线粒体通透性转换孔在失血性休克大鼠血管低反应性中的作用

发布时间：2018-03-26 14:36

本文选题：失血性休克　切入点：血管低反应性　出处：《第三军医大学》2013年硕士论文

【摘要】：失血休克、脓毒症等临床重症患者在经历缺血缺氧、内毒素释放、肠道细菌移位等多重打击后，在失代偿期容易出现严重的组织损伤，出现全身炎症反应综合症(SIRS)、多器官功能障碍(MODS)甚至发生多器官功能衰竭(MOF)。探究其原因，除了全身炎症失控和能量代谢障碍以外，休克后血管反应性降低也是另一重要原因。血管低反应性即血管对血管舒缩药物的失敏。血管低反应性不仅影响着休克的发生发展，还制约着休克的复苏和预后。目前认为休克后血管低反应性的发生机制与钙失敏(calcium desensitization)、肾上腺素能受体失敏、血管平滑肌细胞(VSMC)膜超极化有关。线粒体通透性转换孔(MPTP)是线粒体内膜上的一个重要的非选择性通道，研究表明MPTP在细胞死亡中发挥了重要作用，并参与了多种疾病的发生，例如MPTP开放参与了缺血再灌注损伤；MPTP通过线粒体凋亡途径参与了休克后血管高渗透性的发生。失血性休克后血管组织MPTP是否异常开放？MPTP是否参与了失血性休克后血管低反应性的发生？MPTP开放通过Caspase依赖的凋亡途径参与了细胞凋亡和疾病发生，研究表明Caspase能激活Rho激酶并参与心脏肥大和心衰的发生，而本实验室证实Rho激酶参与调控休克后血管钙敏感性及血管反应性，MPTP是否通过Caspase和Rho激酶从而调节血管钙敏感性及血管反应性？目前还未见相关报道。据此，本研究利用大鼠失血性休克模型，以肠系膜上动脉(SMA)为研究对象，利用MPTP特异性关闭剂环孢素A(CsA)和开放剂苍术苷(ATR)为工具药，探讨了MPTP在失血性休克后血管低反应性中的作用。具体内容包括两大部分：(1)MPTP是否参与失血性休克后血管低反应性的发生；(2)失血性休克后MPTP调节血管反应性的机制。主要实验方法：取正常及失血性休克大鼠肠系膜上动脉(SMA)，采用离体血管环张力测定技术，用血管环对梯度浓度去甲肾上腺素(NA)的收缩反应测定血管反应性，在去极化状态下(120mmol/L K+)，用血管环对梯度浓度Ca2+的收缩反应测定血管的钙敏感性，用分光光度法测定线粒体在540nm处的吸光度变化反映MPTP的开放和关闭状态，用分光光度法测定SMA405nm处的吸光度反映Caspase-3的活性，用western blotting测定SMA肌球蛋白磷酸酶调节亚单位(MYPT)磷酸化水平表示Rho激酶活性。实验分两部分：第一部分实验观察失血性休克大鼠血管反应性变化和钙敏感性变化及MPTP开放的变化，以及通过观察MPTP特异性开放剂ATR和关闭剂CsA是否可以通过MPTP的开放关闭来调节血管反应性及钙敏感性，以证实MPTP在失血性休克血管低反应性形成中的作用。第二部分实验先在体观察休克后Caspase-3活性和Rho激酶活性的变化，以及ATR和CsA对其的影响，以证实MPTP与Caspase-3和Rho激酶的关系。离体实验，缺氧处理血管环测定血管反应性及钙敏感性及MPTP开放状态，以模拟失血性休克模型；在ATR孵育处理下观察Rho激酶特异性抑制剂Y27632、Caspase-3特异性抑制剂Z-DEVD-FMK对Rho激酶、血管钙敏感性及血管反应性的影响，以明确MPTP是否通过Caspase-3、Rho激酶调节血管反应性及钙敏感性。主要研究结果： 1．失血性休克后肠系膜上动脉血管(SMA)反应性和钙敏感性存在双相变化，表现为休克早期(10min-0.5h)SMA血管环对NE和钙的反应性升高，量-效曲线左移，最大收缩力(Emax)升高(p0.05或p0.01)；随着时间的延长，血管环对NE和钙的反应性逐渐下降，表现为休克中晚期(1h-4h)，其量-效曲线明显右移，，最大收缩力(Emax)明显降低(p0.01)。休克早期(10min-0.5h)肠系膜上动脉MPTP的吸光度比值出现下降趋势(表示MPTP开放)，时-效曲线下移，吸光度变化值增大，但无显著性差异(p0.05)；休克中晚期(1h-4h)肠系膜上动脉MPTP的吸光度比值明显下降，时-效曲线下移，吸光度变化值显著增大(p0.05或p0.01)。说明失血性休克中晚期血管MPTP存在异常开放。 2．