三甲胺与甲萘醌联用对VSMCs增殖及HUVECs内胆固醇含量的影响

Effects of trimethylamine combined with menadione on proliferation and migration of VSMCs and cholesterol content in HUVECs

目的探究三甲胺(trimethylamine,TMA)与甲萘醌单独用药及联合用药对血管平滑肌细胞(vascular smooth muscle cells,VSMC)的增殖、迁移能力及人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)内胆固醇含量的影响。方法首先采用MTT法检测不同浓度TMA对VSMC及HUVEC增殖的影响;划痕实验和Transwell检测TMA与甲萘醌单独用药及联合用药对VSMC迁移能力的影响;DCFH-DA荧光探针法检测VSMC内活性氧(reactive oxygen species,ROS)水平;Western blot法检测VSMC中MMP9、PCNA、SM22α的蛋白水平;ELISA法检测HUVEC内总胆固醇(cholesterol,TC)、高密度脂蛋白胆固醇(high-density lipoprotein cholesterol,HDL-C)和低密度脂蛋白胆固醇(low-density lipoprotein cholesterol,LDL-C)的含量。结果不同浓度的TMA对VSMC及HUVEC的生长并无明显的影响。与单独用药相比,TMA与低剂量的甲萘醌联合用药可明显促进VSMC迁移与增殖,提高胞内ROS水平,上调VSMC内MMP9及增殖型标志基因PCNA的蛋白表达,同时下调VSMC内分化型标志基因SM22α的蛋白表达。与TMA和低剂量的甲萘醌联合用药组相比,N-乙酰-L-半胱氨酸(N-acetyl-L-cysteine,NAC)、TMA和甲萘醌联合处理组可明显降低VSMC内ROS水平,下调VSMC内MMP9及PCNA的蛋白表达,上调VSMC内SM22α的蛋白表达。TMA与低剂量的甲萘醌联合用药还可以明显提高HUVEC内TC、HDL-C及LDL-C的含量。结论TMA自身对于VSMC和HUVEC并无显著影响,加入甲萘醌后,联合用药会诱导VSMC表型转化,提高HUVEC内胆固醇含量。同时NAC可缓解TMA与低剂量甲萘醌联合用药对VSMC造成的影响。提示联合用药可能通过影响ROS水平增加动脉粥样硬化发生的风险。

Objective To investigate the effects of trimethylamine(TMA)or/and menadione on the proliferation and migration of vascular smooth muscle cells(VSMCs)and the content of cholesterol in Human umbilical vein endothelial cells(HUVECs).Methods The cell viabilities of VSMCs and HUVECs treated with different concentrations of TMA were assessed by MTT;The effects of TMA,menadione or combinations of both on the cell migration ability of VSMCs were detected by wound healing;The level of reactive oxygen species(ROS)in VSMCs was detected by DCFH-DA fluorescence probe;The protein levels of MMP9,PCNA,SM22αin VSMCs were detected by Western blotting;The contents of total cholesterol(TC),high density lipoprotein cholesterol(HDL-C)and low density lipoprotein cholesterol(LDL-C)in HUVECs were detected by ELISA.Results Different concentrations of TMA had no significant impact on the viability of VSMCs and HUVECs.Compared to the usage of TMA or menadione alone in VSMCs,the cell migration and growth rate,the level of intracellular ROS,the protein levels of MMP9 and proliferative marker gene PCNA exhibited the markedly increase in the combination group(TMA+menadione).The protein level of differentiation marker gene SM22αwas significantly decreased following treatment with TMA plus menadione comparing to that of TMA or menadione treated alone.Compared to the combination group(TMA+menadione),NAC,TMA and menadione significantly reduced the level of ROS,decreased the protein levels of MMP9 and PCNA and markedly increased the protein level of SM22αin VSMC.The contents of TC,HDL-C and LDL-C were elevated in the combination group,compared to the single treatment group.Conclusion The TMA alone has no significant effect on VSMCs and HUVECs.After the combination with menadione,the phenotypic transformation of VSMCs is induced and the content of cholesterol is increased in HUVECs.At the same time,NAC can alleviate the effects of TMA combined with menadione on VSMCs.The results suggest that the combination of TMA and menadione may increase the risk of atherosclerosis by influencing the ROS pathway.