法舒地尔通过Rho/ROCK信号通路对高糖培养基中人类系膜细胞炎症和纤维化的影响

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

作者信息

Ma Dong-wei, Wang Qiu-yue, Ma Xiao-yu, Li Jing, Guan Qing-hua, Fu Yu

机构信息

Department of Endocrinology, The First Hospital, China Medical University, Shenyang 110001, China.

出版信息

Zhonghua Nei Ke Za Zhi. 2011 Jul;50(7):580-4.

PMID:22041269

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-I and 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.

摘要

目的

研究法舒地尔在高糖环境下对人肾小球系膜细胞（HMCs）炎症和纤维化中Rho/ROCK信号通路的抑制作用。

方法

将同步化的HMCs分为以下几组：（1）正常葡萄糖对照组（NG，5.5 mmol/L葡萄糖）；（2）高糖组（HG，30 mmol/L葡萄糖）；（3）甘露醇组（Man，5.5 mmol/L葡萄糖 + 24.5 mmol/L甘露醇）；（4）高糖 + 法舒地尔组（HG + F，法舒地尔浓度分别为25、50和100 μmol/L）。分别在0、12、24、36、48和72 h收集上清液和细胞，采用实时PCR法检测细胞中RhoA、ROCK-I、结缔组织生长因子（CTGF）mRNA的浓度变化，然后用ELISA法检测上清液中纤连蛋白（FN）、CTGF、TNFα的蛋白含量。

结果

（1）高糖培养的HMCs中RhoA、ROCK-I和CTGF mRNA表达显著高于正常组，且有一定的时间依赖性。此外，Man组与NG组比较无统计学意义。（2）在高糖情况下，不同浓度法舒地尔预处理后再与高糖培养24 h或48 h，HG + F组中RhoA、ROCK-I、CTGF mRNA表达较HG组显著降低，且有一定的浓度依赖性。（3）高糖以时间依赖性方式增加HMCs的FN、CTGF、TNFα蛋白分泌，但正常葡萄糖和甘露醇无此作用。（4）不同浓度法舒地尔预处理后再与高糖培养12、24、36、48、72 h，与HG组比较，FN、CTGF、TNFα蛋白分泌显著减少。