文章摘要
姜 玲,汤玉蓉,熊文婕,俞 汀,郑永娉,沈小雪,林 琳.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[J].南京医科大学学报,2020,(2):174~179
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
投稿时间:2019-06-17  
DOI:10.7655/NYDXBNS20200205
中文关键词: 雌二醇  RhoA/ROCK通路  平滑肌  结肠
英文关键词: estradiol  RhoA/ROCK pathway  smooth muscle  colon
基金项目:国家自然科学基金青年基金(81400599)
作者单位
姜 玲 南京医科大学第一附属医院消化科 江苏 南京 210029 
汤玉蓉 南京医科大学第一附属医院消化科 江苏 南京 210029 
熊文婕 南京医科大学第一附属医院消化科 江苏 南京 210029 
俞 汀 南京医科大学第一附属医院消化科 江苏 南京 210029 
郑永娉 南京医科大学第一附属医院消化科 江苏 南京 210029 
沈小雪 南京医科大学第一附属医院消化科 江苏 南京 210029 
林 琳 南京医科大学第一附属医院消化科 江苏 南京 210029 
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中文摘要:
      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p?MYPT1、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)。ERα siRNA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。
英文摘要:
      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/ERα and α?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),ERα selective agonist propyl pyrazole triol(PPT)and ERβ selective agonist diarylpropiolnitrile(DPN)on RhoA/ROCK pathway expression were observed. The ERα and ERβ siRNAs 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 and α?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 p?MYPT1,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 ERα siRNA 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.
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