肠道菌群通过去乙酰化酶3激活单磷酸腺苷活化的蛋白激酶/雷帕霉素信号通路对脓毒症心肌炎和心肌细胞线粒体损伤的影响

Effects of intestinal flora on septic myocarditis and myocardial cell mitochondrial damage by activating adenosine monophosphate activated protein kinase/rapamycin signaling pathway through deacetylase 3

目的探讨正常肠道粪菌移植(FMT)对脓毒症心肌炎和心肌细胞线粒体损伤的改善作用与机制。方法将40只SD大鼠随机分为盲肠结扎穿孔术(CLP)建立的脓毒症模型组(CLP组)、假手术对照组(sham组)、FMT治疗组(CLP+FMT组)及FMT联合去乙酰化酶3(SIRT3)抑制剂(3-TYP)治疗组(CLP+FMT+3-TYP组),每组各10只。采用HE染色观察大鼠心肌组织的病理变化,采用ELISA检测大鼠血液中IL-1β、IL-6、肿瘤坏死因子(TNF)-α的表达水平,采用Western blot检测大鼠心肌组织中IL-1β、NOD样受体热蛋白结构域相关蛋白3(NLRP3)、去乙酰化酶3(SIRT3)、单磷酸腺苷活化的蛋白激酶(AMPK)、磷酸化AMPK(p-AMPK)、雷帕霉素(mTOR)及p-mTOR表达水平,采用透射电镜观察大鼠心肌细胞线粒体形态,采用流式细胞术分析大鼠心肌细胞的线粒体膜电位变化。结果与sham组比较,CLP组、CLP+FMT组、CLP+FMT+3-TYP组心肌组织SIRT3和p-mTOR表达水平均显著降低,而心肌组织p-AMPK和血液中IL-1β、IL-6及TNF-α的表达水平均显著增加;与CLP组比较,CLP+FMT组心肌组织SIRT3和p-mTOR的表达水平均显著增加,而心肌组织p-AMPK和血液中IL-1β、IL-6及TNF-α的表达水平均显著降低;与CLP+FMT组比较,CLP+FMT+3-TYP组心肌组织SIRT3和p-mTOR的表达水平均显著降低,而心肌组织中p-AMPK和血液中IL-1β、IL-6及TNF-α的表达水平均显著增加;与sham组比较,CLP组线粒体膜电位的红/绿荧光强度比值显著下降;与CLP组比较,CLP+FMT组线粒体膜电位的红/绿荧光强度比值显著增加;与CLP+FMT组比较,CLP+FMT+3-TYP组线粒体膜电位的红/绿荧光强度比值显著下降(P<0.05)。结论肠道菌群通过SIRT3激活AMPK/mTOR信号通路对脓毒症心肌炎和心肌细胞线粒体损伤均有改善作用。

Objective To investigate the effect and mechanism of normal intestinal fecal microbiota transplantation(FMT)on myocardial inflammation and myocardial mitochondrial damage in sepsis.Methods Forty SD rats were randomly divided into sepsis model established by cecal ligation and perforation(CLP)group(CLP group),sham operation control group(sham group),FMT treatment group(CLP+FMT group)and FMT combined with deacetylase 3(SIRT3)inhibitor(3⁃TYP)treatment group(CLP+FMT+3⁃TYP group),each group had 10 animals.HE staining was used to observe the pathological changes of rat myocardial tissue,ELISA was used to detect the expression levels of IL⁃1β,IL⁃6 and TNF⁃αin rat blood.The expression levels of IL⁃1β,nod⁃like receptor heat protein domain associated protein 3(NLRP3),SIRT3,adenosine monophosphate activated protein kinase(AMPK),phosphorylated AMPK(p⁃AMPK),rapamycin(mTOR)and p⁃mTOR in rat myocardial tissue were detected by western blotting.The mitochondrial morphology of rat cardiomyocytes was observed by transmission electron microscopy.The mitochondrial membrane potential of rat cardiomyocytes was analyzed by flow cytometry.Results Compared with sham group,the expression levels of SIRT3 and p⁃mTOR in myocardial tissue of CLP group,CLP+FMT group and CLP+FMT+3⁃TYP group were significantly decreased,while the expression levels of p⁃AMPK in myocardial tissue and IL⁃1β,IL⁃6 and TNF⁃αin blood were significantly increased;Compared with CLP group,the expression levels of SIRT3 and p⁃mTOR in myocardium of CLP+FMT group were significantly increased,while the expression levels of p⁃AMPK in myocardium and IL⁃1β,IL⁃6 and TNF⁃αin blood were significantly decreased;Compared with CLP+FMT group,the expression levels of SIRT3 and p⁃mTOR in myocardium of CLP+FMT+3⁃TYP group were significantly decreased,while the expression levels of p⁃AMPK in myocardium and IL⁃1β,IL⁃6 and TNF⁃αin blood were significantly increased;Compared with sham group,the ratio of red/green fluorescence intensity of mitochondrial membrane potential decreased significantly in CLP group;Compared with CLP group,the ratio of red/green fluorescence intensity of mitochondrial membrane potential in CLP+FMT group was significantly increased;Compared with CLP+FMT group,the ratio of red/green fluorescence intensity of mitochondrial membranepotential in CLP+FMT+3⁃TYP group was significantly decreased(P<0.05).Conclusion The activation of AMPK/mTOR signaling pathway by intestinal flora through SIRT3 can improve both septic myocarditis and myocardial mitochondrial damage.