线粒体ALDH2对高糖处理的乳鼠心肌成纤维细胞中自噬相关蛋白影响

Effects of mitochondrial aldehyde dehydrogenase 2 on autophagy-associated proteins in neonatal rat myocardial fibroblasts cultured in high glucose

目的探讨自噬是否介导乙醛脱氢酶(ALDH)2对高糖处理的乳鼠心肌成纤维细胞的作用。方法无菌条件下取出生3d内SD大鼠乳鼠心脏,剪碎并用胰蛋白酶和胶原酶2混合酶消化成单个细胞,经差速贴壁后并传代培养,当传至3代时进行处理。应用免疫荧光检测培养的乳鼠心肌成纤维细胞内波形蛋白以此鉴定原代培养心肌成纤维细胞纯度;实验涉及如下分组:5.5 mmol/L糖对照组(F)、30 mmol/L高糖组(FH)、30 mmol/L高糖加乙醛脱氢酶2激动剂(Alda-1)组(FHA)、30 mmol/L高糖加乙醛脱氢酶2抑制剂(Daidzin)组(FHD)、30 mmol/L高糖加乙醛脱氢酶2激动剂(Alda-1)和乙醛脱氢酶2抑制剂(Daidzin)(FHAD);Western blot检测ALDH2、微管相关蛋白1轻链3B亚基(LC3B)、Beclin-1的蛋白表达;羟脯胺酸试剂盒测定细胞培养液中羟脯胺酸含量;CCK-8试剂盒测定心肌成纤维细胞增殖能力。结果与F组相比,FH组ALDH2蛋白表达降低,Beclin-1、LC3B蛋白表达降低,细胞培养上清液内羟脯胺酸含量、细胞数目增高;与FH组相比,FHA组Beclin-1、LC3B蛋白表达、ALDH2蛋白表达增加,细胞数目、细胞内羟脯胺酸含量降低,与此同时FHD组ALDH2蛋白及Beclin-1、LC3B表达降低,细胞数目、细胞内羟脯胺酸含量增高。结论线粒体ALDH2抑制高糖诱导的乳鼠心肌成纤维细胞增殖作用,其机制可能与ALDH2对自噬相关蛋白Beclin-1、LC3B的上调作用有关。

Objective To investigate whether autophagy mediates the effects of aldehyde dehydrogenase 2(ALDH2)on the proliferation of neonatal rat cardiac fibroblasts cultured in high glucose.Methods Cardiac fibroblasts were isolated from neonatal(within 3 days)SD rats and subcultured.The fibroblasts of the third passage,after identification with immunofluorescence staining for vimentin,were treated with 5.5 mmol/L glucose(control group),30 mmol/L glucose(high glucose group),or 30 mmol/L glucose in the presence of Alda-1(an ALDH2 agonist),daidzin(an ALDH2 2 inhibitor),or both.Western blotting was employed to detect ALDH2,microtubule-associated protein 1 light chain 3 B subunit(LC3 B)and Beclin-1 in the cells,and a hydroxyproline detection kit was used for determining hydroxyproline content in cell culture medium;CCK-8 kit was used for assessing the proliferation ability of the cardiac fibroblasts after the treatments.Results Compared with the control cells,the cells exposed to high glucose exhibited obviously decreased expressions of ALDH2,Beclin-1 and LC3 B and increased cell number and hydroxyproline content in the culture medium.Treatment of the high glucose-exposed cells with Alda-1 significantly increased Beclin-1,LC3 B,and ALDH2 protein expressions and lowered the cell number and intracellular hydroxyproline content,whereas the application of daidzin resulted in reverse changes in the expressions of ALDH2,Beclin-1 and LC3 B,viable cell number and intracellular hydroxyproline content in high glucose-exposed cells.Conclusion Mitochondrial ALDH2 inhibits the proliferation of neonatal rat cardiac fibroblasts induced by high glucose,and the effect is possibly mediated by the up-regulation of autophagy-related proteins Beclin-1 and LC3 B.