益母草碱调节RhoA/ROCK信号通路对冠心病大鼠主动脉内皮细胞损伤的影响

Effects of Leonurine on Aortic Endothelial Cell Injury in Rats with Coronary Heart Disease by Regulating the RhoA/ROCK Signaling Pathway

目的:研究益母草碱(Leo)通过调节Ras同源基因家族成员A(RhoA)/Rho相关的卷曲螺旋激酶(ROCK)信号通路对冠心病(CHD)大鼠主动脉内皮细胞损伤的影响及机制。方法:将造模成功的75只CHD大鼠随机分为模型组(CHD组)、Leo低、中、高剂量组(Leo-L组、Leo-M组、Leo-H组)和Leo高剂量组+RhoA激活剂LPA组(Leo-H+LPA组),每组15只。Leo-L、Leo-M、Leo-H组分别灌胃15、30、60mg·kg^(-1)·d^(-1)的Leo,Leo-H+LPA组灌胃60mg·kg^(-1)·d^(-1)的Leo,另需腹腔注射1mg·kg^(-1)·d^(-1)的LPA,Control组和CHD组以相同方式给予等量生理盐水,均连续干预4周。超声心动图测定心功能指标;全自动生化分析仪测定总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)水平;酶联免疫吸附测定(ELISA)检测白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)、一氧化氮(NO)、内皮素1(ET-1)、内皮细胞特异性分子-1(EMS1)、血管内皮细胞粘附分子1(VCAM-1)水平;苏木精-伊红(HE)染色观察主动脉内皮损伤;流式细胞仪检测主动脉内皮细胞凋亡;免疫印迹法(Western blot)检测RhoA/ROCK信号通路相关蛋白表达。结果:与Control组相比,CHD组主动脉壁增厚且染色不均匀,细胞膨大,排列紊乱,内皮粗糙;左室舒张末期内径(LVEDD)、左室收缩末期内径(LVESD)、TC、TG、LDL-C、IL-6、TNF-α、ET-1、VCAM-1和EMS1水平、细胞凋亡率及RhoA、ROCK1和ROCK2蛋白表达显著升高(P<0.05),左室射血分数(LVEF)、缩短分数(FS)、HDL-C、NO水平显著降低(P<0.05)。与CHD组相比,Leo-L组、Leo-M组、Leo-H组主动脉染色均匀,动脉壁结构清晰,细胞排列紧密且形态较正常,内膜较平滑;LVEDD、LVESD、TC、TG、LDL-C、IL-6、TNF-α、ET-1、VCAM-1和EMS1水平、细胞凋亡率及RhoA、ROCK1和ROCK2蛋白表达逐渐降低(P<0.05),LVEF、FS、HDL-C、NO水平逐渐升高(P<0.05)。Leo-H+LPA组逆转了Leo对CHD大鼠的保护作用(P<0.05)。结论:Leo可能通过抑制RhoA/ROCK信号通路改善CHD大鼠心功能,降低血脂,抑制炎症,进而减轻CHD大鼠主动脉内皮细胞损伤。

Objective:To investigate the effect and mechanism of leonurine(Leo)on aortic endothelial cell injury in rats with coronary heart disease(CHD)by regulating the Ras homolog gene family member A(RhoA)/Rho-associated coiled-coil containing protein kinase(ROCK)signaling pathway.Methods:Seventy-five successfully modeled CHD rats were randomly divided into five groups:model group(CHD group),low,medium,and high dose Leo groups(Leo-L,Leo-M,Leo-H groups),and Leo-H dose group combined with RhoA activator LPA group(Leo-H+LPA group),with 15 rats in each group.The Leo-L,Leo-M,and Leo-H groups were gavaged with 15,30,and 60mg·kg^(-1)·d^(-1)of Leo,respectively.The Leo-H+LPA group was gavaged with 60mg·kg^(-1)·d^(-1)of Leo and intraperitoneally injected with 1mg·kg^(-1)·d^(-1)of LPA.The Control and CHD groups were administered equal volumes of normal saline in the same manner for four weeks.Cardiac function indicators were measured by echocardiography.Total cholesterol(TC),triglycerides(TG),high-density lipoprotein cholesterol(HDL-C),and low-density lipoprotein cholesterol(LDL-C)levels were measured using an automatic biochemical analyzer.Enzyme-linked immunosorbent assay(ELISA)was used to detect levels of interleukin-6(IL-6),tumor necrosis factor-alpha(TNF-α),nitric oxide(NO),endothelin-1(ET-1),endothelial cell-specific molecule-1(EMS1),and vascular cell adhesion molecule-1(VCAM-1).Hematoxylin and eosin(HE)staining was used to observe aortic endothelial damage.Flow cytometry was used to detect apoptosis of aortic endothelial cells.Western blot analysis was used to detect the expression of proteins related to the RhoA/ROCK signaling pathway.Results:Compared with the Control group,the CHD group showed significant aortic wall thickening,uneven staining,cell swelling,disordered arrangement,and rough endothelium;significant increases in left ventricular end-diastolic diameter(LVEDD),left ventricular end-systolic diameter(LVESD),TC,TG,LDL-C,IL-6,TNF-α,ET-1,VCAM-1,EMS1 levels,cell apoptosis rate,and RhoA,ROCK1,and ROCK2 protein expression(P<0.05);and significant decreases in left ventricular ejection fraction(LVEF),fractional shortening(FS),HDL-C,and NO levels(P<0.05).Compared with the CHD group,the Leo-L,Leo-M,and Leo-H groups showed uniform aortic staining,clear arterial wall structure,tightly arranged and morphologically normal cells,and smoother endothelium;significant decreases in LVEDD,LVESD,TC,TG,LDL-C,IL-6,TNF-α,ET-1,VCAM-1,EMS1 levels,cell apoptosis rate,and RhoA,ROCK1,and ROCK2 protein expression(P<0.05);and signif-icant increases in LVEF,FS,HDL-C,and NO levels(P<0.05).The Leo-H+LPA group reversed the pro-tective effects of Leo on CHD rats(P<0.05).Conclusion:Leonurine may improve cardiac function,reduce blood lipids,and inhibit inflammation in CHD rats by inhibiting the RhoA/ROCK signaling pathway,thereby alleviating aortic endothelial cell injury.