动力相关蛋白1在脓毒症心肌损伤中的作用及其机制研究

发布时间：2018-03-15 01:23

本文选题：脓毒症　切入点：心功能损伤　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景脓毒症(sepsis)是由感染引起的全身炎症反应综合征,脓毒症的全球发病率很高。严重脓毒症患者会出现不同程度的心功能下降,并增加患者死亡风险。线粒体功能紊乱是脓毒症时引起心肌细胞不可逆转的损害的主要原因。近年来研究表明,心脏线粒体分裂融合的动态平衡状态在维持心脏线粒体正常生理状态中具有重大的作用。而大量研究表明,在心肌缺血再灌注损伤、心力衰竭、高血压、糖尿病心肌病、动脉粥样硬化等多种心血管疾病的发生发展中,线粒体分裂融合的动态平衡紊乱起了至关重要的作用。动力相关蛋白1(dynamin-related peptide 1,Drp1)是参与线粒体分裂过程中的一种关键蛋白。Drp1是否参与了脓毒症心肌损伤作用尚不明了。目的本实验将从整体和细胞水平入手研究脓毒症时Drp1在心肌损伤中的作用和作用机制;并探讨在脓毒症时,IL-6和TNF-α哪个是导致Drp1改变的主要炎症因子。方法1)SD大鼠腹腔注射LPS建立脓毒症模型,测定大鼠生存率。2)心脏超声检查测定大鼠心功能。3)提取心肌组织总蛋白和线粒体蛋白,测定各组分中Drp1、Drp1 S637、Drp1 S616、CaMKⅡ、p-JNK、Rac1/Cdc42、RhoA 蛋白水平。4)ELISA法测定大鼠血清中炎症因子(LPS、TNF-α、IL-6)浓度。5)透射电镜观察大鼠心脏线粒体形态。6)激光共聚焦显微技术观察心肌细胞线粒体形态。结果1)SD大鼠腹腔注射不同浓度LPS(5、10、20mg/kg)后,每搏输出量和心输出量开始下降;线粒体片段化程度增加。2)SD大鼠给予不同浓度LPS(5、10、20mg/kg)腹腔注射后6h,心肌组织总Drp1水平无明显变化,但线粒体Drp1量随着LPS浓度的增高明显增加。提示,脓毒症心肌组织中Drp1发生了线粒体转位。3)SD大鼠腹腔注射LPS(20mg/kg)后,大鼠死亡率达70%;而Drp1的特异性抑制剂Mdivi-1(1 mg/kg,i.p.)可明显抑制LPS诱导的心肌每搏输出量和心输出量的下降,并降低大鼠死亡率。4)不同浓度LPS注射后6h,发现心肌组织中磷酸化Drp1 S637蛋白水平无明显变化,但磷酸化Drp1 S616蛋白水平明显增加。5)SD大鼠腹腔注射LPS(20mg/kg)后,血清中炎症因子LPS、TNF-α、IL-6浓度明显增加。LPS(0.01、0.1 或 1 μg/mL)、TNF-α(5、10 或 20ng/mL)、IL-6(5、10或20ng/mL)孵育H9C2细胞48h后,可浓度依赖性降低细胞的生存率。6)LPS(1 μg/mL)、IL-6(20ng/ml)对 H9C2 细胞总 Drp1 和线粒体 drp1 水平无明显影响。TNF-α(20ng/ml)对H9C2细胞Drp1总量无明显影响,但TNF-α孵育细胞后,Drp1 Ser616位点的磷酸化和线粒体Drp1水平明显增加。7)TNF-α(20ng/ml)处理H9C2细胞后,可明显增加CaMKⅡ的表达。但CaMKⅡ的抑制剂KN-93不能明显抑制TNF-α诱导的Drp1的磷酸化和线粒体转位,同时对TNF-α诱导的心肌细胞死亡也没有明显影响。8)TNF-α(20 ng/ml)处理 H9C2 细胞后,对细胞 p-JNK、Rac1/Cdc42 无明显影响,但RhoA蛋白表达明显增加。ROCK1和ROCK2的特异性抑制剂Y-27632和Fasudil可明显抑制TNF-α的Drp1的磷酸化和线粒体转位,同时有对抗TNF-α降低心肌细胞生存率的作用。激光共聚焦结果显示,Y-27632和Fasudil同时可抑制TNF-α诱导的线粒体片段化增多。结论本实验结果表明脓毒症心肌损伤与Drp1线粒体转位有关,其转位机制可能是由于Drp1 S616发生了磷酸化。TNF-α可能是脓毒症心肌损伤时引起Drp1 S616磷酸化的主要炎症因子,而RohA/ROCK通路可能参与了 TNF-α诱导的Drp1 S616磷酸化和线粒体转位。
[Abstract]:Background sepsis (sepsis) is a systemic inflammatory response syndrome caused by infection, sepsis, the global incidence rate is very high. In patients with severe sepsis can appear heart function decreased in different degrees, and increase the risk of death in patients. Mitochondrial dysfunction is a major cause of myocardial cell irreversible damage in sepsis the study shows that in recent years, the dynamic equilibrium state of heart mitochondrial fission and fusion plays an important role in maintaining the normal physiological state of heart mitochondria. While a large number of studies show that in myocardial ischemia reperfusion injury, heart failure, high blood pressure, diabetic cardiomyopathy, atherosclerosis and other cardiovascular diseases, disorder of dynamic balance of mitochondrial fission fusion has played a crucial role. Dynamin related protein 1 (dynamin-related peptide 1, Drp1) is a key in mitochondrial fission process Protein.Drp1 is involved in myocardial injury in sepsis remains unclear. Objective: this experiment will start on sepsis Drp1 in myocardial injury and the mechanism of the whole and cellular level; and to explore in sepsis, IL-6 and TNF- alpha which is the main inflammatory factors lead to changes in Drp1 method. 