线粒体自噬-NLRP3炎症小体轴介导丹酚酸B对心肌缺血损伤的保护作用

发布时间：2018-03-07 08:36

  本文选题：丹酚酸B　切入点：心肌损伤　出处：《南京中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的:观察丹酚酸B(salvianolic acid B,Sal B)对大鼠心肌缺血损伤的保护作用。从NLRP3炎症小体角度出发探讨丹酚酸B保护损伤心肌细胞的可能机制。为靶向炎症小体进行疾病的治疗及指导临床提供重要的参考。研究方法:研究共分为三章:第一章、异丙肾腺素(isoproterenol,ISO)诱导Sprague-Dawley(SD)大鼠复制心肌缺血损伤模型。通过心电图中T-波的变化值,大鼠血清心肌酶(CK、GOT和LDH)、氧化指标(MDA和T-SOD)和炎症因子IL-1β含量,心肌组织HE和TUNEL染色研究Sal B对缺血损伤心肌的保护作用;进一步应用免疫组化、western blot和real-time PCR检测心肌组织中炎症小体NLRP3相关蛋白和mRNA表达;探讨心肌缺血损伤与NLRP3炎症小体的相关性。第二章、建立H_2O_2诱导H_9C_2细胞氧化应激损伤模型和LPS+ATP联合诱导H_9C_2细胞炎性损伤模型。应用MTT法检测细胞存活率,试剂盒检测细胞上清液中氧化指标和炎症因子的含量观察Sal B对心肌细胞损伤的保护作用;应用western blot和免疫荧光研究Sal B对心肌细胞损伤过程中NLRP3炎症小体的影响;进一步应用TUNEL和Calciem-AM染色研究Sal B对细胞焦亡的影响。第三章、利用LPS+ATP联合诱导促进H_9C_2细胞中NLRP3炎症小体的激活,通过探针检测ROS和线粒体膜电位,western bolt分析SIRT1、p-Foxo3a和线粒体自噬相关蛋白,研究Sal B抑制NLRP3炎症小体的可能机制,进一步通过自噬抑制剂3-MA验证自噬在NLRP3炎症小体激活中的作用。研究结果:第一章结果显示:Sal B可以明显减轻心肌组织坏死、炎细胞浸润和减少DNA损伤,明显降低心电图中T波值(P0.05,P0.01)。与模型组比较,Sal B各剂量组的CK值、GOT值和IL-1β值,Sal B中、高剂量组的MDA值及中剂量组的LDH值均明显降低(P0.05,P0.01);而Sal B中、高剂量组的T-SOD值明显升高(P0.05,P0.01);Sal B给药组的心脏组织中NLRP3、ASC、caspase-1 P20和IL-1β蛋白和mRNA表达明显降低,呈一定的剂量依赖性(P0.05,P0.01)。第二章结果显示:经MTT法检测,确定H_2O_2合适的造模浓度为l00μM,时间为2h;SalB合适的预处理浓度为1、5、25μM,时间为24h。与H_2O_2模型组比较,给予不同剂量的SalB可以提高细胞的存活率,保持细胞形态的稳定性。与模型组比较,给予SalB可以明显降低H_2O_2刺激的细胞上清液中LDH、MDA水平,升高T-SOD的水平(P0.05,P0.01),呈一定的剂量依赖性。Western bolt、免疫荧光和Elisa结果显示,Sal B可以抑制H_9C_2细胞氧化损伤和炎性损伤中NLRP3炎症小体的激活和IL-lβ的表达和分泌。TUNEL和Calcein-AM染色结果表明,Sal B可以保护细胞膜的完整性,减轻DNA损伤,从而抑制细胞焦亡。第三章结果显示:在LPS+ATP联合诱导作用下,Sal B可以促进H_9C_2细胞中SIRT1、p-Foxo3a和MnSOD表达的升高(P0.05,P0.01),抑制细胞ROS和线粒体膜电位的升高,促进自噬标志蛋白LC3Ⅱ和Beclin-1以及线粒体自噬调控蛋白Parkin的表达,抑制P62和PINK1的表达(P0.05,P0.01)。在自噬抑制剂3-MA的作用下,Sal B(51μM)促进H_9C_2细胞线粒体自噬的作用被减弱,抑制NLRP3炎症小体激活的作用也明显被减弱。但是3-MA并没有影响Sal B对SIRT1和p-Foxo3a的促进作用。研究结论:1.Sal B可以抑制缺血心脏组织中NLRP3炎症小体激活,从而减轻炎症反应达到保护心脏的目的。2.心肌细胞氧化应激损伤时会诱导NLRP3炎症小体的激活,丹酚酸B可以保护H_9C_2细胞氧化应激损伤,抑制NLRP3炎症小体的激活。3.Sal B抑制H_9C_2细胞中NLRP3炎症小体活化的可能机制是激活SIRTl-FOXO3a-Parkin轴,改善心肌细胞线粒体自噬,减少受损线粒体的积累,从而抑制NLRP3激活信号的释放。
[Abstract]:Study objective: To observe the effect of salvianolic acid B (salvianolic acid B, Sal B) protective effect on myocardial ischemia reperfusion injury in rats. The NLRP3 inflammasome perspective to explore the possible mechanism of salvianolic acid B to protect the damaged myocardial cells. Provide important references for the treatment of disease and guide the clinical target to inflammasome research. Methods: the study is divided into three chapters: the first chapter, isoproterenol gland hormone (isoproterenol, ISO) induced by Sprague-Dawley (SD) copy the model of myocardial ischemia reperfusion injury in rats. The changes of T- wave in ECG, rat serum myocardial enzymes (CK, GOT and LDH), oxidation index (MDA and T-SOD) the content of IL-1 and inflammatory factor beta, myocardial tissue HE and TUNEL staining to study the protective effects of Sal B on myocardial ischemia injury; the further application of immunohistochemistry, the expression of inflammatory corpuscle NLRP3 related protein and mRNA detection of myocardial Western blot and real-time PCR; to investigate the myocardial ischemia damage Correlation between injury and NLRP3 inflammasome. In the second chapter, the establishment of H_2O_2 induced oxidative stress damage in H_9C_2 cells and LPS+ATP H_9C_2 cells model of inflammatory injury model induced by the combination. The cell viability was measured by MTT method, to observe the content of cell supernatant kit in the oxidation index of inflammatory factor and the protective effect of Sal B on myocardial cell injury; effect the application of Western blot and Sal B immunofluorescence studies on myocardial cell damage in the process of NLRP3 inflammasome; affect the further application of TUNEL and Calciem-AM staining of Sal B on pyroptosis. In the third chapter, the use of LPS +ATP to promote the activation of H_9C_2 cells induced by the combination of NLRP3 inflammasome, detected by ROS and mitochondrial membrane potential probe, Western bolt analysis of SIRT1, p-Foxo3a and mitochondrial autophagy related protein, study the possible mechanism of Sal B inhibiting NLRP3 inflammasome, further through autophagy inhibitors 3-MA楠岃瘉鑷�鍣�鍦∟LRP3鐐庣棁灏忎綋婵，

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