氧化苦参碱对高糖诱导乳鼠原代心肌成纤维细胞转分化的作用及机制  

Effect and mechanism of oxymatrine on myocardial fibroblasts transdifferentiation of suckling mice induced by high glucose

作　　者：张宇菲 罗红[1,2] 肖红[1,2] 陶玲[1,2] 沈祥春[1,2] 常楚瑞[1,2] ZHANG Yufei;LUO Hong;XIAO Hong;TAO Ling;SHEN Xiangchun;CHANG Churui(Key Laboratory of Optimal Utilization of Natural Medicine Resources,School of Pharmaceutic Sciences,Guizhou Medical University,Guiyang 550025,Guizhou,China;The High Efficacy Application of Natural Medicinal Resources Engineering Center of Guizhou Province,Guizhou Medical University,Guiyang 550025,Guizhou,China)

机构地区：[1]贵州医科大学天然药物资源优效利用重点实验室,贵州贵阳550025 [2]贵州医科大学贵州省特色天然药物资源高效利用工程中心,贵州贵阳550025

出　　处：《贵州医科大学学报》2024年第3期329-339,共11页Journal of Guizhou Medical University

基　　金：国家自然科学基金(U1812403-4-4);贵州省科技创新基地国家自然科学基金(82060729);贵州省科技创新基地(黔科合中引地〔2023〕003);贵州省科技计划项目(黔科合基础-ZK〔2022〕338)。

摘　　要：目的探讨氧化苦参碱(OMT)对高糖(HG)诱导乳鼠原代心肌成纤维细胞(CFBs)增殖和转分化的作用及机制。方法Sprague-Dawley(SD)乳鼠20只,取乳鼠心脏的心尖部分分离与培养原代CFBs,取对数生长期CFBs细胞分为空白(Control)组、40 mmol/L甘露醇(Mannitol)组、不同浓度(30、35、40、45及50 mmol/L)HG组及不同浓度(25、50、100、200、400 mg/L)OMT组,采用噻唑蓝比色法(MTT)法检测细胞的增殖情况并确定后续实验OMT的保护浓度;按前述OMT的保护浓度结果,取对数生长期CFBs细胞分为空白(Control)组、40 mmol/L甘露醇(Mannitol)组、40 mmol/L HG(HG)组、40 mmol/L HG+50 mg/L OMT(OMT低剂量)组、40 mmol/L HG+100 mg/L OMT(OMT中剂量)组及40 mmol/LHG+200 mg/L OMT(OMT高剂量)组,采用天狼星红和苏木素-伊红(HE)染色法观察各组细胞的胶原纤维表达及形态变化,采用羟脯氨酸(Hyp)试剂盒检测法和免疫荧光染色法检测各组细胞Hyp及α-平滑肌肌动蛋白(α-SMA)的表达,采用流式细胞术检测各组细胞的周期分布比例,采用Western blot检测CFBs中α-SMA、结缔组织生长因子(CTGF)、Ⅰ型胶原(CollagenⅠ)、Ⅲ型胶原(CollagenⅢ)、血管内皮生长因子A(VEGFA)及缺氧诱导因子-1α(HIF-1α)蛋白的表达。结果与HG组相比,50、100及200 mg/L OMT组CFBs细胞活力下降(P<0.05);与HG组相比,50、100及200 mg/L OMT组CFBs细胞形态发生变化,细胞数量变少;与HG组相比,50、100及200 mg/L OMT组CFBs内Hyp含量减少,细胞在S期分布比例降低(P<0.05);与Control组相比,HG组CFBs细胞数量增多,α-SMA表达增多;与HG组相比,50、100及200 mg/L OMT组CFBs细胞数量减少,α-SMA表达减少;与HG组相比,50、100及200 mg/L OMT组HIF-1α、VEGFA、α-SMA、CTGF、CollagenⅠ、CollagenⅢ及FN表达下调(P<0.05)。结论OMT可抑制乳鼠CFBs增殖并诱导细胞转分化,其机制可能与调节HIF-1α信号相关。Objective To investigate the effect and mechanism of oxymatrine(OMT)on the proliferation and transdifferentiation of neonatal rat cardiac fibroblasts(CFBs)induced by high glucose(HG).Methods Twenty SD suckling mice were removed the apex of the heart,and the primary CFBs were isolated and cultured.The CFBs cells in logarithmic growth phase were divided into the control group,the 40 mmol/L mannitol group,HG groups with different concentrations(30,35,40,45,and 50 mmol/L)and OMT groups with different concentrations(25,50,100,200,and 400 mg/L).The optical density(OD)of the cells in each group was detected by methyl thiazol tetrazolium(MTT)method 24 h and 48 h after treatment,.The inhibition rate of cell proliferation was calculated.CFBs in logarithmic growth phase were treated with 50,100,and 200 mg/L OMT,respectively.The morphological structure of CFBs was observed by HE staining.Sirius red picric acid staining was used to observe the fiber deposition of cells.Hydroxyproline(Hyp)content in each group was detected by Hyp kit assay.The expression ofα-smooth muscle actin(α-SMA)was detected by immunofluorescence staining.The cycle distribution ratio of CFBs in each group was detected by flow cytometry.The expression levels of hypoxia-inducible factor-1α(HIF-1α),vascular endothelial growth factor A(VEGFA)and myocardial fibrosis-related proteinsα-smooth muscle actin(α-SMA),connective tissue growth factor(CTGF),typeⅠcollagen(CollagenⅠ),typeⅢcollagen(CollagenⅢ)and fibronectin(FN)in CFBs at 24 h were detected by Western blot.After CFBs were treated with HIF-1αinhibitor YC-1 for 24 h,the protein expression levels of HIF-1α,VEGFA,α-SMA,CTGF,CollagenⅠ,and CollagenⅢwere detected by Western blot.Results Compared with HG group,MTT assay showed that the cell viability of CFBs in 50,100,and 200 mg/L OMT groups decreased(P<0.05);HE staining and Sirius red picric acid staining revealed that the morphology of CFBs cells in 50,100 and 200 mg/L OMT groups changed and the number of CFBs cells decreased;the hydroxyproli

关 键 词：细胞增殖 糖尿病心肌病 缺氧诱导因子-1α 乳鼠 氧化苦参碱 高糖 心肌成纤维细胞 转分化 

分 类 号：R965[医药卫生—药理学] R972[医药卫生—药学]