MPTP的开放剂ATR(5mg/kg)可使休克0.5h后肠系膜上动脉MPTP的吸光度时-效曲线下移，吸光度变化值显著增加(p0.05)，并使SMA血管环对NE和钙的量-效曲线右移，使NE和Ca2+的Emax降低(p0.05或p0.01)；而MPTP的关闭剂CsA(10mg/kg)则可使休克2.0h后肠系膜上动脉MPTP的吸光度时-效曲线上移，吸光度变化值显著降低(p0.05)，并使SMA血管环对NE和钙的量-效曲线左移，使NE和Ca2+的Emax升高(p0.05或p0.01)，提示MPTP在失血性休克血管低反应性和钙失敏的发生中起重要作用。 3.失血性休克后Rho激酶活性出现双相变化，表现为休克早期(10min-0.5h)MYPT磷酸化水平升高(代表Rho激酶活性升高)(p0.05或p0.01)，而休克晚期(4h)MYPT磷酸化水平降低(p0.05)；休克早期(10min)Caspase-3活性无显著变化，休克中晚期(1h-4h)Caspase-3活性增加(p0.01)；MPTP开放剂ATR可使休克0.5h后SMA的caspase-3活性增加(p0.05)和MYPT磷酸化水平降低，而MPTP的关闭剂CsA则可使休克2.0h后caspase-3活性降低(p0.01)和MYPT磷酸化水平升高，说明MPTP调节失血性休克后血管组织caspase-3活性和Rho激酶活性。 4．缺氧处理SMA10min、0.5h后，SMA血管环对NE和钙的反应性升高(P 0.05)，缺氧处理1.0h、2.0h，SMA血管环对NE和钙的反应性降低(P 0.05)，MPTP的吸光度变化值显著增加(P 0.05)，说明缺氧处理可模拟失血性休克。在ATR(20μmol/L)孵育下,给予Rho激酶抑制剂Y27632(10-5mol/L)显著降低SMA血管环对NE和钙的反应性(P0.05)，给予Caspase-3抑制剂Z-DEVD-FMK(100μmol/L)显著升高SMA血管环对NE和钙的反应性(P0.05)，同时MYPT磷酸化水平升高(P0.05)，提示MPTP通过Caspase-3、Rho激酶调节血管反应性和钙敏感性。结论： 1.失血性休克血管MPTP存在异常开放；MPTP在失血性休克血管低反应性和钙失敏的发生中起重要作用。 2. MPTP对失血性休克Caspase-3活性和Rho激酶活性有重要的调节作用，MPTP对Caspase-3活性有正向调节作用，对Rho激酶活性有负向调节作用。 3. MPTP可通过Caspase-3、Rho激酶调节血管反应性及钙敏感性。
[Abstract]:In addition to systemic inflammatory reaction and energy metabolism disorder , the mechanism of hyporeactivity is not only the development of shock , but also the resuscitation and prognosis of shock . The study shows that MPTP plays an important role in cell death and participates in the occurrence of various diseases , such as MPTP is open to ischemia - reperfusion injury .
MPTP is involved in the occurrence of vascular hyporeactivity following hemorrhagic shock . Does the MPTP participate in the occurrence of hyporesponsiveness after hemorrhagic shock ? MPTP is involved in the occurrence of apoptosis and heart failure through caspase - dependent apoptotic pathway . The study shows that Caspase can activate rho kinase and participate in cardiac hypertrophy and heart failure .

