗休克治疗后生命仍难以纠正。导致重症休克高死亡的原因很复杂,目前仍然不清楚,因此查明导致高死亡率的发病机制对临床上指导重症休克的治疗具有重要的意义。我们的前期实验证实,血管平滑肌细胞(ASMCs) ATP含量下降,激活ATP敏感性钾通道,促使细胞膜超极化和L型钙通道抑制,导致血管对去甲肾上腺素的反应性下降和顽固性低血压。在这种急性重症休克条件下(失血到MAP30mmHg2小时,回输血液2小时),除了微循环障碍带来血管平滑肌细胞(ASMCs)氧气和营养物质供应不足以外,还查明血管平滑肌细胞线粒体损伤带来ATP的合成障碍引起低血管反应性和顽固性低血压。
重症休克是一个全身性缺血-再灌注损伤,它导致线粒体器质性损伤可能是急性休克晚期一个体内普遍存在的现象,参与了重症休克难治期(微循环衰竭期)的发生。为了证实以上设想,本研究对重症休克大鼠的血管平滑肌细胞、大脑神经元和肝细胞的线粒体功能进行了系列研究,从线粒体结构、功能、代谢及损伤机制方面查明线粒体功能不全在重症休克发病中的作用。
本研究的另一个目的是查明新的线粒体保护剂,并使用线粒体保护剂从实验治疗的角度,确证线粒体损伤在重症休克发生中的作用。白藜芦醇和环孢霉素A是国际公认的线粒体保护剂。白藜芦醇通过螯合氧化反应所需的二价铁抑制氧化应激,又能够抑制线粒体复合物Ⅲ减少氧自由基生成,发挥保护线粒体的作用。和白藜芦醇一样,环孢霉素A通过作用于亲环素D抑制线粒体通透转变孔开放发挥保护线粒体的作用。虎杖苷(Polydatin,PD)的化学名称是白藜芦醇苷(白藜芦醇结合了一个分子的葡萄糖),因此虎杖苷很有可能具有线粒体保护的作用。本研究一方面用三种线粒体保护剂的保护效应进行相互比较,另一方面用三种脏器细胞的变化与全身休克抢救效果相互对照,查明线粒体保护剂对重症休克治疗的重要性,为临床重症休克的治疗提供新途经。
第一部分血管平滑肌线粒体功能不全在重症休克中的作用
一、重症休克时血管平滑肌细胞发生了线粒体损伤
1.重症休克时血管平滑肌细胞线粒体超微结构损伤：透射电镜结果显示假手术组血管平滑肌细胞线粒体呈现椭圆状,嵴结构完整,但是在休克组线粒体呈圆球状,嵴断裂空泡化,明显肿胀,提示重症休克时血管平滑肌细胞细胞线粒体超微结构损伤。
2.重症休克时血管平滑肌细胞线粒体内膜跨跨膜电位降低：采用JC-1荧光探针流式细胞仪测定线粒体跨膜电位。休克组线粒体去极化的血管平滑肌细胞为80.34±9.01%,明显高于假手术组的13.44±7.73%(P=0.000),提示重症休克时血管平滑肌细胞可能存在线粒体质子泵功能损伤或通透转变孔开放,带来跨膜离子梯度的下降。
3.重症休克时血管平滑肌细胞ATP含量降低：休克组血管平滑肌细胞ATP含量降低到正常水平(假手术组)的17.61±7.87%(P=0.000),提示重症休克时血管平滑肌细胞线粒体功能不全。
4.重症休克时血管平滑肌细胞膜KATP的激活和细胞超极化：休克组血管平滑肌细胞膜KATP电流相对假手术组显著增加,在电压为+80mV时,KATP电流密度由假手术组的7.0±2.2pA/p)F增加到休克组的15.7±7.3pA/pF(P=0.001),提示重症休克时血管平滑肌细胞ATP含量降低。和KATP的变化一样,细胞跨膜电位由假手术组的-31.7±5.3mV增加到休克组的-49.7±5.3mV(P=0.000),显示重症休克时血管平滑肌细胞超极化。
5.重症休克时血管平滑肌细胞过氧化脂质(LPO)含量增加：实验结果显示血管平滑肌细胞过氧化脂质从假手术组的10.01±1.56nmol升高到休克组的15.00±2.23nmol(P=0.000),提示重症休克时血管平滑肌细胞内氧自由基激活。
6.重症休克时血管平滑肌细胞溶酶体膜稳定性降低：采用吖叮橙摄取技术评价溶酶体稳定性。休克组血管平滑肌细胞溶酶体吖叮橙荧光显著低于假手术组,提示重症休克时血管平滑肌细胞溶酶体膜通透。流式细胞仪定量分析结果显示休克组“苍白细胞”(溶酶体受损的细胞)由假手术组的28.27±5.19%增加到51.6±4.8%(P=0.000),提示重症休克时大量的血管平滑肌细胞存在溶酶体损伤。
7.重症休克时血管平滑肌细胞线粒体通透转变孔开放：采用钙黄绿素—氯化钴技术,共聚焦显微镜观察发现休克组血管平滑肌细胞线粒体荧光显著低于假手术组。流式细胞仪分析结果显示血管平滑肌细胞线粒体荧光由假手术组的156.6±11.8降低到休克组的48.8±7.1(P=0.000),提示重症休克时血管平滑肌细胞线粒体通透转变孔开放,可能伴随线粒体结构和功能损伤。
综上所述,重症休克通过氧化应激诱导的溶酶体-线粒体轴引起血管平滑肌细胞线粒体损伤。我们查明此时血管平滑肌细胞缺氧,不仅与微循环障碍有关,还可能存在细胞病性缺氧,本质是线粒体损伤,因此保护线粒体可能是重症休克治疗的一个新靶点。
二、查明新的线粒体保护剂-虎杖苷对血管平滑肌细胞的保护效应
在3种保护剂中,环孢霉素A、白藜芦醇和虎杖苷不同程度地抑制重症休克引起的线粒体肿胀等结构破坏,其中虎杖苷的保护效果最显著,线粒体结构基本恢复正常；在环孢霉素A、白藜芦醇和虎杖苷治疗后,线粒体去极化的血管平滑肌细胞由休克组的80.34±9.01%分别降低到为75.38±18.33%、53.69±17.10%和31.57±6.12%,同时血管平滑肌细胞ATP含量由休克组正常水平的17.61±7.89%分别增加到32.69±5.45%、62.12±11.49%和90.71±7.47%；在3种线粒体保护剂治疗后重症休克激活的KATP得到部分抑制,其中虎杖苷效果最显著,在电压钳制在+80mV时,休克虎杖苷治疗组电流密度由休克组的15.7±7.3pA/pF减少至9.4±4.2pA/pF。和KATP的变化一样,休克虎杖苷治疗组的细胞跨膜电位由休克组的-49.7±5.3mV降低到的-36.9±7.2mV；在3种保护剂治疗后血管平滑肌细胞LPO含量均有下降,其中在休克虎杖苷治疗组下降程度最显著,由休克组的15.00±2.23nmol降低到10.42±0.99nmol(P=0.000);3种线粒体保护剂抑制了重症休克时血管平滑肌细胞溶酶体红色吖叮橙荧光和线粒体绿色钙黄绿素荧光强度减弱,其中虎杖苷作用效果最显著,在环孢霉素A、白藜芦醇和虎杖苷治疗后,“苍白细胞”由休克组的51.63±4.77%,分别减少到42.05±2.81%、47.46±3.06%和36.85±3.84%,同时血管平滑肌细胞线粒体绿色钙黄绿素荧光由休克组48.8±7.1恢复到环孢霉素A治疗组的59.7±13.4,白藜芦醇治疗组的62.3±24.8和虎杖苷治疗组的79.6±8.6。总之,3种线粒体保护剂通过抑制重症休克时氧化应激和随后的溶酶体膜通透及线粒体通透转变孔开放发挥了保护血管平滑肌细胞线粒体的作用,其中虎杖苷作用效果最强,血管平滑肌细胞内的ATP含量增加到正常水平的90.71±7.47%。因此虎杖苷是重症休克治疗中理想的线粒体保护剂。
第二部分大鼠多脏器实质细胞线粒体在重症休克中发生的变化
重症休克时,血管平滑肌细胞、神经元和肝细胞发生线粒体损伤。1.重症休克时线粒体有类似的形态学改变,即线粒体肿胀,嵴排列紊乱,断裂呈空泡,基质低电子密度,提示重症休克诱导多脏器线粒体结构破坏。2.细胞内ATP含量降低,以血管平滑肌细胞最为显著,ATP含量降低到正常水平的17.61±7.87%,提示重症休克时多脏器线粒体功能不全。3.线粒体跨跨膜电位降低,提示重症休克时多脏器线粒体电子传递链功能不全和线粒体通透转变孔开放。4.有类似的发病过程,即LPO含量和“苍白”细胞增加,线粒体通透转变孔开放,提示氧化应激诱导的溶酶体损伤和随后的线粒体通透转变孔开放参与了重症休克时多脏器线粒体损伤。
第三部分线粒体保护剂对重症休克线粒体的保护作用
一、线粒体保护剂抑制了重症休克线粒体结构损伤：在线粒体保护剂治疗后,3种细胞(血管平滑肌细胞、神经元和肝细胞)线粒体肿胀减轻,嵴断裂减少,基质电子密度增加,提示线粒体保护剂抑制重症休克诱导的线粒体结构损伤,在虎杖苷治疗组最为显著,线粒体结构基本恢复正常。
