ERK1/2 MAPK通路在TIMP-1抑制高糖诱导的大鼠肾小球系膜细胞凋亡中的作用

The role of ERK1/2 MAPK pathway in the inhibition of TIMP-1 on apoptosis in rat mesangial cells induced by high glucose

目的探讨细胞外信号调节激酶(ERK1/2)丝裂原激活蛋白激酶(MAPK)通路在基质金属蛋白酶1组织抑制剂(TIMP-1)抑制高糖诱导的大鼠肾小球系膜细胞凋亡中的作用。方法将人正义、反义TIMP-1用脂质体法转染大鼠肾小球系膜细胞,并将细胞分为正常对照组、未转染组、空载体组、正义转染组和反义转染组。各组细胞分别用高糖(25 mmol/L)和(或) MEK(ERK1/2通路中的MAPK激酶)特异性抑制剂PD98059(50μmol/L)刺激24 h。应用透射电镜观察细胞内部结构变化。Western印迹观察磷酸化(p)的ERK1/2、P90rsk、Bad及总的P90rsk、Bad、Bcl-2蛋白的表达。结果加入PD98059后,各组细胞内可见凋亡小体形成、细胞皱缩、核固缩、染色体边集及膜泡样突变等现象,与未转染组比较,以反义转染组最明显,凋亡细胞数目最多;正义转染组细胞形态改变不十分明显,凋亡细胞数目相对较少。此外,加入PD98059后, ERK1/2、P90rsk、Bcl-2及Bad的磷酸化明显减少(P<0．05),ERK1/2和P90rsk的磷酸化以正义转染组最明显(P<0．01);总P90rsk、总Bad表达量在各组之间无显著差异。结论ERK1/2通路在TIMP-1抑制高糖诱导的大鼠肾小球系膜细胞凋亡过程中起重要作用。

Objective To investigate the mechanism of ERK1/2 signal pathway in the inhibition of TTMP-1 on apoptosis of rat glomerular mesangial ceils induced by high glucose. Methods The human TIMP-1 sense, antisense plasmid were transferred into rat mesangial ceils through liposome. The celts were divided into control group, non-transfected group,veotor group,sense group and antisense group. After cultured in high glucose （HG 25 retool/L） with the presence of PD98059（50 μmol/L）for 24 hours, cell structure was observed by electron micrecope. Western blot was performed to test the expression of phospho-ERK1/2, phospho-P90rsk, phospho-Bad, Bad, P90rsk and Bcl-2. Results Electron microcope showed that PD98059 caused a significant increase of apoptosis bodies in every group of rat mesangial cells. In contrast with the control group, the expression levels of pbospbo-ERK1/2, pbospbo-P90rsk, pbospbo-Bad and Bcl-2 were inhibited by antisense TIMP-1 and PD98059（P〈 0.05）. Among these groups,the expression levels of total Bad and total P9Orsk protein were not different. Conclusion TIMP-1 exerts its anti-apoptoic effect, at least in part, by activation of ERK1/2 pathway.