多聚二磷酸腺苷(ADP)核糖聚合酶-1(PARP-1)在高糖致大鼠肾脏系膜细胞细胞外基质沉积中的作用

Poly(ADP-ribose) polymerase-1 mediates high glucose-induced extracellular matrix accumulation in rat mesangial cells

目的探讨多聚二磷酸腺苷(ADP)核糖聚合酶-1(PARP-1)在高糖(25 mM)致大鼠肾脏系膜细胞细胞外基质沉积中的作用。方法①体外培养大鼠肾脏系膜细胞株(MCs 1097),使用高糖(25 mM)处理大鼠肾脏系膜细胞,部分实验组中应用PARP-1抑制剂PJ34(3×10-6M)进行干预处理。②RT-PCR和Western blot检测PARP-1、纤维粘连蛋白(FN)、胶原Ⅳ(COLⅣ)的mRNA及蛋白表达。③高通量比色测定法检测PARP-1的活性。结果①高糖显著诱导大鼠肾脏系膜细胞PARP-1 mRNA和蛋白的表达,分别较低糖对照组增加72.3%和68.4%(P<0.05),PJ34可明显抑制高糖诱导的PARP-1 mRNA和蛋白的过度表达,mRNA和蛋白分别为高糖组的31.8%和55.5%(P<0.05)。同时,高糖显著诱导大鼠肾脏系膜细胞PARP-1激活,高糖组为低糖对照组活性的1.77倍(P<0.05),PJ34可显著抑制高糖诱导的PARP激活,活性降低为高糖组的67.8%(P<0.05)。②高糖显著增加COLⅣ和FN表达,PJ34显著抑制高糖诱导的COLⅣ和FN mRNA过度表达(分别为高糖组的68.2%和54.7%(P<0.05);COLⅣ和FN蛋白水平的变化与RNA水平变化相一致,PJ34可显著抑制高糖诱导的COLⅣ和FN蛋白表达(分别为高糖组的54.0%及66.6%(P<0.05)。结论高糖可以诱导培养的大鼠肾脏系膜细胞内PARP激活,PARP-1表达增加,PJ-34预处理可以明显降低高糖诱导的大鼠肾脏系膜细胞内PARP的激活以及下游FN及COLⅣ的高表达,提示PARP1参与了肾脏系膜细胞细胞外基质沉积的发生发展过程。

Objective To investigate the role of Poly（ADP-ribose） Polymerase-1（PARP-1） in high glucose-induced accumulation of extracellular matrix（ECM） in rat mesangial cells（RMCs）.Methods RMCs were treated with or without high glucose（25 mmol/L）.In some experimental groups,cells were pre-treated with PARP-1 inhibitor PJ34（3×10-6mol/L）.RT-PCR was employed to detect the mRNA expression for ADP-ribose polymerase-1（PARP-1）,fibronectin（FN）.collagen Ⅳ（COL Ⅳ）,and all those proteins expression were examined by Western blot.The activity of PARP-1 was examined by Colorimetric assay.Results high glucose significantly stimulated both PARP-1 mRNA and protein overexpression in RMCs.the mRNA and protein expression of PARP-1 were up-regulated by 72.3% and 68.4%（P 0.05）.PJ34 reduced high glucose-induced upregulation of PARP-1 mRNA and protein to 31.8% and 55.5%（P 0.05）,compared with high glucose stimulation group.At the same time,high glucose obviously stimulated PARP-1 activation,the PARP-1 activity of high glucose group was upregulated by 1.77 fold（P 0.05）,compared with control cells.PJ34 effectively inhibited the activation of PARP-1 by 32.2%（P 0.05）,compared with the cells treated with high glucose alone.Also,high glucose increased COL Ⅳ and FN mRNAs.Compared with high glucose stimulation group,PJ34 suppressed high glucose-induced upregulation of COL Ⅳ and FN mRNA by 31.8% and 45.3%,respectively（P 0.05）.The changes in COL Ⅳ and FN were confirmed at protein level.the inhibition rate of COL Ⅳ and FN protein expression were 54.0% and 66.6%,respectively,compared with high glucose stimulation group（P 0.05）.Conclusion Our data suggest that PARP-1 mediates high glucose-induced accumulation of ECM in rat mesangial cells and thus may represent a potential therapeutic target in the management of glomerular disease.