线粒体自噬对高糖诱导小鼠胚胎成纤维细胞活化作用的影响  

Effect of mitophagy on high glucose-induced activation of mouse embry⁃onic fibroblasts

作　　者：周喆 王德琼 张黎 张钟健 李霜 杨永健 ZHOU Zhe;WANG Deqiong;ZHANG Li;ZHANG Zhongjian;LI Shuang;YANG Yongjian(School of Medicine,Southwest Jiaotong University,Chengdu 610083,China;Department of Cardiology,General Hospi-tal of Western Theater Command of PLA,Chengdu 610083,China)

机构地区：[1]西南交通大学医学院,四川成都610083 [2]解放军西部战区总医院心血管内科,四川成都610083

出　　处：《中国病理生理杂志》2023年第5期893-901,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81800338);西部战区总医院院管课题面上项目(No.417311V2Q)。

摘　　要：目的:探究线粒体自噬对高糖(HG)诱导小鼠胚胎成纤维细胞系NIH 3T3活化作用的影响与机制。方法:体外培养NIH 3T3细胞,用40.0 mmol/L葡萄糖诱导NIH 3T3细胞,构建糖尿病心肌病细胞模型,24 h后检测细胞α-平滑肌肌动蛋白(α-SMA)、I型胶原(Col-I)、p62和微管相关蛋白1轻链3-II(LC3-II)蛋白表达水平。培养NIH 3T3细胞,分别设置正常糖(NG;5.5 mmol/L葡萄糖)组、HG(40.0 mmol/L葡萄糖)组、NG+3-甲基腺嘌呤(3-MA;自噬抑制剂,0.2 mmol/L)组和HG+3-MA组,处理细胞24 h。采用JC-1活细胞染色检测各组细胞线粒体膜电位;Ki-67免疫荧光染色检测各组细胞增殖情况;线粒体自噬检测试剂盒检测各组细胞线粒体自噬情况;Western blot检测各组细胞α-SMA、Col-I、p62、LC3-I和LC3-II蛋白表达水平,并比较LC3-II与LC3-I的比值。结果:JC-1法检测线粒体膜电位结果显示,与NG组比较,HG组NIH 3T3细胞的线粒体损伤减轻;自噬抑制剂3-MA处理后,NG+3-MA组和HG+3-MA组线粒体膜电位明显降低;与HG组比较,HG+3-MA组线粒体膜电位降低。Ki-67免疫荧光检测结果显示,与NG组比较,HG组NIH 3T3细胞的Ki-67阳性率明显升高;自噬抑制剂3-MA处理后,NIH 3T3细胞的Ki-67阳性率降低。Mitophagy检测试剂盒结果显示,与NG组相比,HG组的线粒体自噬通量明显增强;与HG组比较,HG+3-MA组线粒体自噬通量明显降低。Western blot检测结果显示,与NG组比较,HG组NIH 3T3细胞中α-SMA、Col-I、LC3-I和LC3-II蛋白相对表达水平均明显升高,而p62蛋白相对表达水平降低;与HG组比较,HG+3-MA组α-SMA、Col-I、LC3-I和LC3-II相对表达水平均明显降低,p62蛋白相对表达水平升高;与NG组比较,HG+3-MA组中α-SMA蛋白相对表达水平降低。结论:HG刺激NIH 3T3细胞增殖及活化,合成过多的胶原蛋白等细胞外基质蛋白,其作用机制可能与HG诱导线粒体自噬增强有关。AIM:To investigate the effect of mitophagy on the high glucose(HG)-induced activation of mouse embryonic fibroblast cell line NIH 3T3 and its mechanism.METHODS:The NIH 3T3 cells were cultured and induced by 40.0 mmol/L glucose to establish the cell model of diabetic cardiomyopathy.After 24 h of treatment,the protein expres⁃sion levels ofα-smooth muscle actin(α-SMA),collagen type I(Col-I),p62 and microtubule-associated protein 1 light chain 3-II(LC3-II)in NIH 3T3 cells were detected.The NIH 3T3 cells were cultured and divided into normal glucose(NG;5.5 mmol/L glucose)group,HG(40.0 mmol/L glucose)group,NG+3-methyladenine(3-MA;an autophagy inhib⁃itor,0.2 mmol/L)group and HG+3-MA group.The cells were treated for 24 h.The JC-1 live cell staining was used to de⁃tect the mitochondrial membrane potential in each group.The Ki-67 immunofluorescence staining was used to detect the cell proliferation in each group.Mitophagy detection kit was used to detect mitochondrial autophagy.Western blot was used to detect the protein expression levels ofα-SMA,Col-I,p62,LC3-I and LC3-II in each group,and the ratio of LC3-II to LC3-I was compared.RESULTS:The results of mitochondrial membrane potential measured by JC-1 staining showed that the mitochondrial damage of NIH 3T3 cells in HG group was lower than that in NG group,the mitochondrial membrane potential in NG+3-MA group and HG+3-MA group significantly decreased after treatment with autophagy inhibi⁃tor 3-MA,and the mitochondrial membrane potential in HG+3-MA group was lower than that in HG group.The results of Ki-67 immunofluorescence showed that the Ki-67 positive rate of NIH 3T3 cells in HG group was significantly higher than that in NG group,while the Ki-67 positive rate of NIH 3T3 cells decreased after treatment with autophagy inhibitor 3-MA.The results of mitophagy detection kit showed that the mitophagy flux in HG group was significantly increased compared with NG group,and that in HG+3-MA group was significantly decreased compared with HG group.The results of West

关 键 词：3-甲基腺嘌呤 糖尿病心肌病 线粒体自噬 心脏成纤维细胞 

分 类 号：R587.2[医药卫生—内分泌] R363.2[医药卫生—内科学]