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目的探讨17β-雌二醇(17β-estradiol,E2)通过快速激活诱导型一氧化氮合成酶(iNOS)的活性,调节细胞外信号调节激酶(mitogen-activated protein kinase,p42/44 MAPK)表达抑制血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖的机制。方法在培养的VSMCs的基础上,采用放射免疫测定法(RIA)和Western blot检测E2预处理前后胎牛血清(fetal calf serum,FCS)对VSMCs中诱导型i NOS的活性及磷酸化p42/44 MAPK蛋白和p42/44MAPK总蛋白表达的影响。采用比色法检测E2预处理后VSMCs中NO含量的变化。结果 E2预处理5 min,即可逆转FCS诱导的iNOS活性下降,此效应在E2预处理15 min时达到高峰,处理30 min后此效应减弱。基因转录抑制剂放线菌素D(actinomycin D,Act D)预处理后对此过程无影响,而三苯氧胺预处理可明显逆转E2诱导的iNOS活性增加。比色法检测上清液中NO的含量显示,E2预处理15 min,NO的含量明显增加。Western blot的结果显示,E2预处理15 min,可明显抑制磷酸化p42/44 MAPK蛋白的表达,而对总p42/44 MAPK蛋白的表达无影响。NO合酶(NOS)抑制剂L-硝基左旋精氨酸甲酯(L-NAME)预处理,可逆转E2诱导的磷酸化p42/44 MAPK的表达下降。结论 E2可通过快速激活i NOS增加NO释放,下调磷酸化p42/44 MAPK的活性,抑制VSMCs增殖。
Objective To investigate the inhibitory effect of 17β-estradiol (E2) on the expression of mitogen-activated protein kinase (p42 / 44 MAPK) through rapid activation of inducible nitric oxide synthase (iNOS) Mechanism of proliferation of vascular smooth muscle cells (VSMCs). Methods On the basis of cultured VSMCs, radioimmunoassay (RIA) and Western blot were used to detect the activity of inducible iNOS and the phosphorylation of p42 / 44 in VSMCs before and after E2 pretreatment with fetal calf serum (FCS) MAPK protein and p42 / 44MAPK protein expression. The contents of NO in VSMCs were detected by colorimetric method. Results E2 preconditioning for 5 min reversed the decrease of iNOS activity induced by FCS. The effect peaked at 15 min of E2 pretreatment and decreased after 30 min of treatment. Actinomycin D (Act D) pretreatment had no effect on this process, while tamoxifen preconditioning could obviously reverse the increase of E2-induced iNOS activity. Colorimetric detection of NO in supernatant showed that E2 pretreatment 15 min, NO content increased significantly. Western blot results showed that E2 pretreatment for 15 min significantly inhibited the phosphorylation of p42 / 44 MAPK protein, but had no effect on the total p42 / 44 MAPK protein expression. Pretreatment with NO synthase (NOS) inhibitor L-NAME reversed the decrease of E2-induced phosphorylated p42 / 44 MAPK expression. Conclusion E2 can increase NO release by rapidly activating iNOS, down-regulate the activity of phosphorylated p42 / 44 MAPK and inhibit the proliferation of VSMCs.