摘要
目的:研究高浓度葡萄糖是否通过蛋白激酶C(PKC)βⅠ和RhoA信号通路介导大鼠肾小球系膜细胞血管内皮生长因子(VEGF)表达上调。方法:体外培养SD大鼠肾小球系膜细胞,给予葡萄糖和各种抑制剂处理细胞,提取细胞蛋白,采用Western免疫印迹检测PKCβⅠ及RhoA蛋白的表达量变化,同时提取RNA,采用实时定量PCR观察VEGF mRNA浓度变化。结果:高浓度葡萄糖(30mmol/L)诱导肾小球系膜细胞VEGF mRNA表达增加,而甘露醇对照组VEGF mRNA表达水平未见明显改变(P>0.05)。高浓度葡萄糖刺激系膜细胞RhoA和PKCβⅠ发生活化,RhoA-GTP蛋白和胞膜PKCβⅠ蛋白均呈时间依赖性增加。使用Rho激酶特异性抑制剂(HA-1077)预处理细胞后,VEGF mRNA表达量与高糖组相比明显下调(P<0.05)。进一步使用广谱PKC抑制剂(PMA)、传统PKC抑制剂(G6976)以及PKCβ特异性抑制剂(LY333531)预处理细胞后,VEGF mRNA和RhoAGTP蛋白表达量与高糖组相比均受到抑制(P<0.05)。结论:高浓度葡萄糖可能通过间接活化PKCβⅠ来激活RhoA信号通路,从而上调系膜细胞VEGF的表达,该信号通路在糖尿病肾病的病理过程中发挥着重要作用。
Objective：To investigate the role of PKCβⅠand RhoA signaling pathway in high glucose induced VEGF upregulation in rat mesangial cells.Methods：Rat mesangial cells were cultured in vitro exposure to either glucose or various inhibitors.After treatment,total proteins and RNA were extracted.PKCβⅠand RhoA expressions were detected by Western blotting and VEGF mRNA expressions were determined with real-time PCR.Results：Cellular VEGF mRNA levels increased after treatment with 30mmol/L glucose.Mannitol group showed no changes（P0.05）.High glucose led to PKCβⅠand RhoA activation.Both RhoA-GTP and cell membrane PKCβⅠproteins were on time-dependence increase.After pretreatment with Rho-kinase specific inhibition（HA-1077）,VEGF mRNA levels significantly decreased,compared with high glucose group（P0.05）.Besides,in the presence of the broad PKC inhibitors（PMA）or conventional PKC inhibitors（G6976）prevented high glucose-induced VEGF mRNA and RhoA-GTP proteins upregulation.These were blocked by the PKCβ specific inhibitors（LY333531）（P0.05）.Conclusion：High glucose may indirectly activate PKCβⅠin mesangial cells,leading to downstream RhoA signaling pathway activation and VEGF induction.This pathway plays a central role in the pathogenesis of diabetic nephropathy.
出处
《武汉大学学报（医学版）》
CAS
2015年第2期170-174,180,共6页
Medical Journal of Wuhan University
基金
国家自然科学基金资助项目(编号:81070573
81370819)
武汉市卫计委科研基金项目(编号:WX13C17)
