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丹酚酸B抑制血管紧张素Ⅱ诱导心肌成纤维细胞增殖与分化的作用 被引量:5

Effect of Salvianolic Acid B in Inhibiting Angiotensin Ⅱ -induced Proliferation and Differentiation of Rat Cardiac Fibroblast
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摘要 目的:研究丹酚酸B(salvianolic acid B,Sal-B)对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导的新生SD大鼠心肌成纤维细胞(cardiac fibroblasts,CFs)增殖与分化的影响,探讨Sal-B是否具有对抗心肌纤维化的作用。方法:胰酶消化,差速贴壁分离纯化CFs,抗波形蛋白免疫细胞化学法鉴定CFs。建立AngⅡ诱导的CFs增殖分化模型。实验分为空白组(无血清DMEM),模型组(1×10^(-6)mol·L^(-1)AngⅡ),Sal-B低剂量组(2.5×10^(-5)mol·L^(-1)Sal-B+1×10^(-6)mol·L^(-1)AngⅡ)及Sal-B高剂量组(5×10^(-5)mol·L^(-1)Sal-B+1×10^-6mol·L^(-1)AngⅡ)共4组。Sal-B预保护1 h,加入AngⅡ共同作用24 h后,采用噻唑蓝(MTT)法分析Sal-B对AngⅡ诱导CFs增殖的细胞存活率,采用试剂盒(消化法)测定羟脯氨酸含量,蛋白质免疫印迹(Western blot)分析α-平滑肌肌动蛋白(α-SMA),Ⅰ型胶原(collagenⅠ,ColⅠ)的表达情况。结果:MTT结果显示,与空白组比较,模型组AngⅡ显著诱导CFs异常增殖(P〈0.01),与模型组比较,Sal-B低、高剂量显著抑制AngⅡ诱导的CFs增殖(P〈0.01);羟脯氨酸含量测定结果显示,与空白组比较,模型组羟脯氨酸含量显著升高(P〈0.01),与模型组比较,Sal-B低、高剂量组含量均显著降低(P〈0.01);Western blot结果显示,与空白组比较,模型组α-SMA,ColⅠ蛋白表达显著上调(P〈0.01),与模型组比较,Sal-B低、高剂量组α-SMA,ColⅠ蛋白表达明显下调(P〈0.05,P〈0.01)。结论:Sal-B可抑制AngⅡ诱导的CFs增殖,减少α-SMA,ColⅠ蛋白的表达,对AngⅡ体外诱导心肌纤维化进程具有抑制作用。 Objective: To investigate the inhibitory effect of salvianolic acid B (Sal-B) on proliferation and differentiation of cardiac fibroblasts (CFs) induced by angiotensin Ⅱ (Ang Ⅱ ) of neonatal Sprague Dawley rats, and clarify the anti-fibrotic mechanism of Sal-B in vitro. Method: Primary CFs were harvested from 1-3-day- old neonatal rats by 0.08% trypsin digesting, and then separated and purified by differential attachment. The CFs were identified by anti-vimentin immunocytochemistry. Proliferation and differentiation of the CFs model were reproduced by Ang U. The CFs was randomly divided into 4 groups as follows: normal group (serum-free DMEM) , model group (1 ×10^-6 mol.L-1Ang 11 ) , Sal-B low-dose group (2.5 × 10^-5 mol.L-1 Sal-B + 1 × 10^-6 mol.L-1Ang 11 ) and high-dose group (5 × 10^-2 mol.L-1Sal-B + 1 × 10^-6 mol.L-1Ang Ⅱ ). The CFs were pretreated with Sal-B for 1 h, and then co-cultured with Ang Ⅱ for 24 hours. The inhibition of Sal-B on CFs proliferation was measured by 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. The content of hydroxyproline was detected by commercial kit. The expressions of alpha-smooth muscle actin (α-SMA) and collagen Ⅰ (Col Ⅰ) were detected by Western blot. Result: The results of MTT assay suggested that compared with the normal group, Ang Ⅱ in model group significantly induced abnormal CF proliferation (P 〈 0. 01); Compared with model group, both low and high-dose Sal-B significantly inhibited Ang Ⅱ-induced CFs proliferation (P 〈 0.01 ). The content of hydroxyproline increased in model group (P 〈 0.01 ) compared with the normal group, but decreased (P 〈 0.01) after being co-cultured with Sal-B compared with model group. Western blot revealed that the expressions of α-SMA and Col I significantly increased in the model group (P 〈 0.01 ) , lowdose and high-dose Sal-B suppressed Ang Ⅱ-induced up-regulation of α-SMA and Col Ⅰ(P 〈 0. 05,
作者 黄海烽 王春花 赵玲璐 罗红 徐旖旎 陶玲 张敏 沈祥春 HUANG Hai-feng, WANG Chun-hua, ZHAO Ling-lu, LUO Hong, XU Yi-ni, TAO Ling, ZHANG Min , SHEN Xiang-chun ( Guizhou Medical University, Guiyang 550025, China)
机构地区 贵州医科大学
出处 《中国实验方剂学杂志》 CSCD 北大核心 2017年第13期128-132,共5页 China Journal of Experimental Traditional Medical Formulae
基金 国家自然科学基金项目(81560588) 贵州省科学技术研究重点项目(黔科合JZ字[2015]2002号) 教育部新世纪优秀人才支持计划项目(NCET-13-0747) 贵州省高等学校科技创新人才团队建设项目(黔教合人才团队字[2014]31) 贵州省高层次创新型人才百层次人才项目(黔科合人才[2015]4029号) 大学生创新创业项目(201410660017) 贵州省科技创新团队项目([2015]4025号)
关键词 血管紧张素Ⅱ 心肌纤维化 心肌成纤维细胞 丹酚酸B 增殖 分化 angiotensin Ⅱ myocardial fibrosis cardiac fibroblast salvianolic acid B proliferation differentiation
作者简介 黄海烽,在读硕士,从事天然药物药理学研究,Tel:18798846265,E-mail:615686558@qq.com [通讯作者]张敏,硕士,副教授,从事心血管系统药物药理研究,Tel:0851—88174007,E-mail:1071632869@qq.com; [通讯作者]沈祥春,博士,教授,从事心血管系统药物药理、功能天然产物化学生物学研究,Tel:0851-88416149,E-mail:shenxiangchun@126.com
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