线粒体融合蛋白2对棕榈酸处理的胰岛β细胞的影响及机制研究

Effect and mechanism of mitofusin 2 on palmitic acid-treated mouse pancreatic β cells

目的探讨线粒体融合蛋白2(Mfn2)对棕榈酸(PA)处理的小鼠胰岛β细胞的影响及机制。方法使用浓度梯度的PA分别处理小鼠胰岛β细胞MIN6细胞,通过CCK8法检测细胞活性,并确定后续实验的PA浓度。通过转染慢病毒构建Mfn2敲低和Mfn2过表达稳定转染细胞系,将MIN6细胞分为Mfn2敲低空载对照(ShNC-Mfn2)组、敲低Mfn2(Sh-Mfn2)组、Mfn2过表达空载对照(OENC-Mfn2)组、Mfn2过表达(OE-Mfn2)组、ShNC-Mfn2+PA组、Sh-Mfn2+PA组、OENC-Mfn2+PA组、OE-Mfn2+PA组。使用小牛血清白蛋白作为PA对照和PA分别处理细胞。使用流式细胞仪检测各组细胞凋亡率、细胞内活性氧簇(ROS)和线粒体膜电位水平,酶标仪检测细胞内ATP水平,实时荧光定量聚合酶链反应与Western blotting法检测Mfn2及凋亡相关蛋白[B细胞淋巴瘤2(Bcl-2)、半胱氨酸依赖的天冬氨酸蛋白酶3(Caspase-3)、Bcl-2相关X蛋白(Bax)]的mRNA和蛋白表达量。两组间比较采用t检验,多组间比较采用单因素方差分析。结果与0 mmol/L PA相比,PA浓度为0.125、0.250、0.500、0.750、1.000 mmol/L时细胞存活率明显下降(P<0.01),选择细胞存活率大概为50%的PA浓度即0.5 mmol/L为后续实验浓度,此时细胞存活率为43.53%±0.56%。MIN6细胞加入0.5 mmol/L PA后,Mfn2 mRNA和蛋白表达量均明显下降(P<0.01)。与ShNC-Mfn2组相比,Sh-Mfn2组Mfn2 mRNA及蛋白表达水平下降(P<0.05);与OENC-Mfn2组相比,OE-Mfn2组Mfn2 mRNA及蛋白表达水平升高(P<0.01)。与ShNC-Mfn2+PA组相比,Sh-Mfn2+PA组的ROS水平显著升高(P<0.01),ATP水平及线粒体膜电位显著下降(P<0.05);与OENC-Mfn2+PA组相比,OE-Mfn2+PA组的ROS水平降低,ATP水平及线粒体膜电位明显升高(P<0.05)。与ShNC-Mfn2+PA组相比,Sh-Mfn2+PA组细胞凋亡率升高(P<0.01);与OENC-Mfn2+PA组相比,OE-Mfn2+PA组细胞凋亡率降低(P<0.01)。与ShNC-Mfn2+PA组相比,Sh-Mfn2+PA组Bax和Caspase-3的mRNA与蛋白表达量显著升高,Bcl-2的mRNA与蛋白表达量显著降低(均P<0.05);与OENC-Mfn2+PA组相比,OE-Mfn2+PA组Bax和Caspase-3的mRNA与蛋白显著降低,Bcl-2的mRNA与蛋白表达量显著升高(均P<0.05)。结论Mfn2可以改善PA诱导的MIN6细胞脂毒性损伤,过表达Mfn2对脂毒性下的MIN6细胞有保护作用,敲低Mfn2会加重脂毒性对MIN6细胞的损伤。

Objective To investigate the effect and mechanism of mitofusin 2(Mfn2)on palmitic acid(PA)-treated mouse pancreatic β cells.Methods Mouse pancreatic β-cell MIN6 cells were treated with PA in a concentration gradient,and cell viability was detected by CCK8 method,and the PA concentration in subsequent experiments was determined.Mfn2 knockdown and Mfn2 overexpression stable cell lines were constructed by lentivirus transfection using MIN6 cells.Each group was named as Mfn2 knockdown empty-load control(ShNC-Mfn2)group,Mfn2 knockdown(Sh-Mfn2)group,Mfn2 overexpression empty-load control(OENC-Mfn2)group,Mfn2 overexpression(OE-Mfn2)group,ShNC-Mfn2+PA group,Sh-Mfn2+PA group,OENC-Mfn2+PA group,and OE-Mfn2+PA group.Cells were treated with bovine serum albumin as PA control and PA.Flow cytometry was used to detect the apoptosis rate,intracellular reactive oxygen species(ROS)level and mitochondria membrane potential level in each group.Microplate reader was used to detect the intracellular ATP level.Real-time fluorescence quantitative polymerase chain reaction and Western blotting were used to detect Mfn2 and apoptosis-related protein[Caspase-3,B cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax)]mRNA and protein expression.The t-test was used for comparison between two groups,and the one-way analysis of variance(ANOVA)was used for comparison between multiple groups.Results Compared with 0 mmol/L PA,the cell viability was significantly decreased when the PA concentrations were 0.125,0.250,0.500,0.750,and 1.000 mmol/L(P<0.01).The PA concentration with a cell viability of about 50%(0.5 mmol/L)was selected as the follow-up experiment.At this time,the cell survival rate was 43.53%±0.56%.After adding 0.5 mmol/L PA to MIN6 cells,the mRNA and protein expressions of Mfn2 were significantly decreased(P<0.01).Compared with the ShNC-Mfn2 group,the levels of mRNA and protein of Mfn2 in the Sh-Mfn2 group decreased(P<0.05);compared with the OENC-Mfn2 group,the mRNA and protein levels of Mfn2 in the OE-Mfn2 group increased(P<0.01).Compared with the ShNC-Mfn2+PA group,the ROS in the Sh-Mfn2+PA group was significantly increased(P<0.01),and the ATP and mitochondrial membrane potential levels were significantly decreased(P<0.05).Compared with the OE-Mfn2+PA group,the ROS level was decreased,and the ATP level and mitochondrial membrane potential were significantly higher(P<0.05).Compared with ShNC-Mfn2+PA group,the apoptosis rate of Sh-Mfn2+PA group was increased(P<0.01).Compared with OENC-Mfn2+PA group,the apoptosis rate of OE-Mfn2+PA group was decreased(P<0.01).Compared with the ShNC-Mfn2+PA group,the mRNA and protein levels of Bax and Caspase-3 in the Sh-Mfn2+PA group were significantly increased,and the mRNA and protein levels of Bcl-2 were significantly decreased(P<0.05).Compared with the OENC-Mfn2+PA group,the mRNA and protein levels of Bax and Caspase-3 in the OE-Mfn2+PA group were significantly decreased,and the mRNA and protein levels of Bcl-2 were significantly increased(P<0.05).Conclusions Mfn2 could ameliorate the PA-induced lipotoxicity of MIN6 cells,and overexpression of Mfn2 had a protective effect on MIN6 cells under lipotoxicity,in addition,knockdown of Mfn2 aggravated the lipotoxicity of MIN6 cells.