In this study , the effects of MPTP on the hyporesponsiveness of hemorrhagic shock were investigated by using the rat hemorrhagic shock model with MPTP - specific closing agent cyclosporin A ( CsA ) and open agent ( ATR ) . The specific contents included two parts : ( 1 ) whether MPTP was involved in the occurrence of hyporeactivity after hemorrhagic shock ;
( 2 ) MPTP after hemorrhagic shock regulates the mechanism of vascular reactivity .

Main experimental methods :

The effects of MPTP on the activity of Caspase - 3 and the activity of Caspase - 3 were measured by means of spectrophotometry . The effects of MPTP on the activity of Caspase - 3 and the activity of Caspase - 3 were measured .
The effects of the specific inhibitor Y27632 , Caspase - 3 specific inhibitor Z - DEVD - FMK on rho kinase , calcium sensitivity and vascular reactivity were observed under ATR incubation .

Key findings :

1 . There was a biphasic change in the reactivity and calcium sensitivity of SMA in the mesenteric artery after hemorrhagic shock . The response to NE and Ca increased at the early stage of shock ( 10 min - 0.5 h ) , and the maximal contraction force ( Emax ) increased ( p < 0.05 or p0.01 ) .
In the early stage of shock ( 1h - 4h ) , the maximal contractile force ( Emax ) decreased significantly ( P 0.01 ) , while the maximum contraction force ( Emax ) decreased significantly ( P 0.01 ) .
In the middle and late stages of shock ( 1h - 4h ) , the absorbance ratio of MPTP in the mesenteric artery decreased significantly , the time - effect curve moved down , and the absorbance changed significantly ( p < 0.05 or p0.01 ) . The abnormal opening of MPTP in the middle and late stage of hemorrhagic shock was described .

2 . MPTP ( 5 mg / kg ) could decrease the absorbance of MPTP in mesenteric artery after 0.5 h shock , and the change in absorbance was significantly increased ( p < 0.05 ) , and the amount of NE and Ca 2 + in SMA blood vessel was decreased ( p < 0.05 or p0.01 ) .
However , CsA ( 10 mg / kg ) of MPTP decreased the absorbance of MPTP on mesenteric artery after shock 2.0h . The change in absorbance was significantly decreased ( p < 0.05 ) , and the Emax of NE and Ca 2 + increased ( p < 0.05 or p0.01 ) . It was suggested that MPTP played an important role in hyporeactivity of hemorrhagic shock and loss of calcium .

3 . After hemorrhagic shock , there were two phase changes after hemorrhagic shock , which showed that the phosphorylation level of MYPT increased in the early stage of shock ( 10 min - 0.5 h ) ( p0.01 or p0.01 ) , while the phosphorylation level of MYPT decreased ( p . 05 ) at the late stage of shock ( 4h ) ;
Caspase - 3 activity was not significantly changed in the early stage of shock ( 10min ) , and the activity of Caspase - 3 in the middle and late stage of shock was increased ( p0.01 ) .
The activity of caspase - 3 and the phosphorylation of MYPT decreased after shock 0.5h , and the activity of caspase - 3 decreased ( p0.01 ) and the level of MYPT phosphorylation increased , indicating that MPTP regulated the activity of caspase - 3 and the activity of caspase - 3 after hemorrhagic shock .

4 . The response of SMA to NE and Ca was significantly increased ( P0.05 ) . The response of SMA to NE and Ca was significantly increased ( P0.05 ) . At the same time , the expression of Caspase - 3 inhibitor Z - DEVD - FMK ( 100 渭mol / L ) increased significantly ( P0.05 ) .

Conclusion :

1 . There was an abnormal opening of MPTP in hemorrhagic shock .
MPTP plays an important role in the hyporeactivity of hemorrhagic shock and the occurrence of calcium loss .

2 . MPTP plays an important role in regulating the activity of Caspase - 3 in hemorrhagic shock . MPTP has a positive regulatory effect on Caspase - 3 activity .

3 . MPTP can regulate vascular reactivity and calcium sensitivity through Caspase - 3 and rho kinase .

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R631.4

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