二、线粒体保护剂抑制重症休克线粒体跨膜电位降低：在3种线粒体保护剂治疗后,线粒体去极化的细胞在血管平滑肌细胞由休克组的80.34±9.01%,分别降低到75.38±18.33%、53.69±17.10%和31.57±6.12%,在神经元由休克组的65.86±10.88%,分别降至59.46±8.23%、56.32±13.70%和26.22±7.73%,在肝细胞分别由休克组的37.21±4.98%分别降至23.09±4.06%、25.12±3.31%和12.49±3.06%,提示线粒体保护剂可能抑制了重症休克诱导的线粒体电子传递链损伤和通透转变孔开放,其中虎杖苷作用效果明显优于环孢霉素A和白藜芦醇。
三、线粒体保护剂改善了重症休克线粒体ATP合成功能：在3种线粒体保护剂治疗后,细胞内ATP含量在血管平滑肌细胞由休克组正常水平的17.61±7.89%分别增加到32.69±5.45%、62.12±11.49%和90.71±7.47%,在神经元由正常水平的44.14±13.81%升高到正常水平(假手术组)的54.93±13.79%、63.74±16.06%和89.57±9.21%,在肝细胞由休克组正常水平的48.84±3.84%分别增加地正常水平(假手术组)的63.03±6.81%,57.56±7.08%,87.17±17.29%,提示线粒体保护剂显著改善了重症休克时3种细胞线粒体功能,尤其是虎杖苷,在3种线粒体保护剂治疗组中虎杖苷治疗组具有最佳的ATP含量。
四、线粒体保护剂降低了重症休克LPO含量：3种线粒体保护剂治疗后,细胞内的LPO含量过氧化脂质含量出现不同程度的下降,其中休克虎杖苷治疗组下降最显著,在血管平滑肌细胞由休克组正常水平的149.85±22.27%下降到假手术组的104.00±9.89%,在神经元由休克组正常水平的139.93±17.67%降低到正常水平的102.25±9.43%,在肝细胞由正常水平的211.87±21.05%降低到正常水平(假手术组)的102.78±19.61%,提示虎杖苷显著抑制了重症休克诱导的氧化应激和随后的溶酶体和线粒体损伤。
五、线粒体保护剂改善了重症休克溶酶体稳态：在环孢霉素A、白藜芦醇和虎杖苷治疗后,“苍白细胞”减少,其中虎杖苷治疗组减少最显著,在血管平滑肌细胞由休克组的51.63±4.77%减少到36.85±3.84%,在神经元由休克组的18.89±2.03%减少到10.12±1.00%,在肝细胞由休克组的30.14±5.73%减少到5.79±1.28%,提示虎杖苷可以显著降低重症休克诱导的溶酶体膜通透。
六、线粒体保护剂抑制了重症休克线粒体通透转变孔开放：在线粒体保护剂(环孢霉素A、白藜芦醇和虎杖苷)治疗后,线粒体钙黄绿素荧光出现不同程度的恢复,其中虎杖苷的作用效果最明显,在血管平滑肌细胞细胞由休克组正常水平的31。16±0.05%增加到50.83±5.49%,在神经元由休克组正常水平的60.37±10.06%增加到正常水平的91.32±18.57%,在肝细胞由休克组正常水平的23.68±2.41%恢复到46.69±2.89%,提示虎杖苷有效地抑制了重症休克诱导的线粒体通透转变孔开放。
七、线粒体保护剂改善了重症休克的血管反应性、血压：休克大鼠在经输血给药治疗2小时后,休克组去甲肾上腺素阈值增加到失血前的29.3倍,平均动脉压降至47.23±11.28mmHg;在环孢霉素A和白藜芦醇治疗组,去甲肾上腺素阈值分别降至失皿丽的10.4借相11.8倍,同时半均动脉监升至55.23±9.92mmHg和57.10±15.74mmHg；但是在虎杖苷治疗组去甲肾上腺素阈值为失血前的4.8倍,平均动脉压为89.38±16.31mmHg,提示线粒体保护剂不同程度地纠正了重症休克血管低反应性和顽固性低血压,其中虎杖苷的作用效果最显著。
八、线粒体保护剂降低重症休克大鼠大脑皮层NADH水平：在输血再灌2小时末,休克组大鼠大脑皮层NADH水平相当失血前增加了38.58±6.52%,提示重症休克大鼠大脑皮层线粒体电子传递链功能障碍。在3种线粒体保护剂中,只有虎杖苷治疗组大脑皮层NADH水平明显降低,降低到15.03±3.06%,提示虎杖苷改善了重症休克大鼠大脑皮层线粒体电子传递链功能。
九、线粒体保护剂延长了重症休克大鼠的存活时间：休克组大鼠存活时间仅仅为5.4±2.6h,24小时存活率为0；环孢霉素A和白藜芦醇治疗后,存活时间分别延长了2.05倍和1.95倍,但24小时存活率仍然为0；在虎杖苷治疗组大鼠存活时间延长至休克组的4.35倍,24小时存活率显著提高到5/8,提示虎杖苷显著延长休克大鼠存活时间。
综上所述,线粒体保护剂通过抑制重症休克时氧化应激和随后发生的溶酶体膜通透及线粒体通透转变孔开放发挥了保护多脏器线粒体的作用,纠正重症休克顽固性低血压和血管低反应性,延长重症休克大鼠的存活时间,提示采用线粒体保护剂保护多脏器线粒体成为重症休克治疗的新疗法,其中虎杖苷作用效果最显著,休克虎杖苷治疗组大鼠的存活时间由休克组的5.4±2.6h延长到23.7±3.7h,24小时存活率延长到5/8。因此,使用虎杖苷保护线粒体是重症休克治疗的理想选择,为临床重症休克治疗提供了新思路。
结论
1.重症休克时血管平滑肌细胞线粒体参与了重症休克顽固性低血压和低血管反应性的发生,最终导致重症休克高死亡率和难治性。
2.查明重症休克时血管平滑肌细胞线粒体损伤与氧化应激诱导的溶酶体-线粒体轴有关。
3.重症休克时不仅存在血管平滑肌细胞线粒体损伤,大脑神经元和肝细胞线粒体损伤也参与了重症休克的发生和发展,提示多脏器线粒体损伤可能是重症休克普遍存在的现象。
4.使用线粒体保护剂可以减轻重症休克时上述线粒体的损伤,在3种检测的药物中,以虎杖苷的保护效应最为明显。据此本文提出重症休克的治疗除了已知的改善微循环措施外,还要针对实质细胞线粒体的新靶点进行治疗,并且找到具有我国自主知识产权的新型的线粒体保护药-虎杖苷。
High mortality is a leading element of human health that following acute severe hemorrhagic shock even after transfusion and therapy. The mechanism of high morbidity in acute severe hemorrhagic shock is relatively unknown. Therefore, to find out the mechanism of high mortality in severe shock is very critical. In previous study, we showed that low ASMC ATP levels took place in relation to acute severe hemorrhagic shock, activation of ATP-sensitive potassium channels (KATP), hyperpolarization with inhibition of L-type calcium channels, as well as norepinephrine (NE) stimulated influx of Ca2+. The consequence of reduced influx of Ca2+are depressed vaso-responsiveness and persistent hypotension. Besides insufficient of oxygen and nutrients for microcirculation dysfunction in this condition of acute severe shock (bleeding2h and refusion2h), mitochondrial injury of arterial smooth muscle cells (ASMCs) led to low ATP level, which might result in difficult treatment of severe shock even after transfusion.
