Rho/Rho激酶信号通路在低氧致肺纤维化中的作用及法舒地尔的干预效应  

Fasudil attenuates hypoxia-induced pulmonary fibrosis via suppressing Rho/Rock signal pathway

作　　者：高艳[1] 谢敏[1] 石俊青[2] 

机构地区：[1]四川大学华西医院呼吸内科,成都610041 [2]成都市第一人民医院呼吸内科,成都610041

出　　处：《第三军医大学学报》2010年第22期2378-2382,共5页Journal of Third Military Medical University

基　　金：四川省科技厅攻关项目(0040205301469)~~

摘　　要：目的观察人胚肺成纤维细胞低氧培养24h对致纤维化相关因子表达的影响及不同浓度法舒地尔(6、12、25μmol/L)的干预作用。方法以人胚肺成纤维细胞(MRC-5)为研究对象,体外构建以HIF-1α蛋白的高表达为特征的低氧微环境,低氧(37℃、5%CO2、2%O2、93%N2)培养0、6、12、24h,Westernblot法检测各低氧时间点RhoA、ROCK1、CTGF蛋白的表达及以低氧24h为对照组,观察低氧24h下不同浓度法舒地尔对结缔组织生长因子(connective tissue growth factor,CTGF)蛋白表达的影响;RT-PCR、MTT法分别检测低氧24h下不同浓度法舒地尔对人胚肺成纤维细胞(MRC-5)CTGF mRNA表达和细胞活力的影响,均以低氧24h为对照组。以常氧(21%O2,74%N2,5%CO2)24h及低氧24h为对照,ELISA法测定低氧24h下不同浓度法舒地尔对细胞上清Ⅰ型胶原(ColⅠ)表达的影响。结果①随着低氧作用时间延长,RhoA、ROCK1、CTGF蛋白表达上调并逐渐增强。②低氧24h下不同浓度法舒地尔(6、12、25μmol/L)对CTGF蛋白及mRNA水平均有抑制作用(P<0.05,P<0.01)。③与低氧24h对照组比较,低氧24h下不同浓度法舒地尔(6、12、25μmol/L)均对细胞上清ColⅠ的合成及细胞增殖活性均有抑制作用(P<0.01)。结论低氧可能是通过激活Rho/Rho激酶信号通路促发CTGF的高表达进而引发肺成纤维细胞细胞外基质表达增加等一系列促纤维化反应,而法舒地尔通过抑制Rho/Rho激酶信号通路来干预低氧致纤维化因子的转录和表达。Objective To investigate the effect of hypoxic condition on the expressions of RhoA, ROCK1and connective tissue growth factor （CTGF） in human fetal fibroblasts, and the beneficial effects of fasudil in the hypoxia-induced pulmonary fibrosis. Methods Human fetal fibroblasts （MRC-5 cells） were cultured under hypoxic condition （37 ℃, 5 %CO2, 2%O2 , 93%N2） for 0 , 6, 12, and 24 h respectively. The cells cultured in a condition of 21%O2, 74%N2 and 5% CO2 were used as control. The effect of fasudil was investigated in normal group, hypoxia group and intervention groups （6, 12 or 25 μmol/L fasudil） under the corresponding oxygen condition for 24 h. The protein levels of HIF-1α, RhoA, ROCK1, and CTGF were assayed by Western blotting. RT-PCR was performed to detect CTGF mRNA levels. Cell viability was measured by thiazolyl blue （MTT） assay. The concentration of collagen type Ⅰ （Col Ⅰ） in fibroblast cells’ supernatant were determined by ELISA. Results The protein levels of RhoA, ROCK1 and CTGF were up-regulated by hypoxia in a time-dependent manner. Different concentrations of fasudil all inhibited the expressions of protein and mRNA of CTGF after 24 h of hypoxia （P0.01, 0.05）. Compared with the hypoxia for 24 h, fasudil all inhibited cell proliferation activity and the secretion of Col Ⅰ in fibroblast cells’s supernatant for hypoxia 24 h （P0.01）. Conclusion Hypoxia exerts a potential brotic stimulus probabily through activating Rho/Rock signal pathway, thus up-regulates the expression of CTGF, and finally triggers a series of brotic responses. These changes could be markedly inhibited by fasudil. Fasudil attenuates hypoxia-induced pulmonary fibrosis, which may be due to suppressing the Rho/Rock signal pathway.

关 键 词：法舒地尔 低氧 人胚肺成纤维细胞 ROCK1结缔组织生长因子 

分 类 号：R364.4[医药卫生—病理学] R563.02[医药卫生—基础医学]