二甲双胍对肝星状细胞凋亡的调控作用

Regulatory Effect of Metformin on the Apoptosis of Hepatic Stellate Cells

目的探究二甲双胍对肝星状细胞(HSC)的作用及其可能的抗肝纤维化作用机制。方法将人HSC LX-2分为对照组(PBS)、模型组(PDGF-BB)和药物组(PDGF-BB+Met)。将30只SPF级C57BL/6J小鼠随机分为3组,对照组(n=10,橄榄油)、模型组(n=10,CCl4)和药物组(n=10,CCl4+Met)。用CCK-8试剂盒检测细胞增殖活力,AnnexinV-PE/7-AAD染色法检测细胞凋亡率;蛋白质印迹技术(Western blot)检测α-SMA、Beclin1、PARP、Bax及bcl-2蛋白表达;取小鼠肝脏石蜡包埋,石蜡切片进行Masson染色检测小鼠组织病理学情况。结果二甲双胍能抑制活化的HSC增殖(IC50=42.66),细胞流式检测发现,二甲双胍能促进HSC凋亡(C=5mmol/L,P<0.001);与模型组相比,药物组α-SMA表达量减少(P<0.001);且药物组PARP、Bax表达量升高(P=0.003,P<0.001),Bcl-2表达量明显降低(P<0.001);马松染色(Masson染色)发现,药物组胶原纤维分泌少于模型组(P=0.003)。结论二甲双胍促进HSC的凋亡,从而改善CCl4诱导的肝纤维化。

Objective To investigate the effect of metformin on hepatic stellate cells(HSC)and its possible mechanism of anti-hepatic fibrosis.Methods Human HSC LX-2 was divided into the control group treated with PBS,model group treated with PDGF-BB and drug group treated with PDGF-BB and Metformin.A total of 30 SPF C57 BL/6 Jmice were randomly divided into the control group treated with olive oil,model group treated with CCl4 and drug group treated with CCl4 and Metformin with 10 mice in each group.The proliferation activity was detected by CCK-8 kit,the apoptosis rate was detected by the annexinV-PE/7-AAD staining method,and the protein expression ofα-SMA,Beclin1,PARP,Bax and Bcl-2 were detected by Western blot.The mouse liver was embedded in paraffin and the paraffin sections were stained to detect the histopathological changes by the Masson staining method.Results Metformin inhibited the proliferation of activated HSC(IC50=42.66 mmol/L)and the results of cell flow cytometry showed that metformin promoted the apoptosis of HSC(C=5 mmol/L,P<0.001).Compared with the model group,the expression ofα-SMA decreased significantly(P<0.001),that of PARP and Bax increased significantly(P=0.03 and P<0.001,respectively),and that of Bcl-2 reduced significantly(P<0.001)in the drug group.The Masson staining results showed that the collagen fiber secretion of the drug group was significantly less than that of the model group(P=0.03).Conclusion Metformin promotes the apoptosis of hepatic stellate cells and improves the liver fibrosis induced by CCl4.