Due to whole body ischemia-reperfusion injury in severe hemorrhagic shock, mitochondrial injury might be a common phenomenon in the later stage of acute severe shock and involved in the genesis of refractory period in severe shock. Therefore we systemic researched mitochondrial function of ASMCs, neurons and hepatocytes in severe shocked rats to prove the above hypothesis and to ascertain the role of mitochondrial dysfunction from mitochondrial ultrastructre, function, metabolism and injury mechanism.
Another objective of this study is to ascertain new mitochondrial protector and to determine the role of mitochondrial injury in severe shock using mitochondrial protectors. It is known that Res and CsA are mitochondrial protectors. Resveratrol (Res) improves mitochondrial function by scavenging oxygen free radicals, or prevent their formation through inhibiting mitochondrial electron transporting chain of complex III in many pathological conditions. As an iron-chelator, another function of Res protect lysosoma and inhibit mitochondrial permeability transit pores (mPTP) opening by preventment iron-dependent, Fenton-type reactions with formation of hydroxyl radicals. Cyclosporin A (CsA) inhibits mitochondrial permeability transition pore opening through interaction with CypD and reduce mPTP sensitivity to Ca2+and thereby plays a protective role for mitochondria. Polydatin (PD) is a monocrystalline drug that can be isolated from a traditional Chinese herb(Polygonum cuspidatum). The molecular composition of PD is3,4',5-trihydroxystibene-3-monoglucoside, which is akin to the poly-phenol resveratrol (3,4',5-trihydroxystibene). Therefore Polydatin (PD) might be a mitochondrial protector. This study compares mitochondria protective effects of Polydatin with Res and CsA effects and alteration of three cells (ASMCs, neurons and hepatocytes) in acute severe shock to ascertain whether protection of mitochondrial against injury using an effective mitochondrial protector is necessary, which may provide a noval therapy for severe shock.
Part one:The role of ASMCs mitochondrial against injury in acute severe hemorrhagic shock
1. ASMCs mitochondrial injury is involve in severe shock.
(1) ASMCs mitochondrial ultrastructure injury in severe shock. ASMCs mitochondria from control (sham) animals appeared sausage-shaped with normal cristae. In contrast, mitochondria from shock+NS group were spherical or irregularly shaped, apparently swollen with ruptured and poorly defined cristae, indicating ASMCs mitochondrial ultrastructure injury in severe shock.
(2) Loss of ASMCs mitochondrial transmembrane potential (△Ψm) in severe shock. The mitochondrial transmembrane potential was determined by flow cytometry with JC-1. The shock+NS group contained80.34±9.01%cells with with low△Ψm, which was substantially higher than the value of13.44±7.73%in the control (sham) group (P=0.000), indicating mitochondrial electron transport chain dysfunction or opening of mPTP,which led to loss of ASMCs mitochondrial transmembrane potential in severe shock.
(3) ASMCs mitochondrial dysfunction in severe shock. The mitochondrial function was evaluated using intracellular ATP content. It was again found that severe shock caused significant decrease in the ATP levels of ASMCs to17.6±7.9%of the control value (P=0.000), indicating ASMCs mitochondrial dysfunction in severe shock.
(4) ASMCs hyperpolarization and KATP channel activation in severe shock. The results showed that the KATP current densities increased remarkably in severe shock at voltages ranging from-80to+80mV compared with control values (sham group)(n=10cells for each group). At+80mV, the KATP current density increased from7.0±2.2pA/pF in the sham group to15.7±7.3pA/pF in the shock+NS group (P=0.001), indicating reduced intracellular ATP content in severe shock. Consistent with the alteration of KATP current density, the ASMCs membrane potential significantly increased from-31.7±5.3mV in the control (sham) group (n=25) to-49.7±5.3mV in the shock+NS group (n=29; P=0.000), showing ASMCs hyperpolarization in severe shock.
(4) ASMCs oxdative stress in severe shock. It was found that the LPO level in ASMCs was increased from10.01±1.56nmol in the sham group to15.00±2.23nmol in the shock+NS group (P=0.000), indicating ASMCs oxygen free radical activity in severe shock.
(5) ASMCs lysosomal membrane permeability in severe shock. Fluorescence microscopy of ASMCs from the shock+NS group showed a population of cells with decreased intensity of AO-dependent red, granular fluorescence compared with normal cells. AO fluorescence was quantitative analyzed by flow cytometry showed 51.6±4.8%"pale cells" indicated lysosomal injury in the shock+NS group, which was much higher than the observed value of28.3±5.2%in the control group (sham group)(P=0.000), indicating ASMCs lysosomal membrane permeability in severe shock.
(6) ASMCs mitochondrial permeability transit pores (mPTP) opening in severe shock. Mitochondrial permeability transition pores were evaluated by calcein-Co2+technique using confocal microscopy and flow cytometry. Fluorescence microscopy of ASMCs from shock+NS group showed decreased intensity of calcein dependent mitochondrial fluorescence compared with normal cells (sham group). Calcein fluorescence was quatitative analyzed by flow cytometry showed mean fluorescence intensity (MIF) of156.6±11.8in the sham group and48.9±7.1in the shock+NS group (P=0.000), implying ASMCs mitochondrial permeability transition pores opening in severe shock.
According to this study, oxidative stress through lysosomal-mitochondrial axis led to ASMCs mitochondrial injury in severe shock. Therefore, not only microcirculation dysfunction led to ASMCs cytopathic hypoxia, but mitochondrial injury might also involve in severe shock. Therefore, protection of mitochondrial against injury might be a new therapeutic target for severe shock treatment.
2. To ascertain ASMCs protective effect of new protector-Polydatin in severe shock. CsA, Res and PD were used as mitochondrial protectors in severe shock. CsA, Res and PD inhibited shock-induced mitochondrial ultrastructure injury, especially PD has the best protective effect with almost normal mitochondria; The low△Ψm ASMCs decreased from80.34±9.01%in shock+NS group to75.38±18.33%in the shock+CsA group,53.69±17.10%in the shock+Res group and31.57±6.12%in the shock+PD group and the intracellular ATP content improved from17.6±7.9%of the control value in the shock+NS group to32.7±5.4%,62.1±11.5%and90.7±7.5%in the CsA, Res and PD treated group, respectively; Along the voltage range of-80to+80mV, CsA, Res and PD decreased the KATP current density and the membrane potential compared with the shock+NS group, especially in the shock+ PD, the KATP current density at+80mV decreased from15.7±7.3pA/pF in the shock+NS group to9.4±4.2pA/pF and the membrane potential of ASMCs decreased from-49.7±5.3mV in the shock+NS group to-36.9±7.2mV; The LPO content was reduced in mitochondrial protector-treated group, especially in the shock+PD group, the value was reduced from15.00±2.23nmol in the shock+NS group to10.42±0.99nmol (P=0.000); The cells showed partial preservation of red granular lysosomal fluorescence and green mitochondrial fluorescence in mitochondrial protector-treated group, flow cytometry showed that "pale cells" was reduced from51.6±4.8%in the shock+NS group to42.0±2.8%in the shock+CsA group,47.5±3.1%in the shock+Res group and36.8±3.8%in the shock+PD group and ASMCs mitochondrial mean fluorescence intensity (MIF) were mildly preserved from48.9±7.1in the shock+NS group to59.7±13.4,62.3±24.8,79.6±8.6in the CsA-, Res-and PD-treated groups, respectively. Based on above results,3mitochondrial protectors could protect ASMCs against mitochondrial injury through inhibition of oxygen stress with lysosomal membrane permeability and subsequent mitochondrial permeability transition pores in severe shock, especially PD has the best mitochondrial protective effect with the best ATP level (90.71±7.47%of normal value). Therefore PD is the best choice for protection of mitochondrial against injury in severe shock treatment.
