Division of Nephrology, The Second Xiangya Hospital, Research Institute of Nephrology, Central-South University, Changsha, China.
Lab Invest. 2011 Dec;91(12):1706-16. doi: 10.1038/labinvest.2011.119. Epub 2011 Sep 19.
Norcantharidin (NCTD) was shown in our previous studies to attenuate renal tubulointerstitial fibrosis in rat models with diabetic nephropathy (DN). The aim of this study was to determine the effects of NCTD on the expression of extracellular matrix (ECM) and TGF-β1 in HK-2 cells stimulated by high glucose and on calcineurin (CaN)/NFAT pathway. Whether or not the antifibrotic effect of NCTD on renal interstitium was dependent on its inhibition of CaN pathway was also investigated. Experimental concentrations of NCTD were verified by cytotoxic test and MTT assay. HK-2 cells were transfected with CaN small interference RNA (siRNA). The mRNA and protein expressions of FN, ColIV, TGF-β1, and CaN in HK-2 cells were detected by real-time PCR and western blot. The CaN/NFAT pathway was examined by indirect immunofluorescence and western blot. Our study revealed that NCTD concentrations over 5 mg/l had overt cytotoxicity on HK-2 cells. Meanwhile, both 2.5 and 5 mg/l NCTD inhibited HK-2 cell proliferation (P < 0.05). NCTD inhibited the upregulation of FN, ColIV, and TGF-β1 of HK-2 cells stimulated by high glucose (P < 0.05), and also significantly downregulated the expression of CaN mRNA and protein in HK-2 cells (P < 0.05). In addition, not only was the nuclear translocation of NFATc inhibited, but its protein level in the nucleus was also reduced. Following CaN siRNA transfection, CaN mRNA and protein expression were significantly decreased. In contrast, the protein levels of FN, ColIV, and TGF-β1 increased in HK-2 cells stimulated by high glucose (P < 0.05). However, NCTD treatment downregulated their expression. These results indicated that NCTD could decrease the expression of ECM and TGF-β1 in HK-2 cells stimulated by high glucose, downregulate CaN expression, and block the CaN/NFAT signaling pathway. However, the effect of NCTD on inhibition of the expression of ECM and TGF-β1 was not associated with its inhibition of the CaN/NFAT pathway.
去甲基斑蝥素（NCTD）在我们之前的研究中显示可减轻糖尿病肾病（DN）大鼠模型的肾小管间质纤维化。本研究旨在确定 NCTD 对高糖刺激的 HK-2 细胞细胞外基质（ECM）和 TGF-β1 表达以及钙调神经磷酸酶（CaN）/NFAT 途径的影响。还研究了 NCTD 对肾间质的抗纤维化作用是否依赖于其对 CaN 途径的抑制作用。通过细胞毒性试验和 MTT 测定验证了实验浓度的 NCTD。用 CaN 小干扰 RNA（siRNA）转染 HK-2 细胞。通过实时 PCR 和 Western blot 检测 HK-2 细胞中 FN、ColIV、TGF-β1 和 CaN 的 mRNA 和蛋白表达。通过间接免疫荧光和 Western blot 检测 CaN/NFAT 途径。我们的研究表明，浓度超过 5 mg/L 的 NCTD 对 HK-2 细胞具有明显的细胞毒性。同时，2.5 和 5 mg/L 的 NCTD 抑制高糖刺激的 HK-2 细胞增殖（P < 0.05）。NCTD 抑制高糖刺激的 HK-2 细胞 FN、ColIV 和 TGF-β1 的上调（P < 0.05），并显著下调 HK-2 细胞 CaN mRNA 和蛋白的表达（P < 0.05）。此外，不仅 NFATc 的核易位被抑制，而且其核内蛋白水平也降低。转染 CaN siRNA 后，CaN mRNA 和蛋白表达明显降低。相反，高糖刺激的 HK-2 细胞中 FN、ColIV 和 TGF-β1 的蛋白水平升高（P < 0.05）。然而，NCTD 处理下调其表达。这些结果表明，NCTD 可降低高糖刺激的 HK-2 细胞中 ECM 和 TGF-β1 的表达，下调 CaN 表达，阻断 CaN/NFAT 信号通路。然而，NCTD 抑制 ECM 和 TGF-β1 表达的作用与抑制 CaN/NFAT 途径无关。
