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目的观察平肝潜阳方对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖和迁移的抑制作用及DNA甲基化水平的影响。方法采用组织贴块法培养VSMCs,制备平肝潜阳方含药血清。将培养细胞分为正常组(原代培育的VSMCs)、模型组、叶酸组及平肝潜阳方组(下称中药组)。以AngⅡ作为诱导复制VSMCs增殖及迁移模型,在AngⅡ诱导VSMCs培育24 h后,叶酸组加入40μg/mL叶酸干预48 h,中药组加入5%中药血清干预48 h。采用CCK-8(Cell Counting Kit)试剂盒绘制VSMCs生长曲线,MTT法测定VSMCs增殖活性,体外细胞划痕实验检测细胞迁移情况,以抗5-甲基胞嘧啶(5-mC)抗体进行免疫荧光检测细胞核中胞嘧啶甲基化水平;Real-time q-PCR方法检测DNA甲基转移酶1(DNA methyltransferase 1,DNMT1)mRNA表达水平。结果 AngⅡ在10-6mol/L浓度下诱导24 h后可促进VSMCs生长。与正常组比较,模型组细胞增殖活性及迁移数量明显增加,DNA甲基化水平明显降低(P<0.05,P<0.01);与模型组比较,叶酸组及中药组VSMCs生长、细胞增殖活性及迁移数量均明显降低,DNA甲基化水平升高(P<0.05,P<0.01);与正常组比较,模型组VSMCs DNMT1 mRNA表达水平降低(P<0.01);与模型组比较,叶酸组及中药组VSMCs DNMT1 mRNA表达明显增强(P<0.01)。结论平肝潜阳方可抑制AngⅡ诱导的VSMCs增殖和迁移,可导致基因组DNA高甲基化,其DNA甲基化水平改变可能与DNMT1表达有关。
Objective To observe the effects of Pinggan Qianyang Recipe on the proliferation and migration of vascular smooth muscle cells (VSMCs) induced by angiotensin Ⅱ (AngⅡ) and its effect on DNA methylation. Methods VSMCs were cultured by tissue patch method to prepare the drug-containing serum of Pinggan Qianyang. Cultured cells were divided into normal group (primary cultured VSMCs), model group, folic acid group and Pinggan Qianyang group (hereinafter referred to as TCM group). Angiotensin II was used to induce VSMC proliferation and migration. After 24 h incubation with AngⅡ, the folic acid group was treated with 40 μg / mL folic acid for 48 h, and the Chinese herbal medicine group was treated with 5% serum for 48 h. The growth curve of VSMCs was drawn by using CCK-8 (Cell Counting Kit) kit. The proliferation activity of VSMCs was determined by MTT assay. The migration of VSMCs was detected by in vitro cell scratch assay. The anti-5-methylcytosine (5-mC) The level of cytosine methylation in the nucleus was detected. The mRNA expression of DNA methyltransferase 1 (DNMT1) was detected by Real-time q-PCR. Results AngⅡ promoted the growth of VSMCs induced by 10-6 mol / L for 24 h. Compared with the normal group, the proliferation and migration of cells in the model group were significantly increased (P <0.05, P <0.01). Compared with the model group, the growth and proliferation activities of VSMCs in the folic acid and traditional Chinese medicine groups and (P <0.05, P <0.01). Compared with normal group, DNMT1 mRNA expression in model group decreased (P <0.01). Compared with model group, folic acid group and The expression of DNMT1 mRNA in Chinese medicine group VSMCs increased significantly (P <0.01). Conclusions Pinggan Qianyang can inhibit the proliferation and migration of VSMCs induced by AngⅡ, leading to hypermethylation of genomic DNA. The change of DNA methylation may be related to the expression of DNMT1.