Part two:The role of multiple organs mitochondrial injury in acute severe hemorrhagic shock**P＜0.01versus sham group.
Mitochondrial injury of ASMCs, neurons and hepatocytes took placed in severe shock.1. Mitochondria in the shock+NS group were apparently swollen with ruptured cristae, indicating severe shock induced multiple organs mitochondrial ultrastracture injury.2. The ATP level were decreased in the shock+NS group, especially in ASMCs, the value decreased to17.61±7.87%of normal cells, indicating multiple organs mitochondrial dysfunction in severe shock.3. Loss of mitochondrial transmembrane potential (△Ψm) was showed in the shock+NS group, indicating multiple organs mitochondrial electron transporting chain dysfunction and mPTP opening in severe shock.4. Mitochondrial injury of ASMCs, neurons and hepatocytes have similar mechanism. LPO level in ASMCs, neurons and hepatocytes increased with lysosomal membrane permeability and subsequent mPTP opening, implying oxidative stress induced lysosomal injury and subsequent mPTP opening might be involved in the genesis of multiple organs mitochondrial injury in severe shock.
Part three:The protective effect of mitochondrial protectors against multiple organs mitochondrial injury in acute severe hemorrhagic shock
1. Mitochondrial protectors attenuate multiple organs mitochondrial ultrastructure injury in severe shock. Transmission electron microscopy (TEM) was used to examine mitochondrial morphology. Control cells showed normal mitochondria with preserved membranes and cristae. In contrast, mitochondria of ASMCs, neurons and hepatocytes from the shock+NS group appeared swollen and irregularly shaped with disrupted and poorly defined cristae. The mitochondrial alterations in the shock+NS group were partially protected by3mitochondrial protectors, especially by PD. It is indicated that mitochondrial protectors might protect multiple organs mitochondrial against injury in severe shock and PD has the best protective effect with almost normal mitochondria.
2. Mitochondrial protectors inhibit loss of multiple organs mitochondrial transmembrane potential (△Ψm) in severe shock. We determined the mitochondrial transmembrane potential (△Ψm) with JC-1. ASMCs with low△Ψm mitochondria decreased from65.86±10.88%in the shock+NS group to59.46±8.23%,56.32±13.70%and26.22±7.73%after treatment with CsA, Res and PD, respectively. Meanwhile, the value decreased from65.86±10.88%in the shock+NS group to59.46±8.23%in the shock+CsA group,56.32±13.70%in the shock+Res group and26.22±7.73%in shock+PD group in neurons, and from37.21±4.98%in the shock+NS group to23.09±4.06%、25.12±3.31%and12.49±3.06 %after treated by CsA,Res and PD in hepatocytes,respectively,indicating inhibition of mitochondrial electron transition chain dysfunction and mitochondrial permeability transition pores opening by mitochondrial protectors in severe shock and PD has the best protective effect.
3.Mitochondrial protectors improve multiple organs mitochondrial function in severe shock.Intracellular ATP content increased from17.61±7.89%in the shock+NS group to32.69±5.45%.62.12±11.49%and90.71±7.47%in ASMCs,and from44.14±13.81%of normal value in the shogk+NS group to54.93±13.79%,63.74±16.06%and89.57±9.21%in the CsA,Res and PD-treated groups, respectively in neurons.Meanwhile,the value also increased from48.84±3.84%in the shock+NS group to63.03±6.81%in shock+CsA group,57.56±7.08%in the shock+Res group and87.17±17.29%in shock+PD group,showing that PD is a better protective effect for multiple organs mitochondrial function than CsA and Res in severe shock.
4.Mitochondrial protectors inhibit multiple organs oxidative stress in severe shock. The LPO levels were significantly increased in shock+NS group compared with the sham group,but reduced among the three protector-treated groups,especially in PD-treated group,LPO levels decreased from149.85±22.27%of normal value to104.00±9.89%in ASMCs and from139.93±17.67%of normal values in the shock+NS group to102.25±9.43%in neurons,meanwhile the value decreased from211.87±21.05%of normal value to102.78±19.61%in hepatocytes,indicating that inhibition of multiple organs oxidative stress with lysosomal and mitochondrial injury by protectors in severe shock,especially by PD.
5.Mitochondrial protectors inhibit multiple organs lysosomal membrane permeability in severe shock.The percentage of "pale cells",which indicated lysosomal membrane permeabllity,decreased after treatment with CsA,Res and PD in severe shock, especially in the shock+PD group.The value decreased dramatically from51.63±4.77%in the shock+NS group to36.85±3.84%in ASMCs.Meanwhile the value reduced from18.89±2.03%in the shock+NS group to10.12±1.00%in neurons and from30.14±5.73%in the shock+NS group to 5.79±1.28%in hepatocytes, indicating inhibition of multiple organs lysosomal membrane permeability in protector-treated groups, especially PD has the best effect.
6. Mitochondrial protectors inhibit multiple organs mitochondrial permeability transit pores (mPTP) opening in severe shock. Mean mitochondrial fluorescence were improved in protector-treated groups and PD showed the best effect. After treated by PD in severe shock, the fluorescence intensity increased from31.16±0.05%of normal value in the shock+NS group to50.83±5.49%in ASMCs, and from60.37±10.06%of normal conditions in the shock+NS group to91.32±18.57%in neurons, meanwhile increased from23.68±2.41%in the shock+NS group to46.69±2.89%in hepatocytes, indicating inhibition of multiple organs mPTP in severe shock by protectors, especially by PD.
7. Mitochondrial protectors improve Vaso-reactivity and MAP in severe shock. The results showed that the NE threshold concentration increased to29.3times of that of the prehemorrhage level at the end of a period, including2h of hemorrhage and2h of treatment and MAP had decreased to47.23±11.28mmHg in the shock+NS group. In the shock+CsA and shock+Res groups, the NE threshold concentration increased to10.4and11.8times of the prehemorrhage level and MAP increased to55.23±9.92and57.10±15.74mmHg, respectively, at the same time points. Meanwhile, in the shock+PD group, the NE threshold concentration increased to4.8times of the prehemorrhage level during the same period, while MAP increased to89.38±16.31mmHg, indicating improvement of shocked rats vaso-reactivity and resistant hypotension in PD treated group.
8. Mitochondrial protectors decrease severe shocked rat cerebral cortex NADH. It was found that NADH level increased by38.57±6.52%compared with the prebleeding value in shock+NS group, implying cerebral cortex mitochondrial electron transporting respiratory chain dysfunction. The value significantly decreased to15.03±3.06%in shock+PD group, indicating improvement of severe shocked rat cerebral cortex mitochondrial electron transporting chain by PD.
9. Mitochondrial protectors prolong shocked rats survival time. A mean survival time was only5.4±2.6h and100%animal death within24h after reinfusion of shed blood in the shock+NS group. The survival time in the shock+CsA and shock+Res groups was prolonged2.05and1.95times that of the shock+NS group, respectively, which was significantly longer than that in shock+NS group (P<0.000), but all shocked animals died within24h. In the shock+PD group, the survival time was significantly prolonged to4.35times that of the shock+NS group, and the24-h survival rate was significantly increased to5/8, it is indicated that the survival time of shocked rat was significantly prolonged by PD.
According to the above results, mitochondrial protectors could protect mitochondrial against injury through inhibition of oxygen stress with subsequent lysosomal membrane permeability and mitochondrial permeability transition pores, finally improve resistant hypotension, low vaso-reactivity and cerebral cortex mitochondrial electron transporting chain function in severe shock, and prolonged the survival time of shocked rats, especially PD has the best effect with survival time (23.7±3.7h) and the24h survival rate (5/8). Therefore protection of mitochondrial against injury using PD is an ideal choice for severe shock treatment, which might provide a novel clinical therapy for severe shock.
Conclusions
1. Mitochondrial injury of arterial smooth muscle cells (ASMCs) might lead to vaso-responsiveness and persistent hypotension with finally high mortality and refractory in severe shock.
2. ASMCs mitochondrial injury might be come from lysosomal-mitochondrial axis and oxidative stress in severe shock.
3. Mitochondrial injury not only appeared in ASMCs, but also in neurons and hepatocytes during severe shock, implying that multiple organs mitochondrial injury might be a common phenomenon in the genesis and development of severe shock.
