α受体介导的雌二醇对大鼠结肠平滑肌细胞RhoA/ROCK通路的影响

Estradiol regulates the expression of RhoA/ROCK pathway in rat colonic smooth muscle cells in an estrogen receptor α-dependent manner

目的:探讨17β-雌二醇(17β-estraial,E2)对大鼠结肠平滑肌细胞(smooth muscle cell,SMC)RhoA/ROCK通路的影响。方法:体外分离培养Sprague-Dawley(SD)大鼠结肠SMCs。细胞免疫荧光双标法观察ROCK1、雌激素受体(ER)α与α肌动蛋白(α-SMA)共表达。不同浓度和不同时间E2刺激后,以反转录实时荧光定量qRT-PCR、Western blot检测RhoA、ROCK1表达。不同浓度ROCK抑制剂Y27632刺激后,Western blot检测E2对其下游蛋白表达情况。观察ER阻断剂ICI182780、牛血清白蛋白结合的E2(BSA-E2)、ERα激动剂PPT及ERβ激动剂DPN在E2影响RhoA、ROCK1表达的作用。构建ERα、ERβ的siRNA,观察E2刺激对转染后SMC中RhoA、ROCK1表达的影响。结果:细胞免疫荧光示结肠SMC上ROCK1与α-SMA共表达。50 nmol/L E2刺激24 h可显著抑制大鼠结肠SMC上RhoA、ROCK1表达(P<0.05)。Y27632可浓度依赖性抑制下游蛋白pMYPT1、p-CPI17、p-MLC表达。E2组及PPT组RhoA、ROCK1表达显著低于其他各组(P<0.05)。ERαsiRNA组可上调RhoA、ROCK1表达(P<0.05)。结论:大鼠结肠SMC上存在ROCK1,E2可抑制RhoA/ROCK通路表达,该作用由雌激素α受体介导。

Objective:The aim of the study was to investigate the effect of 17β-estradiol(E2)on the expression of RhoA/ROCK pathway in rat colon smooth muscle cells(SMC).Methods:Sprague-Dawley rat colonic SMCs were isolated and cultured in vitro.Double immunofluorescence staining was used to observe the expression of ROCK1/ERαandα-actin.After stimulation with E2 at different concentrations and different times,the expression of RhoA and ROCK1 were detected by reverse transcription real-time quantitative PCR(qRT)-PCR and Western blot.After stimulation with different concentrations of ROCK inhibitor Y27632 and 17β-estradiol,the expression of ROCK downstream protein was detected by Western blot.The effects of estrogen receptor(ER)inhibitor ICI182780,bovine serum albumin-bound(BSA-E2),ERαselective agonist propyl pyrazole triol(PPT)and ERβselective agonist diarylpropiolnitrile(DPN)on RhoA/ROCK pathway expression were observed.The ERαand ERβsiRNAs were constructed,and the effects of E2 stimulation on the expression of RhoA and ROCK1 in SMCs were observed.Results:Double immunofluorescence staining showed that ROCK1 andα-SMA co-expressed in rat colon SMC.Treatment with 50 nmol/L E2 for 24 h could significantly inhibit the expression of RhoA and ROCK1(P<0.05).ROCK inhibitor Y27632 inhibited the expression of downstream proteins pMYPT1,p-CPI17 and p-MLC in a concentration-dependent manner.The expressions of RhoA and ROCK1 in E2 group and PPT group were significantly lower than those in other groups(P<0.05).The expressions of RhoA/ROCK1 in ERαsiRNA group were higher than those in control group(P<0.05).Conclusion:ROCK1 is localized in rat colonic SMCs,and E2 inhibits the RhoA/ROCK pathway in an ERα-dependant manner.