论文部分内容阅读
目的:在证实去甲斑蝥素(norcantharidin,NCTD)能减轻糖尿病肾病(DN)大鼠肾间质纤维化和抑制高糖刺激的肾小管上皮细胞细胞外基质表达的基础上,观察NCTD对高糖刺激的肾小管上皮细胞钙调蛋白磷酸酶(calcineurin,CaN)通路的影响,探讨NCTD抗DN肾小管间质纤维化与其抑制CaN的关系。方法:常规培养人肾小管上皮细胞(HK-2),转染CaN siRNA,细胞分五组:(1)正常糖组(D-glucose5.5mmol/L);(2)高糖组(HG,D-glucose30mmol/L);(3)高糖+CaN siRNA组;(4)高糖+CaN siRNA+NCTD(5mg/L)组;(5)高糖+NCTD(5mg/L)组。采用Western-blot、免疫荧光和实时定量PCR,观察NCDT对HK-2细胞CaN/NFAT通路的影响,明确CaN siRNA的干扰效果。采用Western blot,检测NCTD对转染CaN siRNA后的HK-2细胞纤维连接蛋白(FN),胶原蛋白IV(Collagen IV,Col IV)及转化生长因子β1(TGF-β1)蛋白表达的影响。结果:高糖可促进HK-2细胞CaNmRNA及蛋白的表达,NCTD可在基因及蛋白水平抑制CaN的表达(P<0.05)。免疫荧光发现CaN下游活化T细胞核因子(NFATc)在正常对照组中存在于胞质,高糖刺激后细胞核内开始表达,高糖刺激30min后发生明显的核转位,NCTD能在一定程度上抑制核转位的发生,并能减少高糖刺激后核内NFATc蛋白的表达。转染CaN siRNA后,高糖刺激后HK-2细胞中CaN mRNA以及蛋白表达均降低,而FN,Col IV以及TGF-β1蛋白水平表达都明显增强(P<0.05)。NCTD可抑制转染CaN siRNA后高糖刺激的HK-2细胞FN,Col IV和TGF-β1的表达。结论:NCTD能下调肾小管上皮细胞CaN表达,阻断CaN/NFATc信号通路;但NCTD抑制高糖刺激后肾小管上皮细胞细胞外基质的表达,与其阻断CaN/NFATc信号通路无关。
OBJECTIVE: To investigate the effects of NCTD on renal interstitial fibrosis and inhibition of extracellular matrix expression of renal tubular epithelial cells induced by high glucose in diabetic nephropathy (DN) rats. Stimulated renal tubular epithelial cells calcineurin (calcineurin, CaN) pathway to explore the relationship between NCTD anti-DN tubulointerstitial fibrosis and its inhibition of CaN. Methods: Human renal tubular epithelial cells (HK-2) were cultured and transfected with CaN siRNA. The cells were divided into five groups: (1) D-glucose 5.5mmol / L; (3) High glucose + CaN siRNA group; (4) High glucose + CaN siRNA + NCTD group (5mg / L); The effects of NCDT on CaN / NFAT pathway in HK-2 cells were observed by Western-blot, immunofluorescence and real-time quantitative PCR, and the interference effect of CaN siRNA was clarified. The effect of NCTD on the expression of fibronectin (FN), collagen IV (Col IV) and transforming growth factor-β1 (TGF-β1) in HK-2 cells transfected with CaN siRNA was detected by Western blot. Results: High glucose promoted the expression of CaN mRNA and protein in HK-2 cells. NCTD inhibited the expression of CaN at the gene and protein levels (P <0.05). Immunofluorescence showed that NFATc in the downstream of CaN was found in the cytoplasm in the normal control group, and began to be expressed in the nucleus after high glucose stimulation. Significant nuclear translocation occurred 30 minutes after high glucose stimulation, and NCTD could be inhibited to a certain extent Nuclear translocation occurs, and can reduce the expression of nuclear NFATc after high glucose stimulation. After transfection with CaN siRNA, the expression of CaN mRNA and protein in HK-2 cells decreased while the expression of FN, Col IV and TGF-β1 increased significantly (P <0.05). NCTD inhibited the expression of FN, Col IV and TGF-β1 in high glucose-stimulated HK-2 cells after CaN siRNA transfection. CONCLUSION: NCTD can down-regulate the expression of CaN in renal tubular epithelial cells and block the CaN / NFATc signaling pathway. However, NCTD inhibits the expression of extracellular matrix in renal tubular epithelial cells induced by high glucose, which has nothing to do with CaN / NFATc signaling pathway.