4. Mitochondrial protectors could attenuate mitochondrial injury in severe shock and PD has the best protective effect among3protectors in the study. Therefore, we present the conclusion that treatment of severe shock should not only improve microcirculation, but also protect mitochondria against injury with a noval protector-Polydatin in which independent intellectual property rights was owned by our country.
重症休克是一个全身性缺血-再灌注损伤,它导致线粒体器质性损伤可能是急性休克晚期一个体内普遍存在的现象,参与了重症休克难治期(微循环衰竭期)的发生。为了证实以上设想,本研究对重症休克大鼠的血管平滑肌细胞、大脑神经元和肝细胞的线粒体功能进行了系列研究,从线粒体结构、功能、代谢及损伤机制方面查明线粒体功能不全在重症休克发病中的作用。
本研究的另一个目的是查明新的线粒体保护剂,并使用线粒体保护剂从实验治疗的角度,确证线粒体损伤在重症休克发生中的作用。白藜芦醇和环孢霉素A是国际公认的线粒体保护剂。白藜芦醇通过螯合氧化反应所需的二价铁抑制氧化应激,又能够抑制线粒体复合物Ⅲ减少氧自由基生成,发挥保护线粒体的作用。和白藜芦醇一样,环孢霉素A通过作用于亲环素D抑制线粒体通透转变孔开放发挥保护线粒体的作用。虎杖苷(Polydatin,PD)的化学名称是白藜芦醇苷(白藜芦醇结合了一个分子的葡萄糖),因此虎杖苷很有可能具有线粒体保护的作用。本研究一方面用三种线粒体保护剂的保护效应进行相互比较,另一方面用三种脏器细胞的变化与全身休克抢救效果相互对照,查明线粒体保护剂对重症休克治疗的重要性,为临床重症休克的治疗提供新途经。
第一部分血管平滑肌线粒体功能不全在重症休克中的作用
一、重症休克时血管平滑肌细胞发生了线粒体损伤
1.重症休克时血管平滑肌细胞线粒体超微结构损伤：透射电镜结果显示假手术组血管平滑肌细胞线粒体呈现椭圆状,嵴结构完整,但是在休克组线粒体呈圆球状,嵴断裂空泡化,明显肿胀,提示重症休克时血管平滑肌细胞细胞线粒体超微结构损伤。
2.重症休克时血管平滑肌细胞线粒体内膜跨跨膜电位降低：采用JC-1荧光探针流式细胞仪测定线粒体跨膜电位。休克组线粒体去极化的血管平滑肌细胞为80.34±9.01%,明显高于假手术组的13.44±7.73%(P=0.000),提示重症休克时血管平滑肌细胞可能存在线粒体质子泵功能损伤或通透转变孔开放,带来跨膜离子梯度的下降。
3.重症休克时血管平滑肌细胞ATP含量降低：休克组血管平滑肌细胞ATP含量降低到正常水平(假手术组)的17.61±7.87%(P=0.000),提示重症休克时血管平滑肌细胞线粒体功能不全。
4.重症休克时血管平滑肌细胞膜KATP的激活和细胞超极化：休克组血管平滑肌细胞膜KATP电流相对假手术组显著增加,在电压为+80mV时,KATP电流密度由假手术组的7.0±2.2pA/p)F增加到休克组的15.7±7.3pA/pF(P=0.001),提示重症休克时血管平滑肌细胞ATP含量降低。和KATP的变化一样,细胞跨膜电位由假手术组的-31.7±5.3mV增加到休克组的-49.7±5.3mV(P=0.000),显示重症休克时血管平滑肌细胞超极化。
5.重症休克时血管平滑肌细胞过氧化脂质(LPO)含量增加：实验结果显示血管平滑肌细胞过氧化脂质从假手术组的10.01±1.56nmol升高到休克组的15.00±2.23nmol(P=0.000),提示重症休克时血管平滑肌细胞内氧自由基激活。
6.重症休克时血管平滑肌细胞溶酶体膜稳定性降低：采用吖叮橙摄取技术评价溶酶体稳定性。休克组血管平滑肌细胞溶酶体吖叮橙荧光显著低于假手术组,提示重症休克时血管平滑肌细胞溶酶体膜通透。流式细胞仪定量分析结果显示休克组“苍白细胞”(溶酶体受损的细胞)由假手术组的28.27±5.19%增加到51.6±4.8%(P=0.000),提示重症休克时大量的血管平滑肌细胞存在溶酶体损伤。
7.重症休克时血管平滑肌细胞线粒体通透转变孔开放：采用钙黄绿素—氯化钴技术,共聚焦显微镜观察发现休克组血管平滑肌细胞线粒体荧光显著低于假手术组。流式细胞仪分析结果显示血管平滑肌细胞线粒体荧光由假手术组的156.6±11.8降低到休克组的48.8±7.1(P=0.000),提示重症休克时血管平滑肌细胞线粒体通透转变孔开放,可能伴随线粒体结构和功能损伤。
综上所述,重症休克通过氧化应激诱导的溶酶体-线粒体轴引起血管平滑肌细胞线粒体损伤。我们查明此时血管平滑肌细胞缺氧,不仅与微循环障碍有关,还可能存在细胞病性缺氧,本质是线粒体损伤,因此保护线粒体可能是重症休克治疗的一个新靶点。
二、查明新的线粒体保护剂-虎杖苷对血管平滑肌细胞的保护效应
在3种保护剂中,环孢霉素A、白藜芦醇和虎杖苷不同程度地抑制重症休克引起的线粒体肿胀等结构破坏,其中虎杖苷的保护效果最显著,线粒体结构基本恢复正常；在环孢霉素A、白藜芦醇和虎杖苷治疗后,线粒体去极化的血管平滑肌细胞由休克组的80.34±9.01%分别降低到为75.38±18.33%、53.69±17.10%和31.57±6.12%,同时血管平滑肌细胞ATP含量由休克组正常水平的17.61±7.89%分别增加到32.69±5.45%、62.12±11.49%和90.71±7.47%；在3种线粒体保护剂治疗后重症休克激活的KATP得到部分抑制,其中虎杖苷效果最显著,在电压钳制在+80mV时,休克虎杖苷治疗组电流密度由休克组的15.7±7.3pA/pF减少至9.4±4.2pA/pF。和KATP的变化一样,休克虎杖苷治疗组的细胞跨膜电位由休克组的-49.7±5.3mV降低到的-36.9±7.2mV；在3种保护剂治疗后血管平滑肌细胞LPO含量均有下降,其中在休克虎杖苷治疗组下降程度最显著,由休克组的15.00±2.23nmol降低到10.42±0.99nmol(P=0.000);3种线粒体保护剂抑制了重症休克时血管平滑肌细胞溶酶体红色吖叮橙荧光和线粒体绿色钙黄绿素荧光强度减弱,其中虎杖苷作用效果最显著,在环孢霉素A、白藜芦醇和虎杖苷治疗后,“苍白细胞”由休克组的51.63±4.77%,分别减少到42.05±2.81%、47.46±3.06%和36.85±3.84%,同时血管平滑肌细胞线粒体绿色钙黄绿素荧光由休克组48.8±7.1恢复到环孢霉素A治疗组的59.7±13.4,白藜芦醇治疗组的62.3±24.8和虎杖苷治疗组的79.6±8.6。总之,3种线粒体保护剂通过抑制重症休克时氧化应激和随后的溶酶体膜通透及线粒体通透转变孔开放发挥了保护血管平滑肌细胞线粒体的作用,其中虎杖苷作用效果最强,血管平滑肌细胞内的ATP含量增加到正常水平的90.71±7.47%。因此虎杖苷是重症休克治疗中理想的线粒体保护剂。
第二部分大鼠多脏器实质细胞线粒体在重症休克中发生的变化
重症休克时,血管平滑肌细胞、神经元和肝细胞发生线粒体损伤。1.重症休克时线粒体有类似的形态学改变,即线粒体肿胀,嵴排列紊乱,断裂呈空泡,基质低电子密度,提示重症休克诱导多脏器线粒体结构破坏。2.细胞内ATP含量降低,以血管平滑肌细胞最为显著,ATP含量降低到正常水平的17.61±7.87%,提示重症休克时多脏器线粒体功能不全。3.线粒体跨跨膜电位降低,提示重症休克时多脏器线粒体电子传递链功能不全和线粒体通透转变孔开放。4.有类似的发病过程,即LPO含量和“苍白”细胞增加,线粒体通透转变孔开放,提示氧化应激诱导的溶酶体损伤和随后的线粒体通透转变孔开放参与了重症休克时多脏器线粒体损伤。
第三部分线粒体保护剂对重症休克线粒体的保护作用
一、线粒体保护剂抑制了重症休克线粒体结构损伤：在线粒体保护剂治疗后,3种细胞(血管平滑肌细胞、神经元和肝细胞)线粒体肿胀减轻,嵴断裂减少,基质电子密度增加,提示线粒体保护剂抑制重症休克诱导的线粒体结构损伤,在虎杖苷治疗组最为显著,线粒体结构基本恢复正常。
