Objective To investigate the effect of oxidative stress-induced CaMKⅡ activation in endothelial cells on atherosclerosis and its mechanism. Methods Human umbilical vein endothelial cells (HUVECs)were cultured in vitro and divided into the control group,H2O2group,H2O2+KN93 group,and H2O2+KN92 group. The ROS level,cell viability,migration ability,and adhesion ability were determined, and the function of endothelial cells was assessed. RT-PCR was used to determine the expression levels of CaMKⅡ,p-CaMKⅡ,ox-CaMKⅡ,notch-1,NF-κB-p65 and VCAM-1. Thirty 8-week-old male ApoE(-/-) mice were randomly divided into 3 groups:the high-fat diet group (n=10) received high-fat diet and intraperitoneal injection of DMSO;the high-fat diet+KN93 group (n=10) received high-fat diet and intraperitoneal injection of KN93(10 mg-1·kg-1·d-1);and the high-fat diet+KN92 group(n=10)received high-fat diet and intraperitoneal injection of KN92(10 mg-1·kg-1·d-1). The body weight and blood lipid level in each group were recorded. The oil Red O staining of the aorta was performed. The aortic root plaque area and aortic lipid deposition were compared between the groups. Results Compared with the H2O2+KN92 group and H2O2group,the endothelial cell viability,migration ability,and adhesion ability in the H2O2+KN93 group were all improved(P<0.05). The findings of RT-qRCR showed that the mRNA levels of notch-1 and VCAM-1 were down-regulated in the H2O2+KN93 group(P<0.05). The findings of Western blotting showed that KN93 treatment significantly decreased the protein expression levels of ox-CaMKⅡ,p-CaMKⅡ,notch-1,NF-κB-p65 and VCAM-1(P<0.05). The inhibition of notch-1 by DAPT did not affect the protein expression of CaMKⅡ,ox-CaMKⅡ,and p-CaMKⅡ. The aortic root plaque area and aortic lipid deposition in the KN93-treated ApoE-/-mice were significantly decreased compared with those in the control group and KN92 group[(25.15±1.04)% vs(41.60±1.05)% vs(41.83±1.17)%;12.70±0.89% vs(25.28± 1.77)% vs(25.57 ± 0.78)%],respectively. There were no statistically significant differences in the body weight and blood lipid levels between the groups (P>0.05). Conclusion KN93 may ameliorate the endothelial dysfunction through the CaMKⅡotch-1/NF-κB-p65/VCAM-1 pathway,and thereby delay the occurrence and development of atherosclerosis.%目的 探讨氧化应激诱导的内皮细胞钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)激活在动脉粥样硬化中的作用及机制.方法 体外培养的人脐静脉内皮细胞(HUVEC)CRL-1730,实验分4组:对照组、H2O2组、H2O2+KN93组和H2O2+KN92组,测定各组ROS水平、细胞活力、迁移能力、黏附能力,评价内皮细胞功能;RT-PCR测定notch-1、NF-κB、血管黏附分子1(VCAM-1)表达水平;Western Blotting测定CaMKⅡ、p-CaMKⅡ、ox-CaMKⅡ、notch-1、NF-κB-p65、VCAM-1蛋白表达水平.取30只8周龄雄性ApoE(-/-)小鼠,随机分成3组:高脂组(n=10),高脂饮食且腹腔注射DMSO;高脂+KN93组(n=10),高脂饮食且腹腔注射KN93(10 mg-1·kg-1·d-1);高脂+KN92组(n=10),高脂饮食且腹腔注射KN92(10 mg-1·kg-1·d-1).记录各组体质量及血脂水平,进行主动脉根部HE染色,主动脉大体油红O染色,比较各组主动脉根部的斑块面积及主动脉脂质沉积.结果 与H2O2组、H2O2+KN92组比较,H2O2+KN93组内皮细胞活力、迁移能力、黏附能力均有改善(P<0.05);RT-qRCR结果显示,H2O2+KN93组notch-1和VCAM-1 mRNA水平下调(P<0.05);Western Blotting结果显示,KN93处理明显降低了ox-CaMKⅡ、p-CaMKⅡ、notch-1、NF-κB-p65、VCAM-1蛋白表达水平(P<0.05).DAPT抑制notch-1未影响CaMKⅡ、ox-CaMKⅡ、p-CaMKⅡ蛋白表达.KN93处理后的ApoE-/-小鼠主动脉根部的斑块面积(25.15±1.04)%及主动脉脂质沉积(12.70±0.89)%较对照组[(41.60±1.05)%、(25.28±1.77)%]和KN92组[(41.83±1.17)%、(25.57±0.78)%]明显减少,而各组之间小鼠体质量及血脂水平差异无统计学意义(P>0.05).结论 KN93能通过CaMKⅡotch-1/NF-κB-p65/VCAM-1减弱内皮细胞功能紊乱,延缓动脉粥样硬化的发生发展.
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