褪黑素对高糖刺激系膜细胞NF-κB与TGF-β/Smad3通路的影响

Effect of melatonin on NF-κB and TGF-β/Smad3 pathway in mesangial cells stimulated by high glucose

目的探讨褪黑素(MT)对高糖刺激的小鼠肾小球系膜细胞(MCs)核因子-κB(NF-κB)与转化生长因子-β(TGF-β)/Smad3通路的影响。方法将MCs分为甘露醇对照组、正常对照组、正常对照+MT组、高糖组、高糖+不同浓度(10、100、1000)μmol/LMT组。采用CCK-8检测MCs活性;5-乙炔基-2′-脱氧尿苷(EdU)试剂盒检测MCs增殖情况;激光共聚焦法检测MCsNF-κBp65核转移及Ⅳ型胶原(ColⅣ)、纤连蛋白(Fn)表达情况;ELISA法检测细胞上清液中ColⅣ、Fn含量变化;实时荧光定量PCR(qRT-PCR)法检测ColⅣ、FnmRNA表达水平;Westernblot法检测ColⅣ、Fn、核因子κB抑制因子(IκB)、核因子κB抑制因子磷酸化(p-IκB)、TGF-β1、Smad3、p-Smad3蛋白表达。结果与正常对照组比较,高糖组MCs的增殖功能明显增强,同时MCsColⅣ、Fn、p-IκB/IκB、NF-κBp65、TGF-β1、p-Smad3/Smad3表达均明显上调(P<0.05);与高糖组比较,高糖+不同浓度MT组各项指标呈浓度依赖性降低(P<0.05)。结论高糖可以增强MCsNF-κBp65核转移及TGF-β/Smad3信号通路的激活,导致细胞外基质ColⅣ和Fn分泌增加,MT能干预高糖刺激的MCsNF-κBp65核转移,并且抑制TGF-β/Smad3信号通路的激活,降低纤维化因子的释放。

Objective To investigate the effects of melatonin (MT) on nuclear factor-κB(NF-κB) and transforming growth factor-β(TGF-β)/Smad3 pathway in mesangial cells (MCs) stimulated by high glucose. Methods MCs were divided into mannitol control group,normal control group,normal control + MT group,high glucose group,high glucose + different concentrations (10,100, 1 000 ) mol/L MT group.The activity of MCs was detected by CCK-8;the proliferation of MCs was detected by 5-Ethynyl-2’-deoxyuridine (EdU) kit;the expression of NF-κBp65,collageⅣ(Col Ⅳ) and fibronectin (Fn) were detected by laser confocal system;the concentrations of Col Ⅳ and Fn in supernatant were detected by ELISA;the mRNA levels of Col Ⅳ and Fn were detected by qRT-PCR;the expressions of Col Ⅳ,Fn,inhibitor of?NF-κB (IκB),phosphorylated IκB(p-IκB),transforming growth factor-β1 (TGF-β1),Smad3 and p-Smad3 were detected by Western blot. Results Compared with the normal control group,the proliferation function of MCs in high glucose group was significantly enhanced,and the expressions of Col Ⅳ,Fn,p-IκB/IκB,NF-κBp65,TGF-β1 and p-Smad3/Smad3 in MCs were significantly up-regulated ( P <0.05).Compared with the high glucose group,the high glucose + different concentrations of MT group decreased the indexes in a concentration-dependent manner ( P <0.05). Conclusion High glucose can induce the expression of NF-κBp65 and the activation of TGF-β/Smad3 signaling pathway in MCs,which leads to the increase of mesangial extracellular matrix Col Ⅳ and Fn secretion.MT reduces the release of fibrotic factors by interfering with NF-κBp65 nuclear transfer and inhibiting the activation of TGF-β/Smad3 signaling pathways in high glucose-stimulated MCs.