二、线粒体保护剂抑制重症休克线粒体跨膜电位降低：在3种线粒体保护剂治疗后,线粒体去极化的细胞在血管平滑肌细胞由休克组的80.34±9.01%,分别降低到75.38±18.33%、53.69±17.10%和31.57±6.12%,在神经元由休克组的65.86±10.88%,分别降至59.46±8.23%、56.32±13.70%和26.22±7.73%,在肝细胞分别由休克组的37.21±4.98%分别降至23.09±4.06%、25.12±3.31%和12.49±3.06%,提示线粒体保护剂可能抑制了重症休克诱导的线粒体电子传递链损伤和通透转变孔开放,其中虎杖苷作用效果明显优于环孢霉素A和白藜芦醇。
三、线粒体保护剂改善了重症休克线粒体ATP合成功能：在3种线粒体保护剂治疗后,细胞内ATP含量在血管平滑肌细胞由休克组正常水平的17.61±7.89%分别增加到32.69±5.45%、62.12±11.49%和90.71±7.47%,在神经元由正常水平的44.14±13.81%升高到正常水平(假手术组)的54.93±13.79%、63.74±16.06%和89.57±9.21%,在肝细胞由休克组正常水平的48.84±3.84%分别增加地正常水平(假手术组)的63.03±6.81%,57.56±7.08%,87.17±17.29%,提示线粒体保护剂显著改善了重症休克时3种细胞线粒体功能,尤其是虎杖苷,在3种线粒体保护剂治疗组中虎杖苷治疗组具有最佳的ATP含量。
四、线粒体保护剂降低了重症休克LPO含量：3种线粒体保护剂治疗后,细胞内的LPO含量过氧化脂质含量出现不同程度的下降,其中休克虎杖苷治疗组下降最显著,在血管平滑肌细胞由休克组正常水平的149.85±22.27%下降到假手术组的104.00±9.89%,在神经元由休克组正常水平的139.93±17.67%降低到正常水平的102.25±9.43%,在肝细胞由正常水平的211.87±21.05%降低到正常水平(假手术组)的102.78±19.61%,提示虎杖苷显著抑制了重症休克诱导的氧化应激和随后的溶酶体和线粒体损伤。
五、线粒体保护剂改善了重症休克溶酶体稳态：在环孢霉素A、白藜芦醇和虎杖苷治疗后,“苍白细胞”减少,其中虎杖苷治疗组减少最显著,在血管平滑肌细胞由休克组的51.63±4.77%减少到36.85±3.84%,在神经元由休克组的18.89±2.03%减少到10.12±1.00%,在肝细胞由休克组的30.14±5.73%减少到5.79±1.28%,提示虎杖苷可以显著降低重症休克诱导的溶酶体膜通透。
六、线粒体保护剂抑制了重症休克线粒体通透转变孔开放：在线粒体保护剂(环孢霉素A、白藜芦醇和虎杖苷)治疗后,线粒体钙黄绿素荧光出现不同程度的恢复,其中虎杖苷的作用效果最明显,在血管平滑肌细胞细胞由休克组正常水平的31。16±0.05%增加到50.83±5.49%,在神经元由休克组正常水平的60.37±10.06%增加到正常水平的91.32±18.57%,在肝细胞由休克组正常水平的23.68±2.41%恢复到46.69±2.89%,提示虎杖苷有效地抑制了重症休克诱导的线粒体通透转变孔开放。
七、线粒体保护剂改善了重症休克的血管反应性、血压：休克大鼠在经输血给药治疗2小时后,休克组去甲肾上腺素阈值增加到失血前的29.3倍,平均动脉压降至47.23±11.28mmHg;在环孢霉素A和白藜芦醇治疗组,去甲肾上腺素阈值分别降至失皿丽的10.4借相11.8倍,同时半均动脉监升至55.23±9.92mmHg和57.10±15.74mmHg；但是在虎杖苷治疗组去甲肾上腺素阈值为失血前的4.8倍,平均动脉压为89.38±16.31mmHg,提示线粒体保护剂不同程度地纠正了重症休克血管低反应性和顽固性低血压,其中虎杖苷的作用效果最显著。
八、线粒体保护剂降低重症休克大鼠大脑皮层NADH水平：在输血再灌2小时末,休克组大鼠大脑皮层NADH水平相当失血前增加了38.58±6.52%,提示重症休克大鼠大脑皮层线粒体电子传递链功能障碍。在3种线粒体保护剂中,只有虎杖苷治疗组大脑皮层NADH水平明显降低,降低到15.03±3.06%,提示虎杖苷改善了重症休克大鼠大脑皮层线粒体电子传递链功能。
九、线粒体保护剂延长了重症休克大鼠的存活时间：休克组大鼠存活时间仅仅为5.4±2.6h,24小时存活率为0；环孢霉素A和白藜芦醇治疗后,存活时间分别延长了2.05倍和1.95倍,但24小时存活率仍然为0；在虎杖苷治疗组大鼠存活时间延长至休克组的4.35倍,24小时存活率显著提高到5/8,提示虎杖苷显著延长休克大鼠存活时间。
综上所述,线粒体保护剂通过抑制重症休克时氧化应激和随后发生的溶酶体膜通透及线粒体通透转变孔开放发挥了保护多脏器线粒体的作用,纠正重症休克顽固性低血压和血管低反应性,延长重症休克大鼠的存活时间,提示采用线粒体保护剂保护多脏器线粒体成为重症休克治疗的新疗法,其中虎杖苷作用效果最显著,休克虎杖苷治疗组大鼠的存活时间由休克组的5.4±2.6h延长到23.7±3.7h,24小时存活率延长到5/8。因此,使用虎杖苷保护线粒体是重症休克治疗的理想选择,为临床重症休克治疗提供了新思路。
结论
1.重症休克时血管平滑肌细胞线粒体参与了重症休克顽固性低血压和低血管反应性的发生,最终导致重症休克高死亡率和难治性。
2.查明重症休克时血管平滑肌细胞线粒体损伤与氧化应激诱导的溶酶体-线粒体轴有关。
3.重症休克时不仅存在血管平滑肌细胞线粒体损伤,大脑神经元和肝细胞线粒体损伤也参与了重症休克的发生和发展,提示多脏器线粒体损伤可能是重症休克普遍存在的现象。
4.使用线粒体保护剂可以减轻重症休克时上述线粒体的损伤,在3种检测的药物中,以虎杖苷的保护效应最为明显。据此本文提出重症休克的治疗除了已知的改善微循环措施外,还要针对实质细胞线粒体的新靶点进行治疗,并且找到具有我国自主知识产权的新型的线粒体保护药-虎杖苷。
High mortality is a leading element of human health that following acute severe hemorrhagic shock even after transfusion and therapy. The mechanism of high morbidity in acute severe hemorrhagic shock is relatively unknown. Therefore, to find out the mechanism of high mortality in severe shock is very critical. In previous study, we showed that low ASMC ATP levels took place in relation to acute severe hemorrhagic shock, activation of ATP-sensitive potassium channels (KATP), hyperpolarization with inhibition of L-type calcium channels, as well as norepinephrine (NE) stimulated influx of Ca2+. The consequence of reduced influx of Ca2+are depressed vaso-responsiveness and persistent hypotension. Besides insufficient of oxygen and nutrients for microcirculation dysfunction in this condition of acute severe shock (bleeding2h and refusion2h), mitochondrial injury of arterial smooth muscle cells (ASMCs) led to low ATP level, which might result in difficult treatment of severe shock even after transfusion.
