法舒地尔通过Rho/ROCK信号通路对高糖培养人肾小球系膜细胞炎症反应及纤维化的影响

目的 体外实验研究法舒地尔(fasudil)通过抑制Rho/ROCK信号通路对高糖培养下的人肾小球系膜细胞(HMCs)炎症反应及其纤维化的影响.方法 传代培养的HMCs同步化后分组:(1)正常糖浓度对照组(NG,含葡萄糖5.5 mmol/L);(2)高糖组(HG,含葡萄糖30.0 mmol/L);(3)甘露醇渗透压对照组(Man,含5.5 mmol/L葡萄糖+24.5 mmol/L甘露醇);(4)高糖+法舒地尔处理组(HG+F组,法舒地尔浓度分别为25、50、100 μmol/L).培养12、24、36、48、72 h收集上清及细胞,用实时PCR检测细胞中小G蛋白(RhoA)、小G蛋白激酶-Ⅰ(ROCK-Ⅰ)、纤维结缔组织生长因子(CTGF)mRNA浓度的变化,用ELISA方法检测上清中纤维连接蛋白(FN)、CTGF、TNFα的蛋白含量.结果 (1)高糖培养下的HMCs中RhoA、ROCK-Ⅰ、CTGF 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).(2)正常培养的HMCs经不同浓度法舒地尔预处理后,高糖继续培养24 h或48 h,HG+F组与HG组对比,RhoA、ROCK-Ⅰ、CTGF mRNA的表达明显下降(P＜0.05),并有一定的浓度依赖性.(3)高糖呈时间依赖方式增加HMCs对FN、CTGF、TNFα蛋白的分泌(P＜0.05),而NG组和Man组无此作用(P＞0.05).(4)经不同浓度法舒地尔预处理后,高糖继续培养12、24、36、48、72 h后FN、CTGF、TNFα蛋白的分泌较HG组明显降低(P＜0.05).结论 法舒地尔通过抑制高糖激活的HMCs的Rho/ROCK信号转导通路,从而降低下游的炎性因子和细胞因子的分泌,减少HMCs的炎症反应及纤维化,为糖尿病肾病的治疗提供新的依据.

Abstract：

Objective To study the effect of fasudil on inhibiting the Rho/ROCK signaling pathway under high glucose in human mesangial cells (HMCs) inflammation and fibrosis. 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) High glucose + fasudil group ( HG + F, the concentrations of fasudil were 25 ,50 and 100 μmol/L, respectively). Collect the supernatant and cells at 0, 12, 24, 36, 48 and 72 h respectively, and determine the concentration changes of the RhoA, ROCK- Ⅰ, connective tissue growth factor (CTGF)mRNA with real-time PCR method in the cells, then used the ELISA method to check the protein content of the fibronectin ( FN) , CTGF, TNFα in the supernatant. Results ( 1) RhoA, ROCK- Ⅰand CTGF mRNA of the HMCs cultured under the high glucose expressed significantly higher than those in the normal group, and there was certain time-dependence. Besides, there was no statistic significance by comparing Man and NG. (2) Under the high glucose situation, after the fasudil pretreatment with different concentrations and 24 h or 48 h culture with high glucose, RhoA, ROCK- Ⅰ , CTGF mRNA expression was significantly decreased in HG + F, compared with HG, and there was certain concentration-dependence. (3) High glucose increased the FN, CTGF, TNFα protein secretion of HMCs in a time-dependent manner, but normal glucose and mannitol had no such effect. (4) After the fasudil pretreatment with different concentrations and culture with high glucose for 12, 24, 36, 48, 72 h, the FN, CTGF, TNFα protein secretion was significantly reduced compared with HG. Conclusion Fasudil can reduce the secretion of downstream inflammatory factors and cytokines by inhibiting high glucose-activated HMCs Rho/ROCK signaling pathway, and reduce the inflammation and fibrosis of HMCs. This provides a new basis for the therapeutic target in the treatment of diabetic nephropathy.

The effect of fasudil via Rho/ROCK signaling pathway on the inflammation and fibrosis in human mesangial cells in high glucose medium

Objective To study the effect of fasudil on inhibiting the Rho/ROCK signaling pathway under high glucose in human mesangial cells (HMCs) inflammation and fibrosis. 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) High glucose + fasudil group ( HG + F, the concentrations of fasudil were 25 ,50 and 100 μmol/L, respectively). Collect the supernatant and cells at 0, 12, 24, 36, 48 and 72 h respectively, and determine the concentration changes of the RhoA, ROCK- Ⅰ, connective tissue growth factor (CTGF)mRNA with real-time PCR method in the cells, then used the ELISA method to check the protein content of the fibronectin ( FN) , CTGF, TNFα in the supernatant. Results ( 1) RhoA, ROCK- Ⅰand CTGF mRNA of the HMCs cultured under the high glucose expressed significantly higher than those in the normal group, and there was certain time-dependence. Besides, there was no statistic significance by comparing Man and NG. (2) Under the high glucose situation, after the fasudil pretreatment with different concentrations and 24 h or 48 h culture with high glucose, RhoA, ROCK- Ⅰ , CTGF mRNA expression was significantly decreased in HG + F, compared with HG, and there was certain concentration-dependence. (3) High glucose increased the FN, CTGF, TNFα protein secretion of HMCs in a time-dependent manner, but normal glucose and mannitol had no such effect. (4) After the fasudil pretreatment with different concentrations and culture with high glucose for 12, 24, 36, 48, 72 h, the FN, CTGF, TNFα protein secretion was significantly reduced compared with HG. Conclusion Fasudil can reduce the secretion of downstream inflammatory factors and cytokines by inhibiting high glucose-activated HMCs Rho/ROCK signaling pathway, and reduce the inflammation and fibrosis of HMCs. This provides a new basis for the therapeutic target in the treatment of diabetic nephropathy.