二甲双胍抑制高糖状态下大鼠肾小球系膜细胞增殖作用机制的进一步研究

Further research of the mechanism of metformin inhibiting rat mesangial cells proliferation induced by high glucose

目的探讨二甲双胍抑制高糖状态下大鼠肾小球系膜细胞增殖的作用机制。方法体外培养大鼠肾小球系膜细胞(HBZY-1)分为2组,AICAR组分为正常对照(NC)组(葡萄糖浓度5.6 mmol/L)、高糖(HG)组(葡萄糖浓度30 mmol/L)、HG+AICAR组(葡萄糖浓度30 mmol/L+AICAR浓度2.0 mmol/L)、HG+AICAR+二甲双胍组(葡萄糖浓度30 mmol/L+AICAR浓度2.0 mmol/L+二甲双胍浓度200 mmol/L)4个亚组,Compound C组分为NC组、HG组、HG+Compound C组(葡萄糖浓度30 mmol/L+Compound C浓度0.5 mmol/L)、HG+Compound C+二甲双胍组(葡萄糖浓度30 mmol/L+Compound C浓度0.5 mmol/L+二甲双胍浓度200 mmol/L)4个亚组,作用时间均为24 h,MTT法检测各组HBZY-1细胞增殖的情况;如上分组,作用12 h,用Western blot法检测各组细胞内p-单磷酸腺苷激活的蛋白激酶(p-AMPK)和AMPK蛋白表达的变化。结果①与NC组相比,高糖刺激24 h后HBZY-1增殖明显(P均<0.01);与HG组相比,加AICAR干预后HBZY-1增殖明显抑制,加Compound C干预后系膜细胞进一步增殖,均有显著性差异(P均<0.01);与HG+AICAR组相比,加二甲双胍干预后HBZY-1增殖进一步抑制,有显著性差异(P<0.01);与HG+Compound C组相比,加二甲双胍干预后HBZY-1增殖明显抑制,有显著性差异(P<0.01);②与NC组相比,高糖刺激24 h后HBZY-1细胞内p-AMPK表达水平明显下降(P均<0.01);与HG组相比,加AICAR干预后p-AMPK表达水平明显升高,加Compound C干预后p-AMPK表达水平进一步下降,均有显著性差异(P均<0.01);与HG+AICAR组相比,加二甲双胍干预后HBZY-1细胞内p-AMPK表达水平进一步升高,有显著性差异(P<0.01);与HG+Compound C组相比,加二甲双胍干预后p-AMPK表达水平明显升高,有显著性差异(P<0.01)。结论二甲双胍可能通过增加肾小球系膜细胞内p-AMPK的表达水平来抑制HBZY-1的增殖。

Objective It is to investigate the possible mechanism of metformin inhibiting rat mesangial cells proliferation induced by high glucose.Methods The rat mesangial cells（HBZY-1） were cultured in vitro and divided into 2 groups.AICAR group were divided into four subgroups：normal glucose （5.6 mmol/L）,high glucose （30 mmol/L）,high glucose plus AICAR （2.0 mmol/L）,high glucose plus AICAR plus metformin （200 mmol/L） ; Compound C group were divided into four subgroups：normal glucose,high glucose,high glucose plus Compound C （0.5 mmol/L）,high glucose plus Compound C plus metformin （200 mmol/L）,all the application time was 24 h.The proliferations of HBZY-1 cells were detected by micro-tetrazolium salt colorimetric enzyme reaction test （MTT）.The grouping was the same as the step above,all the application time was 12 h.The protein levels of p-AMPK,AMPK were measured by Western blotting.Results ①As compared to normal cells,exposure of HBZY-1 cells to D-glucose at concentration of 30 mmol/L caused significant proliferations at 24 h （P ＜ 0.01）.The addition of AICAR significantly attenuated the high glucose-enhanced proliferations of HBZY-1 cells,the proliferations were further suppressed when metformin added（P ＜ 0.01）.The addition of Compound C significantly promoted the proliferations of HBZY-1 cells,the proliferations were suppressed when metformin added（P ＜ 0.01）.②As compared to normal cells,the protein levels of p-AMPK significantly decreased in high glucose group（P ＜ 0.01）.The addition of AICAR significantly promoted the levels of p-AMPK,and when metformin added,the levels increased more obviously （P ＜ 0.01）.The addition of Compound C significantly further decreased the levels of p-AMPK,the levels were significantly increased when metformin added（P ＜ 0.01）.Conclusion Metformin can significantly reverse high glucose-regulated proliferations of HBZY-1 cells,perhaps through increasing the expression of p-AMPK protein in HBZY-1 cells.