Due to whole body ischemia-reperfusion injury in severe hemorrhagic shock, mitochondrial injury might be a common phenomenon in the later stage of acute severe shock and involved in the genesis of refractory period in severe shock. Therefore we systemic researched mitochondrial function of ASMCs, neurons and hepatocytes in severe shocked rats to prove the above hypothesis and to ascertain the role of mitochondrial dysfunction from mitochondrial ultrastructre, function, metabolism and injury mechanism.
Another objective of this study is to ascertain new mitochondrial protector and to determine the role of mitochondrial injury in severe shock using mitochondrial protectors. It is known that Res and CsA are mitochondrial protectors. Resveratrol (Res) improves mitochondrial function by scavenging oxygen free radicals, or prevent their formation through inhibiting mitochondrial electron transporting chain of complex III in many pathological conditions. As an iron-chelator, another function of Res protect lysosoma and inhibit mitochondrial permeability transit pores (mPTP) opening by preventment iron-dependent, Fenton-type reactions with formation of hydroxyl radicals. Cyclosporin A (CsA) inhibits mitochondrial permeability transition pore opening through interaction with CypD and reduce mPTP sensitivity to Ca2+and thereby plays a protective role for mitochondria. Polydatin (PD) is a monocrystalline drug that can be isolated from a traditional Chinese herb(Polygonum cuspidatum). The molecular composition of PD is3,4',5-trihydroxystibene-3-monoglucoside, which is akin to the poly-phenol resveratrol (3,4',5-trihydroxystibene). Therefore Polydatin (PD) might be a mitochondrial protector. This study compares mitochondria protective effects of Polydatin with Res and CsA effects and alteration of three cells (ASMCs, neurons and hepatocytes) in acute severe shock to ascertain whether protection of mitochondrial against injury using an effective mitochondrial protector is necessary, which may provide a noval therapy for severe shock.
Part one:The role of ASMCs mitochondrial against injury in acute severe hemorrhagic shock
1. ASMCs mitochondrial injury is involve in severe shock.
(1) ASMCs mitochondrial ultrastructure injury in severe shock. ASMCs mitochondria from control (sham) animals appeared sausage-shaped with normal cristae. In contrast, mitochondria from shock+NS group were spherical or irregularly shaped, apparently swollen with ruptured and poorly defined cristae, indicating ASMCs mitochondrial ultrastructure injury in severe shock.
(2) Loss of ASMCs mitochondrial transmembrane potential (△Ψm) in severe shock. The mitochondrial transmembrane potential was determined by flow cytometry with JC-1. The shock+NS group contained80.34±9.01%cells with with low△Ψm, which was substantially higher than the value of13.44±7.73%in the control (sham) group (P=0.000), indicating mitochondrial electron transport chain dysfunction or opening of mPTP,which led to loss of ASMCs mitochondrial transmembrane potential in severe shock.
(3) ASMCs mitochondrial dysfunction in severe shock. The mitochondrial function was evaluated using intracellular ATP content. It was again found that severe shock caused significant decrease in the ATP levels of ASMCs to17.6±7.9%of the control value (P=0.000), indicating ASMCs mitochondrial dysfunction in severe shock.
(4) ASMCs hyperpolarization and KATP channel activation in severe shock. The results showed that the KATP current densities increased remarkably in severe shock at voltages ranging from-80to+80mV compared with control values (sham group)(n=10cells for each group). At+80mV, the KATP current density increased from7.0±2.2pA/pF in the sham group to15.7±7.3pA/pF in the shock+NS group (P=0.001), indicating reduced intracellular ATP content in severe shock. Consistent with the alteration of KATP current density, the ASMCs membrane potential significantly increased from-31.7±5.3mV in the control (sham) group (n=25) to-49.7±5.3mV in the shock+NS group (n=29; P=0.000), showing ASMCs hyperpolarization in severe shock.
(4) ASMCs oxdative stress in severe shock. It was found that the LPO level in ASMCs was increased from10.01±1.56nmol in the sham group to15.00±2.23nmol in the shock+NS group (P=0.000), indicating ASMCs oxygen free radical activity in severe shock.
(5) ASMCs lysosomal membrane permeability in severe shock. Fluorescence microscopy of ASMCs from the shock+NS group showed a population of cells with decreased intensity of AO-dependent red, granular fluorescence compared with normal cells. AO fluorescence was quantitative analyzed by flow cytometry showed 51.6±4.8%"pale cells" indicated lysosomal injury in the shock+NS group, which was much higher than the observed value of28.3±5.2%in the control group (sham group)(P=0.000), indicating ASMCs lysosomal membrane permeability in severe shock.
(6) ASMCs mitochondrial permeability transit pores (mPTP) opening in severe shock. Mitochondrial permeability transition pores were evaluated by calcein-Co2+technique using confocal microscopy and flow cytometry. Fluorescence microscopy of ASMCs from shock+NS group showed decreased intensity of calcein dependent mitochondrial fluorescence compared with normal cells (sham group). Calcein fluorescence was quatitative analyzed by flow cytometry showed mean fluorescence intensity (MIF) of156.6±11.8in the sham group and48.9±7.1in the shock+NS group (P=0.000), implying ASMCs mitochondrial permeability transition pores opening in severe shock.
According to this study, oxidative stress through lysosomal-mitochondrial axis led to ASMCs mitochondrial injury in severe shock. Therefore, not only microcirculation dysfunction led to ASMCs cytopathic hypoxia, but mitochondrial injury might also involve in severe shock. Therefore, protection of mitochondrial against injury might be a new therapeutic target for severe shock treatment.
2. To ascertain ASMCs protective effect of new protector-Polydatin in severe shock. CsA, Res and PD were used as mitochondrial protectors in severe shock. CsA, Res and PD inhibited shock-induced mitochondrial ultrastructure injury, especially PD has the best protective effect with almost normal mitochondria; The low△Ψm ASMCs decreased from80.34±9.01%in shock+NS group to75.38±18.33%in the shock+CsA group,53.69±17.10%in the shock+Res group and31.57±6.12%in the shock+PD group and the intracellular ATP content improved from17.6±7.9%of the control value in the shock+NS group to32.7±5.4%,62.1±11.5%and90.7±7.5%in the CsA, Res and PD treated group, respectively; Along the voltage range of-80to+80mV, CsA, Res and PD decreased the KATP current density and the membrane potential compared with the shock+NS group, especially in the shock+ PD, the KATP current density at+80mV decreased from15.7±7.3pA/pF in the shock+NS group to9.4±4.2pA/pF and the membrane potential of ASMCs decreased from-49.7±5.3mV in the shock+NS group to-36.9±7.2mV; The LPO content was reduced in mitochondrial protector-treated group, especially in the shock+PD group, the value was reduced from15.00±2.23nmol in the shock+NS group to10.42±0.99nmol (P=0.000); The cells showed partial preservation of red granular lysosomal fluorescence and green mitochondrial fluorescence in mitochondrial protector-treated group, flow cytometry showed that "pale cells" was reduced from51.6±4.8%in the shock+NS group to42.0±2.8%in the shock+CsA group,47.5±3.1%in the shock+Res group and36.8±3.8%in the shock+PD group and ASMCs mitochondrial mean fluorescence intensity (MIF) were mildly preserved from48.9±7.1in the shock+NS group to59.7±13.4,62.3±24.8,79.6±8.6in the CsA-, Res-and PD-treated groups, respectively. Based on above results,3mitochondrial protectors could protect ASMCs against mitochondrial injury through inhibition of oxygen stress with lysosomal membrane permeability and subsequent mitochondrial permeability transition pores in severe shock, especially PD has the best mitochondrial protective effect with the best ATP level (90.71±7.47%of normal value). Therefore PD is the best choice for protection of mitochondrial against injury in severe shock treatment.
Part two:The role of multiple organs mitochondrial injury in acute severe hemorrhagic shock**P＜0.01versus sham group.
