缬沙坦对高糖培养的肾小球系膜细胞p38MAPK传导通路的影响

Effects of valsartan on expression of p38MAPK signal transduction pathway in glomerular mesangial cells under high concentration of glucose

目的探讨高糖状态下肾小球系膜细胞中p38丝裂原活化蛋白激酶(p38 MAPK)、其上游因子MAPK激酶3/6(MKK3/6)和下游因子cAMP反应元件结合蛋白1(CREB1)的表达以及血管紧张素受体1拮抗剂(AT1Ra)缬沙坦的影响。方法体外培养大鼠肾小球系膜细胞,分别给予高糖和缬沙坦干预,采用Western bolt检测MKK3/6、p38 MAPK和CREB1及其磷酸化蛋白的表达,逆转录-聚合酶链反应(RT-PCR)检测系膜细胞内TGF-β1和FN mRNA的表达。放免法测定细胞上清液中纤维连接蛋白(FN)和IV型胶原的含量。MTT法检测缬沙坦在不同时间、不同药物浓度对细胞增殖状态的影响。结果①与低糖对照组相比,高糖组系膜细胞p-p38 MAPK、p-MKK3/6和p-CREB1表达明显上调,TGF-β1和FN mRNA的表达增加,FN和IV型胶原含量增加。②缬沙坦组p-p38 MAPK、p-MKK3/6和p-CREB1的表达明显下调,TGF-β1和FN mRNA的表达降低,同时FN和IV型胶原的含量减少。③MTT法检测显示不同浓度的缬沙坦对细胞增殖状态都有所抑制,并随药物浓度的增加而作用增强。结论缬沙坦抑制肾小球系膜细胞TGF-β1的表达和细胞外基质的分泌可能部分是通过影响p38 MAPK传导通路的激活来实现的。

Aim To investigate the effects of valsartan on the expression of TGF-β1 mRNA and the activation of p38 mitogen-activated protein kinase （ p38 MAPK）, mitogen activated protein kinase kinasse3/6 （MKK3/ 6） and cAMP response element-binding proteinl （CREB1） in glomerular mesangial cells under high concentration of glucose. Methods High concentration glucose and valsartan were used to stimulate the cultured rat GMCs in vitro. The protein expressions of p38 MAPK, CREB1, p-p38 MAPK, p-MKK3/6 and p- CREB1 were observed by Western blot analysis. TGF- β1 and fibronectin （FN） mRNA were measured by reverse transcription and polymerase chain reaction （RT- PCR）. The protein synthesis of FN and type Ⅳ collagen in the supernatants of the GMCs was detected by radioimmunoassay. Results Compared with low glucose control group, the expressions of p-p38 MAPK, p-MKK3/6, p-CREB1 protein, TGF-β1 and FN mR-NA, FN and type Ⅳ collagen in the supernatants were significantly increased in GMCs under high concentration glucose medium. The expression levels of p-p38 MAPK, p-MKK3/6 and p-CREB1 were significantly lower in the valsartan group than those in the high concentration glucose group. So did the mRNA of TGF-β1 and FN. The concentration of FN and type Ⅳ collagen in the supernatants in the valsartan group was lower than that in the high concentration glucose control group. Conclusion Valsartan can inhibit over production of TGF-β1 and ECM proteins in GMCs under high concentration of glucose, partly by regulating the phosphorylation of p38 MAPK, MKK3/6 and CREB1.