高糖环境中血管紧张素Ⅱ影响肾小球系膜细胞促纤维化因子的表达及机制

Significance of JAK 2/STAT 3 in angiotensin Ⅱ up-regulation of TGF-β1、CTGF and FN mRNA expression on mesangial cells under hyperglucose

目的:在高糖环境中,用不同浓度血管紧张素II(AngII)刺激肾小球系膜细胞(GMC),观察不同时点细胞内磷酸化信号转导子与转录激活子3(p-STAT3)、转化生长因子β1(TGF-β1)、结缔组织生长因子(CTGF)、纤维连接蛋白(FN)的表达变化及AG-A90对上述指标表达的影响,探讨高糖和AngII共同作用对GMC分泌促纤维化因子及细胞外基质的影响。方法:(1)GMC在高糖环境中培养48h后,加入AngII(0.1μmol/L)孵育不同时间,用免疫细胞化学及Western Blot方法检测p-STAT3的表达,RT-PCR检测GMCTGF-β1、CTGF、FNmRNA的表达。(2)用JAK2/STAT3通路特异性阻断剂AG-490(10μmol/L)对GMC进行预处理后再加入AngII,RT-PCR方法检测大鼠GMCTGF-β1、CTGF、FNmRNA的表达变化。结果:(1)外源性AngII显著增加了GMCp-STAT3表达,刺激5min后p-STAT3表达即开始增多,并向细胞核积聚,30min达高峰,90min后逐渐恢复到刺激前水平。(2)AngII刺激后GMC的TGF-β1、CTGF、FNmRNA表达明显上调,而AG-490预处理显著降低了TGF-β1、CTGF、FNmRNA的高表达。结论:AngII可一过性激活大鼠GMCJAK2/STAT3信号转导通路,并上调TGF-β1、CTGF及FNmRNA表达,而阻断JAK2/STAT3信号转导通路可明显降低AngII诱导的大鼠GMCTGF-β1、CTGF、FNmRNA的高表达。

Objective：To investigate the role of JAK 2/STAT 3 in angiotensin II up-regulating the expression of TGF-β1 ,CTGF,FN mRNA on glomerular mesangial cells （GMC） under hyperglncose. Methodology：GMC cultured under DMEM （HG） to 70% -80% confluence, then treated with angiotensin Ⅱ （0. 1 μmol/L） for different times or preincubation with AG-490 （ a specific inhibitor of JAK 2） then treateted with angiotensin II. At the end of desired periods, the cells were lysed for further detection of TGF-β1 ,CTGF and FN mRNA by RT-PCR, and detection of p-STAT 3 by Western blot and immunohistochemistry. Results： angiotensin Ⅱ could activate phosphorylation of STAT 3 and significantly increase in synthesis of TGF-β1 ,CTGF,FN mRNA under the concentration of high glucose. The over expression of those factors were significantly inhibited by preincubation with AG-490. Conclusion：Angiotensin Ⅱ could increase the expression of TGF-β1 ,CTGF,FN mRNA on GMC under high glucose through the activation of JAK 2/STAT 3 pathway.