Mitochondrial injury of ASMCs, neurons and hepatocytes took placed in severe shock.1. Mitochondria in the shock+NS group were apparently swollen with ruptured cristae, indicating severe shock induced multiple organs mitochondrial ultrastracture injury.2. The ATP level were decreased in the shock+NS group, especially in ASMCs, the value decreased to17.61±7.87%of normal cells, indicating multiple organs mitochondrial dysfunction in severe shock.3. Loss of mitochondrial transmembrane potential (△Ψm) was showed in the shock+NS group, indicating multiple organs mitochondrial electron transporting chain dysfunction and mPTP opening in severe shock.4. Mitochondrial injury of ASMCs, neurons and hepatocytes have similar mechanism. LPO level in ASMCs, neurons and hepatocytes increased with lysosomal membrane permeability and subsequent mPTP opening, implying oxidative stress induced lysosomal injury and subsequent mPTP opening might be involved in the genesis of multiple organs mitochondrial injury in severe shock.
Part three:The protective effect of mitochondrial protectors against multiple organs mitochondrial injury in acute severe hemorrhagic shock
1. Mitochondrial protectors attenuate multiple organs mitochondrial ultrastructure injury in severe shock. Transmission electron microscopy (TEM) was used to examine mitochondrial morphology. Control cells showed normal mitochondria with preserved membranes and cristae. In contrast, mitochondria of ASMCs, neurons and hepatocytes from the shock+NS group appeared swollen and irregularly shaped with disrupted and poorly defined cristae. The mitochondrial alterations in the shock+NS group were partially protected by3mitochondrial protectors, especially by PD. It is indicated that mitochondrial protectors might protect multiple organs mitochondrial against injury in severe shock and PD has the best protective effect with almost normal mitochondria.
2. Mitochondrial protectors inhibit loss of multiple organs mitochondrial transmembrane potential (△Ψm) in severe shock. We determined the mitochondrial transmembrane potential (△Ψm) with JC-1. ASMCs with low△Ψm mitochondria decreased from65.86±10.88%in the shock+NS group to59.46±8.23%,56.32±13.70%and26.22±7.73%after treatment with CsA, Res and PD, respectively. Meanwhile, the value decreased from65.86±10.88%in the shock+NS group to59.46±8.23%in the shock+CsA group,56.32±13.70%in the shock+Res group and26.22±7.73%in shock+PD group in neurons, and from37.21±4.98%in the shock+NS group to23.09±4.06%、25.12±3.31%and12.49±3.06 %after treated by CsA,Res and PD in hepatocytes,respectively,indicating inhibition of mitochondrial electron transition chain dysfunction and mitochondrial permeability transition pores opening by mitochondrial protectors in severe shock and PD has the best protective effect.
3.Mitochondrial protectors improve multiple organs mitochondrial function in severe shock.Intracellular ATP content increased from17.61±7.89%in the shock+NS group to32.69±5.45%.62.12±11.49%and90.71±7.47%in ASMCs,and from44.14±13.81%of normal value in the shogk+NS group to54.93±13.79%,63.74±16.06%and89.57±9.21%in the CsA,Res and PD-treated groups, respectively in neurons.Meanwhile,the value also increased from48.84±3.84%in the shock+NS group to63.03±6.81%in shock+CsA group,57.56±7.08%in the shock+Res group and87.17±17.29%in shock+PD group,showing that PD is a better protective effect for multiple organs mitochondrial function than CsA and Res in severe shock.
4.Mitochondrial protectors inhibit multiple organs oxidative stress in severe shock. The LPO levels were significantly increased in shock+NS group compared with the sham group,but reduced among the three protector-treated groups,especially in PD-treated group,LPO levels decreased from149.85±22.27%of normal value to104.00±9.89%in ASMCs and from139.93±17.67%of normal values in the shock+NS group to102.25±9.43%in neurons,meanwhile the value decreased from211.87±21.05%of normal value to102.78±19.61%in hepatocytes,indicating that inhibition of multiple organs oxidative stress with lysosomal and mitochondrial injury by protectors in severe shock,especially by PD.
5.Mitochondrial protectors inhibit multiple organs lysosomal membrane permeability in severe shock.The percentage of "pale cells",which indicated lysosomal membrane permeabllity,decreased after treatment with CsA,Res and PD in severe shock, especially in the shock+PD group.The value decreased dramatically from51.63±4.77%in the shock+NS group to36.85±3.84%in ASMCs.Meanwhile the value reduced from18.89±2.03%in the shock+NS group to10.12±1.00%in neurons and from30.14±5.73%in the shock+NS group to 5.79±1.28%in hepatocytes, indicating inhibition of multiple organs lysosomal membrane permeability in protector-treated groups, especially PD has the best effect.
6. Mitochondrial protectors inhibit multiple organs mitochondrial permeability transit pores (mPTP) opening in severe shock. Mean mitochondrial fluorescence were improved in protector-treated groups and PD showed the best effect. After treated by PD in severe shock, the fluorescence intensity increased from31.16±0.05%of normal value in the shock+NS group to50.83±5.49%in ASMCs, and from60.37±10.06%of normal conditions in the shock+NS group to91.32±18.57%in neurons, meanwhile increased from23.68±2.41%in the shock+NS group to46.69±2.89%in hepatocytes, indicating inhibition of multiple organs mPTP in severe shock by protectors, especially by PD.
7. Mitochondrial protectors improve Vaso-reactivity and MAP in severe shock. The results showed that the NE threshold concentration increased to29.3times of that of the prehemorrhage level at the end of a period, including2h of hemorrhage and2h of treatment and MAP had decreased to47.23±11.28mmHg in the shock+NS group. In the shock+CsA and shock+Res groups, the NE threshold concentration increased to10.4and11.8times of the prehemorrhage level and MAP increased to55.23±9.92and57.10±15.74mmHg, respectively, at the same time points. Meanwhile, in the shock+PD group, the NE threshold concentration increased to4.8times of the prehemorrhage level during the same period, while MAP increased to89.38±16.31mmHg, indicating improvement of shocked rats vaso-reactivity and resistant hypotension in PD treated group.
8. Mitochondrial protectors decrease severe shocked rat cerebral cortex NADH. It was found that NADH level increased by38.57±6.52%compared with the prebleeding value in shock+NS group, implying cerebral cortex mitochondrial electron transporting respiratory chain dysfunction. The value significantly decreased to15.03±3.06%in shock+PD group, indicating improvement of severe shocked rat cerebral cortex mitochondrial electron transporting chain by PD.
9. Mitochondrial protectors prolong shocked rats survival time. A mean survival time was only5.4±2.6h and100%animal death within24h after reinfusion of shed blood in the shock+NS group. The survival time in the shock+CsA and shock+Res groups was prolonged2.05and1.95times that of the shock+NS group, respectively, which was significantly longer than that in shock+NS group (P<0.000), but all shocked animals died within24h. In the shock+PD group, the survival time was significantly prolonged to4.35times that of the shock+NS group, and the24-h survival rate was significantly increased to5/8, it is indicated that the survival time of shocked rat was significantly prolonged by PD.
According to the above results, mitochondrial protectors could protect mitochondrial against injury through inhibition of oxygen stress with subsequent lysosomal membrane permeability and mitochondrial permeability transition pores, finally improve resistant hypotension, low vaso-reactivity and cerebral cortex mitochondrial electron transporting chain function in severe shock, and prolonged the survival time of shocked rats, especially PD has the best effect with survival time (23.7±3.7h) and the24h survival rate (5/8). Therefore protection of mitochondrial against injury using PD is an ideal choice for severe shock treatment, which might provide a novel clinical therapy for severe shock.
Conclusions
1. Mitochondrial injury of arterial smooth muscle cells (ASMCs) might lead to vaso-responsiveness and persistent hypotension with finally high mortality and refractory in severe shock.
2. ASMCs mitochondrial injury might be come from lysosomal-mitochondrial axis and oxidative stress in severe shock.
3. Mitochondrial injury not only appeared in ASMCs, but also in neurons and hepatocytes during severe shock, implying that multiple organs mitochondrial injury might be a common phenomenon in the genesis and development of severe shock.
4. Mitochondrial protectors could attenuate mitochondrial injury in severe shock and PD has the best protective effect among3protectors in the study. Therefore, we present the conclusion that treatment of severe shock should not only improve microcirculation, but also protect mitochondria against injury with a noval protector-Polydatin in which independent intellectual property rights was owned by our country.
引文
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