活血潜阳祛痰方抑制心肌成纤维细胞增殖的机制研究

Mechanism of Huoxue Qianyang Qutan Recipe in Inhibiting Cardiac Fibroblast Proliferation

目的观察活血潜阳祛痰方(HQQR)对血管紧张素Ⅱ(AngⅡ)诱导的心肌成纤维细胞(CFs)增殖的影响及可能机制。方法分离、培养SD大鼠原代CFs,并进行鉴定。应用AngⅡ诱导CFs建立体外心肌纤维化模型。将CFs分成空白细胞组、AngⅡ组、AngⅡ+HQQR(低剂量)组、AngⅡ+HQQR(高剂量)组和AngⅡ+缬沙坦组,作用48 h。应用CCK-8法检测各组细胞增殖水平,流式细胞仪检测各组细胞ROS水平,Elisa检测各组上清中羟脯氨酸水平,蛋白质免疫印迹(Western blot)检测NADPH氧化酶4(NOX4)、核因子κB p65(NF-κB p65),细胞外信号调节激酶1/2(ERK1/2)和p-ERK1/2的水平,对相关数据进行统计分析。结果HQQR可显著改善AngⅡ诱导CFs增殖。AngⅡ组上清中羟脯氨酸水平显著高于空白组(P<0.05),ROS水平和NOX4蛋白表达量亦显著升高(P<0.05)。AngⅡ刺激CFs后,细胞中NF-κB p65蛋白表达量和ERK1/2磷酸化比值高于空白细胞组(P<0.05)。HQQR治疗可以改善CFs增殖和胶原分泌,降低ROS水平,并显著减少NOX4、NF-κB p65蛋白表达量和ERK1/2磷酸化比值,差异有统计学意义(P<0.05)。结论HQQR可能通过抑制氧化应激、减轻炎症反应来抑制CFs增殖,改善心肌纤维化。

Objective To investigate the impact and underlying mechanisms of Huoxue Qianyang Qutan Recipe(HQQR)on the proliferation of angiotensinⅡ(AngⅡ)induced cardiac fibroblasts(CFs).Methods Primary CFs were isolated and cultured from SD rats and subsequently identified.AngⅡwas used to induce CFs and establish an in vitro cardiac fibrosis model.CFs were divided into a blank cell group,an AngⅡgroup,an AngⅡ+HQQR(low dose)group,an AngⅡ+HQQR(high dose)group,and an AngⅡ+losartan group,treated for 48 hours.The CCK-8 method was used to detect cell proliferation levels in each group,flow cytometry was used to detect ROS levels,ELISA was used to detect hydroxyproline levels in the supernatants of each group,and Western blot was used to detect the levels of NADPH oxidase 4(NOX4),nuclear factor kappa B p65(NF-κB p65),extracellular signal-regulated kinase 1/2(ERK1/2),and p-ERK1/2.Statistical analysis was conducted on the collected data.Results HQQR significantly ameliorated AngⅡ-in-duced CFs proliferation.The hydroxyproline levels in the supernatants of the AngⅡgroup were significantly higher than those in the blank group(P<0.05),with ROS levels and NOX4 protein expression also significantly elevated(P<0.05).After AngⅡstimulation of CFs,the expression of NF-kB p65 protein and the phosphorylation ratio of ERK1/2 in cells were higher than those in the blank cell group(P<0.05).HQQR treatment effectively improved CFs proliferation and collagen secretion,reduced ROS levels,and signifcantly decreased the expression of NOX4,NF-κB p65 protein,and the phosphorylation ratio of ERK1/2,with statistically significant differences(P<0.05).Conclusion HQQR may inhibit CFs proliferation and ameliorate myocardial fibrosis by inhibiting oxidative stress and reducing inflammatory responses.