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

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.

Abstract：

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.

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

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.