Rho/Rock信号通路在TGF-β_1刺激大鼠颈内动脉平滑肌细胞表型转化中的作用及可能机制

The effect and possible mechanism of the Rho/Rock signaling pathway during TGF-β_1-induced ISMCs phenotypic modulation in rats

目的探讨Rho/Rock信号通路在TGF-β_1刺激大鼠颈内动脉平滑肌细胞(ISMC)表型转化中的作用及可能机制。方法 ISMC随机分为两组:空白对照组和TGF-β_1刺激组,以不同浓度(1、5、10、20、50 ng/m L)TGF-β_1刺激细胞24 h,用荧光实时定量PCR法检测转凝蛋白(SM22α)和骨桥蛋白(OPN)mRNA的表达;再以上述得出的最佳TGF-β_1浓度刺激细胞,在不同时间(6、12、24、48 h)收集细胞,采用同样方法检测SM22α、OPN mRNA的表达水平;经无血清DMEM高糖培养基静止培养24 h后,随机分为以下各组:空白对照组、TGF-β_1(10ng/m L)刺激组及TGF-β_1(10 ng/m L)+法舒地尔(Fasudil)(10、30、50μmol/L)组,分别采用FQ-RT-PCR法检测SM22α、OPN、Rho A、Rock-1 mRNA的表达;Western blot法检测SM22α、OPN蛋白表达水平;根据实验结果用透射电镜观察各组细胞超微结构的相应变化。结果 10 ng/m L TGF-β_1刺激大鼠ISMCs 24 h达顶峰。TGF-β_1+Fasudil组SM22αmRNA和蛋白表达水平较TGF-β_1刺激组高(P<0.01),且在Fasudil浓度为30μmol/L时达到高峰,随后下降;TGF-β_1+Fasudil组OPN mRNA表达水平较TGF-β_1刺激组低,呈浓度依赖性,至30μmol/L达最低(P<0.05),后又呈上升趋势。Western blot结果显示TGF-β_1+Fasudil组的OPN蛋白表达水平较TGF-β_1刺激组低,至Fasudil浓度为30μmol/L达最低(P<0.05)。同时FQ-RT-PCR结果显示TGF-β_1刺激组的Rho A和Rock-1 mRNA表达水平较空白对照组高(P<0.01),在Fasudil浓度为30μmol/L时TGF-β_1+Fasudil组的Rho A和Rock-1mRNA表达水平较TGF-β_1刺激组低(P<0.05)。电镜下观察各组细胞显示,空白对照组(×20 000)ISMC高尔基体肥大,线粒体及粗面内质网正常,10 ng/m L TGF-β_1刺激后细胞内线粒体增多,在上述基础上加入30μmol/L Fasudil后电镜下观察细胞又回到正常成熟结构状态。结论 10 ng/m L TGF-β_1刺激大鼠ISMC 24 h可成功刺激其表型转化,且该过程可能通过Rho/Rock信号通路实现;Rho/Rock信号通路抑制剂Fasudil可增加TGF-β_1刺激的SM22α的表达,下调OPN的表达水平,抑制大鼠ISMCs表型转化。

Objective To investigate the effects and possible mechanism of the Rho/Rock signaling pathway on TGF- β_1- induced of rat ISMCs phenotypic modulation. Methods The rat ISMCs were randomly divided into two groups： blank control group and TGF- β_1stimulation group,in which TGF- β_1of different concentrations（ 1,5,10,20 and 50 ng / m L） were given for 24 h. Transgelin（ SM22α） and osteopontin（ OPN） mRNA expression was assessed by fluorescence quantitative real- time polymerase chain reaction（ FQ- RT- PCR）. The derived optimal concentration was applied to stimulate cells,which were collected at different times（ 6,12,24 and 48 h）. After 24 h of serum- free DMEM high glucose medium culture,ISMCs were randomly divided into 3 groups： blank control group,TGF- β_1（ 10ng/m L）stimulation group,TGF- β_1（ 10 ng/m L） ＋ Fasudil（ 10,30 and 50 μmol/L） groups. The expression levels of SM22α,OPN,Rho A,Rock- 1 mRNA were assessed with FQ- RT- PCR; and SM22α and OPN expression levels were assessed using Western Blot. The ultrastructure of the cells was observed by transmission electron microscopy. Results TGF- β_1in 10 ng / m L stimulation of ISMCs reached the summit at 24 h. SM22 mRNA and protein expression levels were significantly increased in TGF- β_1＋ Fasudil group（ P〈0. 01）. Significant reduction in OPN mRNA was observed in TGF- β_1＋ Fasudil group in dose- dependent manners,and reached lowest effects with Fasudil of 30 μmol / L（ P〈0. 05）. So was the protein expression of OPN according to Western blot. According to FQ- RT- PCR,the mRNA expression of Rho A and Rock- 1 and Rock- 1 was significantly increased in TGF- β_1group（ P〈0. 01）,and it was significantly lower in TGF- β_1＋ Fasudil（ 30 μmol / L） group than TGF- β_1group（ P〈0. 05）. Under electron microscope,normal Golgi ISMC hypertrophy,mitochondria and rough endoplasmic reticulum were observed in control group（ × 20 000）,while increased intracellular mitochondria was observed in TGF- β_1group,which was reversed by Fasudil. Conclusion TGF-β_1of 10 ng / m L stimulation in ISMCs for 24 h can be successfully induced phenotype modulation. Rho / Rock signaling pathway inhibitors Fasudil increases of TGF- β_1- induced SM22α expression,loweres OPN expression,thus inhibits ISMCs phenotypic modulation.