去甲斑蝥不依赖钙调蛋白磷酸酶信号通路抑制高糖刺激肾小管上皮细胞细胞外基质的表达

肾脏病与透析肾移植杂志 ›› 2010, Vol. 19 ›› Issue (6): 526-533.

去甲斑蝥不依赖钙调蛋白磷酸酶信号通路抑制高糖刺激肾小管上皮细胞细胞外基质的表达

出版日期:2010-12-28 发布日期:2011-01-04

NCTD inhibits the expression of extracellular matrix in human proximal tubular epithelial cells induced by high glucose in vitro independent of Calcineurin signal pathway

Online:2010-12-28 Published:2011-01-04

摘要/Abstract

摘要：

目的：我们通过体内外实验已证实去甲斑蝥素(Norcantharidin NCTD)能减轻糖尿病肾病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的表达。本研究观察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抗糖尿病肾病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5组：(1)正常糖组(D-Glucose 5.5mmol/L)，(2)高糖组(HG，D-Glucose 30 mmol/L)，(3)高糖+CaN siRNA组, (4)高糖+CaN siRNA +NCTD(5ug/ml)组，(5)高糖+NCTD(5ug/ml)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CaNsiRNA的干扰效果。采用Western blot，检测NCTD对转染CaNsiRNA后的HK-2细胞FN, ColⅣ及TGF-β1蛋白表达的影响。结果：30mM D-葡萄糖可促进HK-2细胞CaN mRNA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NFATc在正常对照组中存在于胞浆，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, ColⅣ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, ColⅣ和TGF-β₁的表达。结论：NCTD能下调肾小管上皮细胞CaN的表达，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。

Abstract:

Objective：To learn the effect of NCTD on Calcineurin (CaN) /NFAT pathway and to explore whether the anti-fibrogenic effect of NCTD on renal interstitium in diabetic nephropathy is dependent of NCDT’s inhibition to CaN pathway. Methodology: HK-2 cells were cultured and transfected with CaN small interference RNA, then divided into 5 groups: (1) Normal glucose group (5.5mmol/L D-Glucose), (2) High glucose group (30 mmol/L D-Glucose), (3) High glucose +CaN siRNA groups: (4) High glucose +CaN siRNA+ NCTD (5ug/ml) group， (5) High glucose + NCTD(5ug/ml) group. The effect of NCTD on CaN /NFAT pathway in HK-2 cells and CaN siRNA transfection efficiency was examined by Western-blot, immunofluorescence and real-time PCR. The effects of NCTD on the protein expression of FN, ColⅣ and TGF-β1 in HK-2 cells transfected by CaNsiRNA were observed by Western blot. Results: The expressions of CaN mRNA and protein in HK-2 cells were elevated when exposed to 30mmol/L glucose, and significant inhibition of the up-regulated expression of CaN was observed after treatment with NCTD（P<0.05）. NFATc, a downstream molecular of CaN, was predominantely visualized in cytoplasm of HK-2 cell in normal control group, whereas it was markedly decreased in the cytoplasm of high glucose group and was found mainly in nucleus, which peaked at 30min, suggesting the translocation of NFATc to the nucleus. NCTD treatment could inhibit the nuclear translocation of NFATc and reduce its protein level in the nucleus. Following CaN siRNA transfection, the mRNA and protein expression of CaN was significantly decreased, however, the protein levels of FN, ColIV and TGF-β were obviously increased（P<0.05）. NCTD treatment could down-regulated the increase of FN, ColIV and TGF-β protein expression in HK-2 cells stimulated by high glucose after transfecting CaN-SiRNA . Conclusions: NCTD could down-regulate CaN expression in HK-2 cells, and block CaN/NFAT signaling pathway. However, the effect of NCTD inhibiting the expression of extracellular matrixs in HK-2 cells induced by high glucose was not associated with its inhibition to CaN/NFAT pathway

李瑛, 陈琼, 刘伏友, 等. 去甲斑蝥不依赖钙调蛋白磷酸酶信号通路抑制高糖刺激肾小管上皮细胞细胞外基质的表达[J]. 肾脏病与透析肾移植杂志, 2010, 19(6): 526-533.

LI Ying, CHEN Qiong, LIU Fu-you, et al. NCTD inhibits the expression of extracellular matrix in human proximal tubular epithelial cells induced by high glucose in vitro independent of Calcineurin signal pathway[J]. Chinese Journal of Nephrology, Dialysis & Transplantation, 2010, 19(6): 526-533.

ISSN 1006-298X CN 32-1425/R