生松素对AngⅡ诱导的大鼠肾小球系膜细胞细胞外基质合成的影响及其机制研究

The effect and mechanism of pinocembrin on synthesis of extracellular matrix in angiotensin Ⅱ-induced rat glomerular mesangial cells

目的探讨生松素(Pb)对血管紧张素Ⅱ(AngⅡ)诱导的大鼠肾小球系膜细胞细胞外基质(ECM)合成的影响及其机制。方法以大鼠肾小球系膜细胞为研究对象,将实验分为以下5组:正常对照(NC)组、1×10-6mol/L AngⅡ(Ng)组、1×10-6mol/L AngⅡ+1‰DMSO(Ng-D)组、1×10-6mol/L AngⅡ+30μmol/L Pb(Ng-Pb)组、1×10-6mol/L AngⅡ+10μmol/L缬沙坦(Ng-Val)组。分别在干预12、24、48 h搜集细胞和上清液,采用Western blot检测Ⅳ型胶原(ColⅣ)、纤连蛋白(FN)、转化生长因子-β1(TGF-β1)、Smad3、磷酸化Smad3(p-Smad3)、核因子κB(NF-κB)、磷酸化NF-κB(pNF-κB)的蛋白水平,酶联免疫吸附法(ELISA)检测上清液中肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)水平,实时荧光定量PCR(qPCR)检测细胞中ColⅣ、FN、TGF-β1、TNF-α、IL-6的mRNA水平。结果 (1)Pb对AngⅡ诱导的系膜细胞合成ECM的影响:与NC组相比,Ng组细胞内ColⅣ和FN的蛋白质和mRNA水平升高(P<0.05),而Ng-Pb组和Ng-Val组细胞内ColⅣ和FN的蛋白质和mRNA水平均较Ng组降低(P<0.05)。(2)Pb对TGF-β1/Smad3通路的影响:与NC组相比,Ng组细胞内TGF-β1的蛋白质和mRNA水平增加,p-Smad3/Smad3水平增加(P<0.05),而Ng-Pb组和Ng-Val组细胞内TGF-β1的蛋白质和mRNA水平以及p-Smad3/Smad3水平均较Ng组降低(P<0.05)。(3)Pb对NF-κb介导的促炎症因子产生的影响:与NC组相比,Ng组p-NF-κb/NF-κb水平增加,IL-6和TNF-α的蛋白质和mRNA水平增加(P<0.05),而Ng-Pb组和Ng-Val组p-NF-κb/NF-κb水平以及IL-6和TNF-α的蛋白质和mRNA水平均较Ng组降低(P<0.05)。结论 Pb可能通过抑制TGF-β1/Smad3信号通路和NF-κb介导的促炎症因子的产生,从而抑制AngⅡ诱导的大鼠系膜细胞合成ECM。

Objective To study the effect of pinocembrin(Pb) on extracellular matrix(ECM) synthesis in angiotensinⅡ(Ang Ⅱ)-induced rat mesangial cells and its mechanism.Methods Rat mesangial cells were divided into the following groups:normal control group(NC),1×10-6 mol/L Ang Ⅱ group(Ng),1×10-6 mol/L Ang Ⅱ+1‰DMSO group(Ng-D),1×10-6mol/L Ang Ⅱ+30 μmol/L Pb group(Ng-Pb) and 1×10-6 mol/L Ang Ⅱ+10 μmol/L valsartan group(Ng-Val).Cells and supernatants were collected at 12,24 and 48 hours after intervention.Collagen Ⅳ(Col Ⅳ),fibronectin(FN),transforming growth factor β1(TGF-β1),Smad3,p-Smad3,nuclear factor kappa B(NF-κb) and p-NF-κb "were detected by Western blot assay.The levels of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) " were detected by ELISA,and the RNA levels of Col Ⅳ,FN,TGF-β1,TNF-α and IL-6-were detected by qPCR.Results(1) The effect of Pb on synthesis of ECM in AngⅡ-induced rat mesangial cells:after Ang Ⅱ stimulation,the protein and mRNA levels of Col Ⅳ and FN in rat mesangial cells were significantly increased(P <0.05),and pre-stimulation with Pb could significantly reduce the protein and mRNA levels of Col Ⅳ and FN(P <0.05).(2) The effect of Pb on the TGF-β1/Smad3 path-way:Ang Ⅱ increased the protein and mRNA levels of TGF-β1,and raised the level of p-Smad3/smad3(P <0.05),while pre-stimulation with Pb could significantly reduce the protein and mRNA level of TGF-β1,and decrease the level of p-Smad3/smad3(P <0.05).(3)Effects of Pb on NF-κB-mediated pro-inflammatory factor production:after Ang Ⅱ stimulation,the level of p-NF-κB/NF-κB increased significantly,the protein and RNA levels of IL-6 and TNF-α increased significantly(P <0.05).Prestimulation with Pb could significantly alleviate the above changes(P <0.05).Conclusion Pb may inhibit synthesis of ECM in Ang Ⅱ-induced rat mesangial cells through inhibiting the TGF-β1/Smad3 pathway and NF-κB-mediated proinflammatory factor production.