小檗碱抑制机械牵张力诱导的小鼠血管平滑肌细胞PKCδ磷酸化及增殖/迁移

目的观察机械牵张力(SS)是否通过激活PKCδ诱导小鼠血管平滑肌细胞(VSMC)增殖和迁移,并进一步探究小檗碱(BBR)对其的影响及作用机制。方法体外常规培养小鼠VSMC分为6组:阴性对照组(NC)、小檗碱组(BBR)、PKCδ抑制剂Sotrastaurin组(Sotras)、SS组、SS+BBR组和SS+Sotras组。静息培养的VSMC分别用BBR或Sotrastaurin或ddH₂O预处理1 h,继而机械牵张力(10%牵张强度)牵拉不同时间或不牵拉作为对照。收集各组VSMC,Western blot检测PKCδ磷酸化水平;免疫荧光法检测VSMC增殖;细胞划痕实验检测VSMC迁移。结果免疫荧光和细胞划痕实验结果显示,与阴性对照组相比,机械牵张力刺激显著提高VSMC中Ki67阳性水平约469%(P<0.05,n=3),划痕宽度缩小约54.9%(P<0.05,n=3),而BBR和Sotrastaurin能明显抑制机械牵张力引起的上述变化,与SS组相比,Ki67阳性水平分别下降约66.9%和80.2%(P<0.05,n=3),划痕宽度增加约79.4%和120.1%(P<0.05,n=3);Western blot结果显示,与阴性对照组相比,机械牵张力刺激可诱导PKCδ磷酸化水平呈时间依赖性升高,以30 min最显著,提升约97.5%(P<0.05,n=3);而BBR可呈浓度依赖性抑制机械牵张力刺激诱导的PKCδ磷酸化水平升高,以200μmol/L效果最显著,降低约37.6%(P<0.05,n=3)。结论BBR可通过抑制PKCδ磷酸化进而阻断机械牵张力诱导的VSMC增殖和迁移。

Berberine inhibited the proliferation/migration of mouse vascular smooth muscle cells induced by mechanical stretch stress via inhibition of PKCδ phosphorylation

Aim To observe whether mechanical stretch stress(SS)induces the proliferation and migration of mouse vascular smooth muscle cells(VSMC)through PKCδactivation,and to further explore the effect and mechanism of berberine(BBR)on the proliferation and migration of VSMC.Methods Mouse VSMC in vitro were divided into six groups:negative control group(NC),berberine group(BBR),PKCδinhibitor Sotrastaurin group(Sotras),SS group,SS+BBR group and SS+Sotras group.The cultured VSMC in each group,which were pretreated with BBR or Sotrastaurin or ddH₂O for 1h,followed by SS(10%tensile strength)for different time or no treatment for control,were collected for the detection of PKCδphosphorylation,proliferation and migration by Western blot,immunofluorescence and scratch assay respectively.Results Immunofluorescence and cell scratch test results showed that compared with NC group,SS stimulation significantly increased Ki67 positive level in VSMC by 469%(P<0.05,n=3),and reduced scratch width by 54.9%(P<0.05,n=3),while BBR and Sotrastaurin could significantly inhibit the changes caused by SS,Ki67 positive level decreased by 66.9%and 80.2%(P<0.05,n=3),and scratch width increased by 79.4%and 120.1%(P<0.05,n=3)compared with SS group,respectively.Meanwhile,Western blot results showed that SS stimulation induced a time-dependent increase in PKCδphosphorylation compared with NC group,with the most significant increase of 97.5%(P<0.05,n=3)at 30 min,which also inhibited by berberine in a concentration-dependent manner,and the effect was the most significant at 200μmol/L,with a decrease of about 37.6%(P<0.05,n=3).Conclusion Berberine inhibits SS-induced vascular smooth muscle cell proliferation and migration by inhibiting PKCδphosphorylation.