[去甲斑蝥素对高糖诱导的HK-2细胞中纤连蛋白、IV型胶原及转化生长因子-β1 mRNA和蛋白表达的影响]

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

作者信息

Chen Qiong, Li Ying, Luo Junhui, Yang Yang, Li Jun, Sun Lin, Xiao Li, Xu Xiangqing, Peng Youming, Liu Fuyou

机构信息

Department of Nephrology, Second Xiangya Hospital,Institute of Nephrology, Central South University, Changsha 410011, China.

出版信息

Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2012 Mar;37(3):278-84. doi: 10.3969/j.issn.1672-7347.2012.03.012.

DOI:10.3969/j.issn.1672-7347.2012.03.012

PMID:22561496

Abstract

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 HK-2 cells induced by high glucose of NCTD was detected by Trypan blue dye exclusive assay. The effect of NCTD on the proliferation of HK-2 cells in high glucose was determined by MTT. 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 by Western 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 (P<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.

摘要

目的

观察去甲斑蝥素（NCTD）对高糖诱导的人肾近端小管上皮（HK）-2细胞中纤连蛋白（FN）、Ⅳ型胶原（Col Ⅳ）及转化生长因子-β1（TGF-β1）mRNA和蛋白表达的影响。

方法

将HK-2细胞用无血清DMEM培养24 h以同步细胞生长，然后将细胞分为4组：C组（5.5 mmol/L D-葡萄糖）、M组（5.5 mmol/L D-葡萄糖 + 24.5 mmol/L甘露醇）、HG组（30 mmol/L D-葡萄糖）、HG + NCTD组（30 mmol/L D-葡萄糖 + 0.5 - 40 mg/L NCTD）。采用台盼蓝拒染法检测NCTD高糖诱导的HK-2细胞毒性。用MTT法测定NCTD对高糖环境下HK-2细胞增殖的影响。培养6、24和48 h后收集细胞提取总RNA和蛋白。采用RT-PCR检测FN、Col Ⅳ和TGF-β1 mRNA的表达，用Western blot分析FN、Col Ⅳ和TGF-β1蛋白。

结果

台盼蓝拒染法显示NCTD浓度超过5 mg/L时对HK-2细胞毒性较大。MTT法检测发现5 mg/L NCTD干扰后高糖环境下HK-2细胞的增殖受到抑制（P<0.05）。5 mg/L NCTD的抑制作用强于2.5 mg/L NCTD。实时PCR和Western blot显示，高糖处理的HK-2细胞中FN、Ⅳ型胶原和TGF-β1的mRNA和蛋白表达增加（P<0.05），而NCTD处理的细胞中这些指标显著受到抑制（P<0.05）。30 mmol/L D-甘露醇对照组这些参数无变化（P>0.05）。