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目的探讨去甲斑蝥素对体外人源性肾小管细胞株(HK-2)上皮细胞间质转分化(epithelial-mesenchymaltransition,EMT)以及信号蛋白Smad2/3的影响。方法体外培养HK-2细胞,分为对照组、TGF-β1组和去甲斑蝥素组(NCTD组)。对照组为无血清DMEM培养,TGF-β1组为TGF-β15 ng.mL-1诱导,NCTD组为不同浓度NCTD(0.5、1.0、2.5μg.mL-1)与TGF-β1(5 ng.mL-1)共同作用,各组作用时间48 h。使用半定量RT-PCR法检测α-平滑肌肌动蛋白(α-SMA)、上皮性钙黏附蛋白(E-cadherin)与Smad2、Smad3 mRNA表达;采用Western blot检测α-SMA、E-cadherin、Smad2/3、p-Smad2/3蛋白质表达。结果与对照组相比,TGF-β1组α-SMA mRNA及其蛋白表达明显上调(P<0.05),E-cadherin mRNA及其蛋白表达显著下调(P<0.05);与TGF-β1组相比,NCTD干预组α-SMA mRNA及其蛋白表达明显下调(P<0.05),而E-cadherin mRNA及其蛋白表达显著上调(P<0.05),且均呈剂量依赖性。与对照组相比,TGF-β1组Smad2、Smad3 mRNA和Smad2/3、p-Smad2/3蛋白表达上调(P<0.05);与TGF-β1组比较,NCTD干预组Smad2、Smad3 mRNA和Smad2/3、p-Smad2/3蛋白表达下调(P<0.05),具有剂量依赖关系。结论去甲斑蝥素具有抑制肾小管上皮细胞EMT的作用,该作用可能与其下调信号蛋白Smad2/3的表达有关。
Objective To investigate the effects of norcantharidin on the epithelial-mesenchymal transition (EMT) and the signaling protein Smad2 / 3 in cultured human renal tubular epithelial cells (HK-2). Methods HK-2 cells were cultured in vitro and divided into control group, TGF-β1 group and norcantharidin group (NCTD group). The control group was cultured in serum-free DMEM, the TGF-β1 group was induced by TGF-β15 ng.mL-1 and the NCTD group was treated with different concentrations of NCTD (0.5,1.0,2.5μg.mL-1) and TGF- -1) together, the role of each group of time 48 h. The expression of α-smooth muscle actin (α-SMA), E-cadherin and Smad2, Smad3 mRNA were detected by semi-quantitative RT-PCR. The expressions of α-SMA, E-cadherin and Smad2 / 3, p-Smad2 / 3 protein expression. Results Compared with the control group, the expression of α-SMA mRNA and protein in TGF-β1 group was significantly increased (P <0.05), while the expression of E-cadherin mRNA and protein was significantly decreased (P <0.05). Compared with TGF-β1 group The mRNA and protein expression of α-SMA in NCTD intervention group were significantly decreased (P <0.05), while the expression of E-cadherin mRNA and protein was significantly increased (P <0.05) in a dose-dependent manner. Compared with the control group, the expressions of Smad2, Smad3 mRNA and Smad2 / 3 and p-Smad2 / 3 in TGF-β1 group were up-regulated (P <0.05) 3, p-Smad2 / 3 protein expression was down-regulated (P <0.05) in a dose-dependent manner. Conclusion Norcantharidin can inhibit the EMT of renal tubular epithelial cells, which may be related to the down-regulation of Smad2 / 3 expression.