高糖通过Rho／ROCK信号通路诱导人肾小球系膜细胞的炎症反应及纤维化

High glucose induced inflammatory response and fibrosis of human mesangial cells via Rho/ROCK signaling pathway

目的 探讨Rho/ROCK信号通路在高糖诱导的人肾小球系膜细胞（HMCs）炎症反应及纤维化中的作用.方法 将传代培养的HMCs同步化后分组：（1）正常糖浓度对照组（NG,5.5 mmol/L葡萄糖）;（2）高糖组（HG,30 mmol/L葡萄糖）;（3）甘露醇渗透压对照组（Man,5.5 mmol/L葡萄糖＋24.5 mmol/L甘露醇）;（4）NG＋Y-27632（10 μmmol/L）组;（5）HG＋Y-27632（10 μmmol/L）组,培养12、24、36、48、72 h后收集上清及细胞,用Western印迹检测RhoA蛋白的活化,用实时PCR检测细胞中RhoA、ROCK-Ⅰ、结缔组织生长因子（CTGF）、肿瘤坏死因子α（TNF-α）mRNA浓度的变化,用ELISA方法检测上清中纤维连接蛋白（FN）、CTGF、TNF-α的蛋白含量.结果 （1）高糖刺激HMCs的RhoA活化,于30 min即可出现活性升高,1 h达到高峰,之后活化的RhoA表达逐渐下降（P=0.02）.（2）高糖培养下的HMCsRhoA、ROCK-Ⅰ、CTGF、TNF-α mRNA的表达较NG组明显升高（P＜0.05）,并有一定的时间依赖性,Man组与NG组相比差异无统计学意义（P＞0.05）.（3）经Y-27632预处理后,在正常糖和高糖浓度培养24 h或48 h后,NG＋Y-27632组和HG＋Y-27632组与未处理组相比RhoA、ROCK-Ⅰ、CTGF、TNF-α mRNA的表达明显下降（P＜0.01）.（4）高糖呈时间依赖方式增加HMCs的FN、CTGF、TNF-α分泌（P＜0.05）.（5）经Y-27632预处理,继续培养12、24、36、48、72 h后NG组和HG组中FN、CTGF、TNF-α蛋白的分泌较处理前明显降低（P＜0.05）.结论 高糖可通过Rho/ROCK信号通路介导HMCs的炎症反应和纤维化,抑制此通路可作为减缓糖尿病肾病发生发展的潜在靶点.

Objective To investigate the role of Rho/ROCK signaling pathway in the process of human mesangial cells （HMCs） inflammation and fibrosis induced by high glucose. Methods Synchronized HMCs were divided into following groups： （ 1 ） Normal glucose control group （ NG, 5.5 mmol/L glucose）; （ 2 ） High glucose group （ HG, 30 mmol/L glucose）; （3） Mannitol group（ Man,5.5 mmol/L glucose＋ 24.5 mmol/L mannitol）; （4） NG ＋Y-27632 group（ 10 μ mmol/L Y-27632 ）; （ 5 ） HG Y-27632 group （ 10 μmmol/L Y-27632 ）. The supernatant and cells were collected at 0,12,24,36,48, and 72 h. Western blot was used to detect the active RhoA and total RhoA,while RhoA, ROCK-Ⅰ, CTGF, and TNF-α mRNA expressions were determined with realtime PGR method in the cells, then ELISA method was used to check protein levels of FN, CTGF, and TNF-α in the supernatant. Results （ 1 ） RhoA activation was stimulated after treatment for with 30 mmol/L glucose, peaked at 1 h, and then decreased （ P = 0. 02）. （2） RhoA, ROCK-Ⅰ, CTGF, and TNF-α mRNA expressions in HMC cultured under high glucose were higher than those in the normal group （ P 〈 0.05 ）, and there was certain time-dependence. Besides, there was no statistical significance between Man and NG groups（ P〉0. 05 ）. （ 3 ） After Y-27632 pretreatment and being cultured with normal glucose and high glucose for24 h or48 h, RhoA, ROCK-Ⅰ, CTGF, and TNF-α mRNA expressions were significantly decreased （ P〈0.01 ） as compared with groups without treatment. （4） High glucose increased FN, CTGF,and TNF-α protein secretion of HMC in a time-dependent manner（ P〈0. 05 ）. （ 5 ） After Y-27632 pretreatment and being cultured with normal and high glucose for 12,24,36,48,72 h, FN, CTGF, and TNF-α protein secretions were significantly reduced（ P〈0.05 ）. Conclusion Rho/ROCK signaling pathway may mediate inflammation and fibrosis induced by high glucose in HMCs, supporting a potential role for inhibitors of Rho/ROCK in the treatment of diabetic nephropathy.