NLRC4炎症小体介导自噬反应在脓毒症心肌功能障碍小鼠中的作用  

The role of NLRC4 inflammasome-mediated autophagy in a mouse model of sepsis-induced myocardial dysfunction

作　　者：蒋大军 蒋万威 周颖 田勇 杨国辉 Jiang Dajun;Jiang Wanwei;Zhou Ying;Tian Yong;Yang Guohui(Affiliated Hospital,Guizhou Medical University,Guiyang 550004,Guizhou Province,China)

机构地区：[1]贵州医科大学附属医院,贵州省贵阳市550004

出　　处：《中国组织工程研究》2023年第23期3667-3673,共7页Chinese Journal of Tissue Engineering Research

基　　金：贵州医科大学附属医院国自然培育基金项目(gyfynsfc-2021-54),项目负责人:杨国辉;贵阳市科技计划项目[筑科合同(2019)9-1-9号],项目负责人:杨国辉。

摘　　要：背景:研究发现炎症小体及自噬在脓毒症患者的免疫功能中扮演了重要角色。但现阶段研究仅局限于探索脓毒症中某一自噬信号通路的变化特点,对脓毒症心肌功能障碍自噬调控的具体机制尚未阐明。目的:探讨核苷酸结合寡聚化结构域样受体蛋白4(NOD-like receptor family,pyrin domain-containing protein 4,NLRC4)炎症小体与自噬水平在小鼠脓毒症心肌功能障碍中的变化,以期为脓毒症心肌功能障碍的发生机制提供理论依据。方法:昆明雄性小鼠48只,随机分为6组,分别为假手术(6,12,24 h)组和盲肠结扎穿孔(6,12,24 h)组。盲肠结扎穿孔组结扎小鼠盲肠远端1/2,22号针头来回穿刺2次,挤出少许肠内容物;假手术组不结扎穿孔,其余操作与盲肠结扎穿孔术相同。观察小鼠术后一般情况,超声测定小鼠心功能,ELISA法检测肿瘤坏死因子α、肌钙蛋白T质量浓度;苏木精-伊红染色法观察心肌病理变化;透射电镜观察心肌线粒体及自噬体变化;实时荧光定量PCR检测心肌组织NLRC4 mRNA的表达,Western blot检测心肌组织炎症因子、LC3、Beclin-1、NLRC4的蛋白表达。结果与结论:(1)小鼠盲肠结扎穿孔术后6 h肿瘤坏死因子α、Beclin-1、LC3-Ⅱ/LC3-Ⅰ比值升高(P<0.05);术后12 h,肿瘤坏死因子α、肌钙蛋白T质量浓度升高,左室射血分数及左室短轴缩短率降低,NLRC4 mRNA及蛋白、白细胞介素1β、白细胞介素18、Beclin-1、LC3-Ⅱ/LC3-Ⅰ比值均升高(P<0.05);术后24 h,NLRC4 mRNA及蛋白表达进行性升高,白细胞介素1β、白细胞介素18维持高表达,Beclin-1、LC3-Ⅱ/LC3-Ⅰ比值降低(P<0.05);(2)心肌病理学改变(苏木精-伊红染色):假手术组及盲肠结扎穿孔术后6h组,心肌纤维正常,未见炎症细胞浸润;盲肠结扎穿孔术后12 h,心肌纤维水肿,炎症细胞浸润;随着时间的延长,心肌纤维排列紊乱,炎症细胞浸润加重,部分心肌纤维出现变性坏死;(3)透射电镜显示:盲肠结扎BACKGROUND:Studies have found that inflammasome and autophagy play important roles in the immune function of patients with sepsis.However,the current research is only limited to exploring the characteristics of changes in a certain autophagy signaling pathway in sepsis,and the specific mechanism of autophagy regulation in sepsis-induced myocardial dysfunction has not been elucidated.OBJECTIVE:To investigate the changes of NOD-like receptor family,pyrin domain-containing protein 4(NLRC4)inflammasome and autophagy levels in a mouse model of sepsis-induced myocardial dysfunction,in order to provide a theoretical basis for the pathogenesis of sepsis-induced myocardial dysfunction.METHODS:Forty-eight clean and healthy Kunming male mice were randomly divided into six groups,sham operation group(6,12,24 hours)and cecal ligation and puncture group(6,12,24 hours)with three groups each.In the cecal ligation and puncture group,the distal half of the cecum of the mice was ligated,and a 22-gauge needle was used for puncturing twice to squeeze out a litile intestinal content.Except for ligation and perforation,other operations in the sham operation group were the same as those in the cecal ligation and perforation group.The general condition of the mice after operation was observed.Mouse cardiac function was measured by VINNO 76LAB ultrasound.Levels of tumor necrosis factorαand troponin T were detected by enzyme-linked immunosorbent assay.Pathological changes of the myocardium were observed by hematoxylin-eosin staining.Changes in myocardial mitochondria and autophagosome were observed under transmission electron microscope.Real-time quantitative polymerase chain reaction was used to detect the expression of NLRC4 mRNA in myocardialtissue,and western blot was used to detect the expression of inflammatory factors,LC3,Beclin-1,and NLRC4 protein in mouse myocardialtissue.RESULTS AND CONCLUSION:Tumor necrosis factorαlevel,Beclin-1 level,and LC3-II/LC3-I ratio were significantly increased in mice at 6 hours after cecal ligation a

关 键 词：脓毒症 脓毒症心肌功能障碍 盲肠结扎穿孔术 免疫紊乱 NLRC4炎症小体 自噬 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R541.9