去甲斑蝥素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  

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

作　　者：李瑛[1,2] 陈琼 刘伏友[1,2] 彭佑铭[1,2] 李军[1,2] 孙林[1,2] 罗俊辉[1,2] 杨阳[1,2] 段绍斌[1,2] 凌光辉[1,2] 刘虹[1,2] 刘映红[1,2] 

机构地区：[1]中南大学湘雅二医院肾脏内科 [2]中南大学肾脏病研究所,长沙410011 [3]上海第十人民医院肾内科,上海200072

出　　处：《肾脏病与透析肾移植杂志》2010年第6期526-533,共8页Chinese Journal of Nephrology,Dialysis & Transplantation

基　　金：湖南省自然科学基金重点项目(10JJ2011);国家高等学校博士学科点专项科研新教师基金(20070533062);中南大学代谢综合征研究中心课题(DY-2008-02-03)

摘　　要：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 learn the effect of NCTD on Caleineurin （CaN） /NFAT pathway and to explore whether the anti - fibrogenie effect of NCTD on renal interstitium in diabetic nephropathy is dependent of NCDT＇s inhibition to CaN pathway. Methodology：HK-2 cells were cuhured and transfeeted with CaN small interference RNA, then divided into 5 groups： （1） Normal glucose group （5.5 mmol/L D-Glucose）, （2） High glucose group （30 mmol/L D-Glucose）, （3） High glucose ＋ CaN siRNA groups： （4） High glucose ＋ CaN siRNA ＋ NCTD （5 rag/L） group, （5） High glucose ＋ NCTD（5 mg/L） 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, Col IV and TGF-I31 in HK-2 cells transfected by CaN siRNA were observed by Western blot. Results：The expressions of CaN mRNA and protein in HK-2 cells were elevated when exposed to 30 mmol/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 30 min, 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, Col IV and TGF-β were obviously increased（ P 〈 0. 05 ）. NCTD treatment could down-regulated the increase of FN, Col IV and TGF-β protein expression in HK-2 ceils stimulated by high glucose after transfecting CaN siRNA. Conclusion： NCTD could down-regulate CaN expression in HK-2 cells, and block CaN/NFAT signaling pathway. H

关 键 词：糖尿病肾病 去甲斑蝥素 肾小管间质纤维化 钙调蛋白磷酸酶 细胞外基质 

分 类 号：R692[医药卫生—泌尿科学]