去甲斑蝥素对高糖刺激的HK-2细胞FN,Col Ⅳ和TGF–β1 mRNA及蛋白表达的影响

Effect of norcantharidin on the expression of FN, Col Ⅳ and TGF-β1 mRNA and protein in HK-2 cells induced by high glucose

目的：观察去甲斑蝥素（NCTD）对高糖刺激的HK-2细胞外基质和TGF-β1表达的影响。方法：常规培养HK-2细胞，无血清DMEM培养基同步培养24h，细胞分为正常葡萄糖组（C，D-glucOSe5．5mmol／L）、甘露醇对照组（M，5．5mmol／LD—glucose＋24．5mmol／Lmannitoll、高糖组（HG，30mmol／LD—glucosel、高糖＋NCTD干预组（30mmol／LD-glucose＋0．5～40mg／LNCTD）。台盼蓝排斥实验检测NCTD对高糖刺激的细胞毒性。MTT法检测NCTD对高糖刺激的细胞增殖的影响。收集培养6，24，48h后细胞总RNA及蛋白，用RT．PCR检测细胞FN，Col1V和TGF-β1mRNA的表达，采用Western印迹检测FN，ColIV和TGF-β1蛋白的表达。结果：不同浓度NCTD作用72h后，浓度超过5mg／L的NCTD对高糖环境下的HK．2细胞有明显的毒性。RT-PCR和Western印迹结果显示：30mmol／LD-葡萄糖可引起HK．2细胞FN，Col1V和TGF-β1mRNA及蛋白水平的升高（P〈0．05），而5mg／LNCTD可抑制高糖刺激的FN，ColIV和TGF-β1的表达（P〈O．05）。相同渗透浓度的D-甘露醇组对上述指标均无影响（P〉O．05）。结论：NCTD能下调HK-2细胞FN，ColIV及TGF-β1的表达。

Objective： To observe the effect of norcantharidin （NCTD） on the expression of mRNA and protein of fibronectin （FN）, collagen IV（Col IV） and transforming growth factor-β1 （TGF-β1） in human kidney proximal tubular epithelial （HK）-2 cells induced by high glucose. Methods： HK-2 cells were incubated with serum-free DMEM for 24 h to synchronize cell growth, and then the cells were divided into 4 groups： Group C （ 5.5 mmol/L D-glucose）, Group M （5.5 mmol/L D-glucose ＋ 24.5 mmol/L-mannitol）, Group HG （30 mmol/L D-glucose）, and Group HG ＋ NCTD （30 mmol/L D-glucose ＋ 0.5-40 mg/L NCTD）. Cytotoxicity of ilK-2 cells induced by high glucose of NCTD was detected by Trypan blue dye exclusive assa）, The effect of NCTD on the proliferation of ilK-2 cells in high glucose was determinedby MTE The cells were collected to extract total RNA and protein at 6, 24 and 48 h after the incubation. The expression of FN, Col IV and TGF-β1 mRNA was examined by RT-PCR, and FN, Col IV and TGF-β1 protein was analyzed byWestem blot. Results： Trypan blue dye exclusive assay showed NCTD concentrations over 5 mg/L were rather toxic in HK-2 cells. The proliferation of HK-2 cells in high glucose was interrupted by interfered with 5 mg/L NCTD as measured by MTT （P〈0.05）. NCTD at 5 mg/L had a stronger inhibitory effect than NCTD at 2.5 mg/L. Real-time PCR and Western blot showed that the mRNA and protein expression of FN, collagen IV and TGF-β1 increased in HK-2 cells treated with high glucose （V〈0.05）, while that in cells treated by NCTD was dramatically inhibited （P〈0.05）. No change in these parameters was detected in the 30 mmol/L D-mannitol control group （P〉0.05）. Conclusion： NCTD can downregulate FN, collagen IV and TGF-β1 mRNA and protein expression in HK-2 cells stimulated by 30 mmol/L D-glucose..