二甲双胍对非酒精性脂肪性肝病细胞模型线粒体途径凋亡及氧化应激的影响  

Effect of metformin on mitochondrial pathway of apoptosis and oxidative stress in cell model of nonalcoholic fatty liver disease

作　　者：吴鹏波[1,2] 宋琪 俞媛洁 于红刚[1,2] 罗和生 谭诗云[1,2] Wu Pengbo;Song Qi;Yu Yuanjie;Yu Honggang;Luo Hesheng;Tan Shiyun(Department of Gastroenterology,Renmin Hospital of Wuhan University,Wuhan 430060,China;Hubei Key Laboratory of Digestive Diseases,Wuhan 430060,China)

机构地区：[1]武汉大学人民医院消化内科,武汉430060 [2]消化系统疾病湖北省重点实验室,武汉430060

出　　处：《中华肝脏病杂志》2020年第1期64-68,共5页Chinese Journal of Hepatology

基　　金：武汉大学自主科研青年教师资助项目(2042017kf0099);湖北省自然科学基金(2018CFB236);湖北省卫生和计划生育委员会面上项目(WJ2017M019)。

摘　　要：目的探讨二甲双胍对非酒精性脂肪性肝病细胞模型线粒体途径凋亡及氧化应激的影响。方法体外用0.6 mmol/L油酸诱导HepG2细胞脂质沉积建立非酒精性脂肪性肝病细胞模型,将HepG2细胞分为对照(Con)组、油酸(OA)组、二甲双胍低剂量组(1 mmol/L)、二甲双胍高剂量组(10 mmol/L)。用油红O染色检测细胞内脂滴分布,试剂盒检测培养上清液丙氨酸转氨酶、天冬氨酸转氨酶的水平。用DCFH-DA法检测HepG2细胞活性氧的生成量;用双染流式细胞技术检测HepG2细胞凋亡情况;蛋白质印迹法检测半胱氨酸天冬氨酸蛋白酶3、B淋巴细胞淋巴瘤相关蛋白、B淋巴细胞淋巴瘤2、细胞质细胞色素C蛋白的表达。数据组间比较采用单因素方差分析。结果油酸可诱导HepG2细胞内脂滴明显增加,低、高剂量二甲双胍均可减少细胞内脂滴堆积,二甲双胍高剂量组比二甲双胍低剂量组作用更加明显;OA组HepG2细胞内天冬氨酸转氨酶、丙氨酸转氨酶明显升高,分别为(43.41±7.11)U/L、(29.56±4.11)U/L;二甲双胍低、高剂量处理后细胞内天冬氨酸转氨酶、丙氨酸转氨酶明显降低,分别为(32.44±4.08)U/L、(19.31±3.03)U/L和(26.00±3.11)U/L、(15.11±4.11)U/L,差异均有统计学意义(P值均<0.05);DCFH-DA法检测结果提示油酸组细胞活性氧荧光强度为41.21%±4.23%,二甲双胍低、高剂量组细胞活性氧荧光强度均降低,分别为27.44%±3.91%和17.55%±5.11%,组间差异均有统计学意义(P值均<0.05);流式细胞术检测分析结果显示,OA组细胞凋亡率明显高于Con组(12.12%±0.72%比3.04%±0.57%,P<0.05),二甲双胍低、高剂量处理HepG2细胞后凋亡均明显降低(8.71%±0.71%,5.71%±0.61%,P<0.05)。蛋白质印迹法检测结果提示与Con组相比,OA组B淋巴细胞淋巴瘤相关蛋白、细胞色素C、半胱氨酸天冬氨酸蛋白酶3蛋白表达均增加,而B淋巴细胞淋巴瘤2降低(P值均<0.05),低、高剂量二甲双胍处理HepG2细胞后B淋�Objective To investigate the effects of metformin on mitochondrial pathway of apoptosis and oxidative stress in cell model of nonalcoholic fatty liver disease.Methods An in vitro cell model of nonalcoholic fatty liver disease was established using 0.6 mmol/L oleic acid to induce lipid accumulation in HepG2 cells.HepG2 cells were divided into control(Con)group,oleic acid(OA)group,and metformin-low(1mmol/L)and high(10mmol/L)dose group.Oil Red O stain was used to detect intracellular lipid droplet distribution.The levels of alanine aminotransferase and aspartate aminotransferase in the culture supernatant were detected by assay kits.DCFH-DA method was used to detect the reactive oxygen species of HepG2 cells.Double staining flow cytometry was used to detect the apoptosis rate of HepG2 cells.Western blot was used to detect caspase-3,B-lymphocyte lymphoma-related protein,B-cell lymphoma 2,and cytochrome c protein.One-way analysis of variance was used to compare the data between groups.Results Oleic acid-induced HepG2 cells were significantly increased with lipid droplets.Low and high-dose metformin had reduced intracellular lipid droplets accumulation.The effect of metformin in the high-dose group was more significant than that in the low-dose group.Aspartate aminotransferase and alanine aminotransferase in HepG2 cells of OA group were significantly increased,which were(43.41±7.11)U/L and(29.56±4.11)U/L,respectively.The intracellular aspartate aminotransferase and alanine aminotransferase were decreased significantly after the treatment with low and high-dose metformin,which were(32.44±4.08)U/L,(19.31±3.03)U/L,(26.00±3.11)U/L and(15.11±4.11)U/L,respectively and the differences were statistically significant(P<0.05).DCFH-DA test results showed that the fluorescence intensity of reactive oxygen species in the oleic acid group was 41.21%±4.23%,while the fluorescence intensity of reactive oxygen species in the low and high-dose metformin groups were reduced to 27.44%±3.91%,and 17.55%±5.11%,respectively and the di

关 键 词：二甲双胍 线粒体 细胞凋亡 

分 类 号：R575[医药卫生—消化系统]