Eupatilin调控血管新生内膜形成的作用和分子机制研究

Role and molecular mechanisms of Eupatilin in the regulation of neointima formation

目的探讨Eupatilin调控血管新生内膜形成的作用和分子机制。方法选择10~12周龄TSC1c/c SM22Cre+/-小鼠,体内实验予以行左侧颈动脉结扎制备左侧颈动脉狭窄小鼠模型后分为2组,每组8只,Sestrin2激动剂组每日予以10 mg/kg Eupatilin灌胃,对照组每日予以Sestrin2激动剂组等容量生理盐水灌胃,共持续28 d。Van Gieson染色检测左颈动脉新生内膜形成。体外实验分离和培养TSC1c/c SM22Cre+/-小鼠源性主动脉血管平滑肌细胞(AVSMCs)。予以不同终浓度Eupatilin干预AVSMCs24 h后,CCK-8检测AVSMCs增殖。予以终浓度为0、100μmol/L Eupatilin干预AVSMCs 24 h后,划痕实验检测AVSMCs迁移能力,蛋白印迹法检测AVSMCs中Sestrin2以及TSC2、S6K1、4E-BP1等分子及其磷酸化水平的表达。结果动物体内实验研究显示:与对照组相比,Eupatilin干预后TSC1c/c SM22Cre+/-小鼠的左颈动脉管腔面积增加(P<0.05)、内膜面积和I/M比值减少(P<0.05)。体外细胞实验研究显示:Eupatilin呈剂量依赖性地抑制AVSMCs的增殖,以100μmol/L Eupatilin的抑制效率最高(P<0.05)。与对照组相比,100μmol/L Eupatilin组AVSMCs迁移距离缩短(P<0.05),AVSMCs中Sestrin2、TSC2磷酸化水平表达升高,p-S6K1表达降低(P<0.05),4E-BP1及其磷酸化水平表达无明显变化(P>0.05)。结论 Eupatilin是抑制血管新生内膜形成以及AVSMCs的增殖和迁移的有效药物,与激活Sestrin2密切相关。

Objective To investigate the role of Eupatilin in regulation of neointima formation and its molecular mechanism.Methods TSC1 c/c SM22 Cre+/-mice aged 10-12 weeks were selected.The left carotid artery stenosis model was made by ligation of left carotid artery in vivo.The mice were divided into two groups.Sestrin2 agonist group(n=8 mice)was given 10 mg/kg Eupatilin intragastrically every day for 28 d.The control group(n=8 mice)was given equal volume saline with Sestrin2 agonist group intragastrically for 28 d.Van Gieson staining was used to detect neointimal formation in left carotid artery.Aortic vascular smooth muscle cells(AVSMCs)derived from TSC1c/c SM22Cre+/-mouse were isolated and cultured in vitro.After 24 h of Eupatilin intervention,the proliferation of AVSMCs was detected by CCK-8.The migration ability of AVSMCs was detected by scratch test,and the expression of Sestrin2,and TSC2,S6 K1,4 E-BP1 and their phosphorylation levels were detected by western blot.Results In vivo animal experiments showed that compared with the control group,the left carotid lumen area of TSC1 c/c SM22 Cre+/-mice after Eupatilin intervention was increased(P<0.05),the intima area and I/M ratio were decreased(P<0.05).In vitro cell experiments showed that Eupatilin inhibited the proliferation of AVSMCs in a dose-dependent manner,and 100μmol/L Eupatilin got the best inhibition efficiency(P<0.05).Compared with the control group,100μmol/L Eupatilin decreased the migration distance of AVSMCs(P<0.05),upregulated the expression of Sestrin2 and the phosphorylation levels of TSC2,and downregulated the expression of p-S6 K1 in AVSMCs(P<0.05),but had no effect on the expression of 4 E-BP1 and its phosphorylation levels(P>0.05).Conclusion Eupatilin is an effective drug to inhibit the neointima formation and the proliferation and migration of AVSMCs,which is closely related to the activation of Sestrin2.