1) SD rats were intraperitoneally injected with LPS to establish the model of sepsis rats were survival.2) echocardiography determination of heart function in rats with.3) to extract the total protein and mitochondrial protein in myocardial tissue, determination of Drp1 components in Drp1 S637, Drp1, S616, CaMK II, p-JNK, Rac1/Cdc42, RhoA).4 protein level determination of inflammatory factors in serum of rats by ELISA (LPS, TNF- alpha, IL-6) concentration of.5).6 rat heart mitochondria were observed by transmission electron microscope) observation of mitochondria myocardial cells by confocal laser microscopy. Results 1) intraperitoneal injection of SD in rats with concentration of LPS (5,10,20mg/kg ), stroke volume and cardiac output decreased; mitochondrial fragmentation increased.2) SD rats were treated with different concentrations of LPS (5,10,20mg/kg) after intraperitoneal injection of 6h, no significant changes in total Drp1 level of myocardial tissue, but the amount of mitochondrial Drp1 with increase of the concentration of LPS increased significantly. The results indicated that the occurrence of mitochondrial translocation of.3 Drp1 in myocardial tissue of sepsis) in SD rats by intraperitoneal injection of LPS (20mg/kg), rat mortality rate was 70%; while the Drp1 specific inhibitor Mdivi-1 (1 mg/kg, i.p.) decreased significantly inhibited LPS induced cardiac stroke volume and cardiac output, and reduce the mortality of rats) in different.4 the concentration of LPS after injection of 6h phosphate in myocardial tissue of Drp1 S637 protein level had no obvious change, but the phosphorylation of Drp1 S616 protein level increased.5) SD rats by intraperitoneal injection of LPS (20mg/kg), serum inflammatory cytokines LPS, TNF- alpha, IL-6 concentration significantly increased.LPS (0.01 0.1, or 1 g/mL), alpha TNF- (5,10 or 20ng/mL), IL-6 (5,10 or 20ng/mL) of H9C2 cells incubated with 48h, a concentration dependent decrease in cell survival rate of.6 (LPS) 1 g/mL), IL-6 (20ng/ml) on H9C2 cell total Drp1 and mitochondrial drp1 levels had no significant effect on.TNF- alpha (20ng/ ml) had no obvious effect on the total H9C2 of Drp1 cells, but TNF- alpha after the cells were incubated in Drp1, phosphorylation of Ser616 and mitochondrial Drp1 levels significantly increased.7) TNF- alpha (20ng/ml) in H9C2 cells after treatment, can significantly increase the expression of CaMK II. But the CaMK II inhibitor KN-93 can not inhibit TNF- alpha induced Drp1 phosphorylation and mitochondrial translocation, while TNF- induced myocardial cell death and no obvious effect of.8 TNF- (ng/ml) alpha 20) after treatment of H9C2 cells, the p-JNK cells had no significant effect on Rac1/Cdc42, but significant increase in the expression of RhoA specific inhibitor Y-2763.ROCK1 and ROCK2 2 and Fasudil can significantly inhibit TNF- alpha Drp1 phosphorylation and mitochondrial translocation, and against TNF- alpha decreased myocardial cell survival rate. Confocal laser scanning results showed that mitochondrial Y-27632 fragments and Fasudil also inhibited TNF- induced increased. Conclusion the experimental results show that the myocardial injury in sepsis associated with Drp1 the mitochondrial translocation, translocation may be due to the occurrence of S616 Drp1 phosphorylation of.TNF- alpha may be mainly caused by the inflammatory cytokines Drp1 S616 phosphorylation in septic myocardial injury in sepsis, and RohA/ROCK pathway may be involved in the TNF- induced Drp1 S616 phosphorylation and mitochondrial translocation.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7;R542.2

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