吴茱萸次碱抑制AngⅡ诱导的血管平滑肌Cx43表达上调

Rutaecarpine prevents up-regulation of Cx43 in vascular smooth muscle cells induced by angiotensin Ⅱ

目的探讨吴茱萸次碱(rutaecarpine,Rut)对血管紧张素Ⅱ(AngⅡ)诱导的缝隙连接蛋白43(Cx43)表达,血管平滑肌细胞(VSMCs)增殖和表型改变的影响及可能机制。方法加入不同浓度的Rut(0.3、1.0、3.0μmol·L^(-1))预处理VSMCs 10 min后,加入AngⅡ(1μmol·L^(-1))共同孵育24 h,应用辣椒素受体(transient receptor potential vanilloid 1,TRPV1)竞争性拮抗剂Capsazepine(CAPZ,10μmol·L^(-1)),探讨TRPV1是否介导Rut的效应。Western blot检测VSMCs中Cx43和NF-κB亚基p65的水平,免疫细胞化学判断p65核转位。CCK-8和Ed U法检测VSMCs增殖,Western blot检测细胞周期蛋白Cyclin D1,确定对细胞周期的影响;Western blot检测收缩型标志蛋白α-SMA和Calponin水平。结果Rut可抑制AngⅡ诱导的Cx43表达上调,并抑制p65核转位,预先给予CAPZ可取消此效应。Rut抑制AngⅡ诱导的VSMCs增殖,表现为抑制VSMCs的DNA合成及细胞活力,还可抑制VSMCs表型转化,以上效应均被CAPZ部分阻断。结论 Rut可下调Cx43表达,从而抑制AngⅡ诱导的VSMCs增殖和表型转换,其机制涉及TRPV1/NF-κB信号途径。

Aim To investigate the effect of rutae-carpine（Rut） on expression of connexin43（Cx43） and the proliferation and phenotypic switch of vascular smooth muscle cells（VSMCs） induced by angiotensin II（Ang Ⅱ）,and its possible mechanism. Methods VSMCs were pretreated with Rut（0. 3,1. 0,3. 0 μmol·L-1） for 10 min,then co-incubated with AngⅡ（1μmol·L-1） for 24 h. The transient receptor potential vanilloid 1（TRPV1） competitive antagonist capsazepine（CAPZ,10 μmol·L-1） was used to investigate whether TRPV1 mediated the effects of Rut. Western blot was used to detect the protein levels of Cx43 and NF-κB p65 in VSMCs,and immunocytochemistry was applied to determine the nuclear translocation of p65.CCK-8 and Ed U were used to detect the proliferation of VSMCs,and flow cytometry was employed to detect cell cycle distribution. Again,Western blot was used to assess the α-SMA and Calponin levels of contractile marker protein. Results Rut prevented the up-regulation of Cx43 and nuclear translocation of p65 induced by Ang Ⅱ,and the effect was blocked by CAPZ. Rut prevented the proliferation of VSMCs induced by AngⅡ,as presented by inhibiting DNA synthesis and cell viability in VSMCs. Rut also prevented phenotypic switch of VSMCs induced by Ang Ⅱ,and the effect could be partially blocked by CAPZ. Conclusion Rut reduces the expression of Cx43 and prevents the proliferation and phenotypic switch of VSMCs induced by AngⅡ,and its mechanism involves the TRPV1/NF-κB signaling pathway.