【作者】 李静；

【导师】 张骏艳；

【作者基本信息】 安徽医科大学 ， 药学， 2022， 硕士

【摘要】 目的:心肌梗死(myocardial infarction,MI)引起的心肌损伤是导致心力衰竭的主要原因。最近研究表明在缺血性心脏病中心肌存在炎性小体和自噬相关水平的变化,本研究旨在探讨自噬与NLRP1炎性小体交互调控在心肌梗死后心脏功能及心肌细胞损伤中的作用及机制研究。方法:体内实验采用永久结扎C57BL/6小鼠左冠状动脉前降支构建急性小鼠心肌梗死(MI)模型,体外实验采用血管紧张素Ⅱ(AngⅡ)诱导H9c2心肌细胞损伤。分别使用自噬激活剂雷帕霉素(Rapa)和自噬抑制剂3-甲基腺嘌呤(3-MA)来激活或抑制心肌细胞自噬,通过心电图和心动超声检测小鼠心功能;TTC染色检测心肌梗死面积;HE染色及Masson染色检测心肌组织病理学变化;透射电镜观察心肌自噬小体及线粒体超微结构变化;试剂盒检测CK-MB、LDH、SOD活力及c Tn I、MDA含量;免疫组化、免疫荧光及蛋白免疫印迹检测p62、Beclin-1、LC3B、NLRP1、caspase-1、ASC、IL-1β蛋白的相对表达量。体外实验进一步采用Muramyl dipeptide(MDP)和NLRP1-si RNA分别激活和沉默H9c2心肌细胞中NLRP1炎性小体表达,观察对AngⅡ诱导H9c2心肌细胞损伤后的细胞自噬水平的影响。结果:调控小鼠MI后心肌自噬水平及体外AngⅡ诱导H9c2心肌细胞后的自噬,通过心电图、超声心动图、TTC染色、组织病理学分析及细胞活力检测等表明Rapa能够改善心梗小鼠心脏功能,减少心肌梗死面积,减轻心肌细胞损伤。而3-MA则能加重心梗小鼠心功能障碍及心肌细胞损伤。透射电镜结果显示Rapa组小鼠心肌内自噬小体增多,并且改善MI引起心肌线粒体外膜破裂,内嵴断裂甚至消失。3-MA组心肌内自噬小体减少,心肌线粒体结构破坏严重。检测结果显示与模型组相比,Rapa组心肌损伤标志物水平降低,心肌损伤有所改善,而3-MA组提示心肌损伤加重。实验结果显示,适当激活小鼠MI后心肌自噬,可抑制小鼠心脏组织NLRP1炎性小体相关蛋白的表达。相反地,抑制自噬可升高NLRP1炎性小体相关蛋白的表达。提示调控自噬可影响小鼠心脏组织及心肌细胞中NLRP1炎性小体的表达情况。进一步通过激活和沉默AngⅡ处理后H9c2心肌细胞中NLRP1炎性小体的表达,结果显示,NLRP1-si RNA干扰后,诱导H9c2心肌细胞中NLRP1炎性小体的表达减少,可激活心肌细胞中自噬,使其自噬相关蛋白表达增加,而使用MDP可增加心肌细胞中NLRP1炎性小体的表达,抑制心肌细胞自噬,使其自噬相关蛋白表达减少。结论:NLRP1炎性小体与自噬在心肌梗死及心肌损伤后具有交互调控关系。适度增加心梗后心肌自噬可减少心肌组织NLRP1炎性小体表达,进而改善心梗后小鼠心脏功能及减轻心肌细胞损伤,而抑制心梗后心肌自噬则增加心肌组织NLRP1炎性小体表达,加重心梗后小鼠心脏功能障碍及心肌细胞损伤。抑制心肌细胞中NLRP1炎性小体,可适当的激活自噬,而激活心肌细胞NLRP1炎性小体,则会抑制心肌细胞自噬。更多还原

【Abstract】 Purpose: Cardiac injury caused by myocardial infarction(MI)is the major cause of heart failure.Recent studies have demonstrated the presence of inflammasomes and autophagy expression in ischemic heart disease,and this study aimed to explore the roles and mechanisms of autophagy and NLRP1 inflammasomes in the reciprocal regulation of myocardial function and cardiomyocyte injury after MI.Methods: In in vivo experiments,an acute mouse myocardial infarction(MI)model was constructed using permanent ligation of C57 BL /6 mice,and in vitro angiotensin(AngⅡ)induced H9c2 cardiomyocyte injury.The autophagy activator Rapamycin(Rapa)and the autophagy inhibitor 3-methyladenine(3-MA)were used to activate or inhibit myocardial autophagy respectively.Mouse cardiac function was detected by ECG and echocardiography.The MI area was measured by TTC staining.Cardiac histopathology was determined by HE and Masson staining.Ultrastructural changes in myocardial autophagosomes and mitochondria were observed by TEM.CK-MB,LDH,SOD activity and c Tn I,MDA content were analysed with a kit;Autophagy and inflammation in cardiomyocytes were assessed via measuring autophagy-associated proteins and NLRP1 inflammasome pathway related proteins.More,in vitro experiments,Muramyl dipeptide(MDP)and NLRP1-si RNA activated and silenced NLRP1 expression in H9c2 cardiomyocytes,respectively,to observe the effect on cardiomyocyte damage and autophagy levels induced by Ang treatment.Results: Effect of regulating myocardial autophagy levels after MI and in vitro AngⅡinduced H9c2 cardiomyocytes by ECG,echocardiography,and cell viability detection showed that Rapa could improve cardiac function with TTC staining,histopathological analysis,and reduced cardiomyocyte injury.However,3-MA can aggravate cardiac dysfunction and cardiomyocyte injury in MI mice.Transmission electron microscopy revealed increased myocardial autophagosomes in the Rapa group,and improved myocardial infarction leading to the rupture of,or even disappearance,of the extramyocardial mitochondrial membrane.In the 3-MA group,myocardial autophagomes decreased and severe damage to the myocardial mitochondrial structure.The results showed decreased levels of myocardial damage markers in the Rapa group compared to the model group,whereas the 3-MA group showed increased myocardial injury.The experimental results showed that appropriately activated myocardial autophagy after myocardial infarction in mice inhibited the expression of the NLRP1 inflammasome-associated protein in the mouse heart tissue.In contrast,inhibition of autophagy increased the expression of NLRP1 inflammasome-associated proteins.It is suggested that regulating autophagy can affect the expression of NLRP1 inflammasome in myocardial tissues and cardiomyocytes of mice.Furthermore,the activation and silencing of H9c2 cardiomyocyte inflammasome NLRP1 expression after Ang Ⅱ treatment indicated that inhibition of H9c2 cardiomyocyte inflammasome NLRP1 expression activates cardiomyocyte autophagy,resulting in increased autophagy-related protein expression,while MDP increases cardiomyocyte NLRP1 expression and decreases autophagy-related protein expression in cardiomyocytes.Conclusion: The NLRP1 inflammasome and autophagy regulate each other after myocardial infarction and myocardial injury.Cardiac autophagy after MI reduces the expression of myocardial tissue inflammasome NLRP1,improves myocardial function and cardiomyocyte injury in mice,while inhibiting myocardial autophagy increases the expression of myocardial tissue inflammasome NLRP1,and aggravates myocardial dysfunction and cardiomyocyte injury in mice after MI.Simultaneous inhibition of the inflammasome NLRP1 in cardiomyocytes activates autophagy,whereas activation of the cardiomyocyte inflammasome NLRP1 suppresses autophagy in cardiomyocytes.更多还原