摘要
探讨天麻钩藤饮对血管紧张素Ⅱ(AngⅡ)诱导血管平滑肌细胞(VSMC)氧化应激的影响及其分子机制。采用组织贴块法进行原代大鼠血管平滑肌细胞培养,α-actin免疫细胞荧光法鉴定VSMC,AngⅡ1×10^(-6) mol·L^(-1)作为刺激因子,天麻钩藤饮灌胃Sprague Dawley(SD)大鼠制备药物血清;将大鼠VSMC分为正常组、模型组、中药组和抑制剂(3-甲基腺嘌呤,3-MA)组;cell counting kit-8(CKK-8)法检测细胞增殖活性,溴脱氧尿苷(BrdU)流式细胞仪检测细胞周期,Transwell实验检测细胞迁移能力;酶联免疫吸附实验(ELISA)检测VSMC中超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和丙二醛(MDA)的活性;采用DCFH-DA荧光探针检测细胞内活性氧(ROS)荧光强度;采用蛋白免疫印迹(Western blot)检测VSMC中PTEN诱导假定激酶1(PTEN-induced putative kinase 1,PINK1)、帕金蛋白(Parkin)、p62及轻链3-Ⅱ(LC3-Ⅱ)蛋白表达。结果显示,天麻钩藤饮含药血清在8%质量分数下干预48 h后可显著抑制VSMC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,SOD、CAT含量显著降低,MDA含量显著升高,ROS荧光强度显著增强,PINK1、Parkin及LC3-Ⅱ蛋白表达显著下降,p62蛋白表达显著升高。与模型组比较,中药组VSMC增殖活性及迁移数量均明显降低,SOD、CAT含量显著增加,MDA含量显著下降,ROS荧光强度显著减弱,PINK1、Parkin及LC3-Ⅱ蛋白表达显著升高,p62蛋白表达显著下降。与中药组比较,加入线粒体自噬抑制剂3-MA可阻断天麻钩藤饮含药血清干预VSMC增殖迁移、线粒体自噬及氧化应激水平,差异具有统计学意义。综上所述,天麻钩藤饮具有良好的抗氧化活性,可抑制细胞增殖和迁移,其作用机制可能与激活线粒体自噬PINK1/Parkin信号通路有关。
This study explored the effect of Tianma Gouteng Decoction on oxidative stress induced by angiotensinⅡ(AngⅡ)in vascular smooth muscle cell(VSMC)and its molecular mechanism.Primary rat VSMC were cultured using tissue block method,and VSMC were identified byα-actin immunofluorescence staining.AngⅡat a concentration of 1×10^(-6)mol·L^(-1)was used as the stimulating factor,and Sprague Dawley(SD)rats were orally administered with Tianma Gouteng Decoction to prepare drug serum.Rat VSMC were divided into normal group,model group,Chinese medicine group,and inhibitor(3-methyladenine,3-MA)group.Cell counting kit-8(CCK-8)assay was used to detect cell proliferation activity.Bromodeoxyuridine(BrdU)flow cytometry was used to detect cell cycle.Transwell assay was used to detect cell migration ability.Enzyme-linked immunosorbent assay(ELISA)was used to detect the activity of superoxide dismutase(SOD),catalase(CAT),and malondialdehyde(MDA)in VSMC.The intracellular reactive oxygen species(ROS)fluorescence intensity was detected using DCFH-DA fluorescent probe.Western blot was used to detect the expression of PTEN-induced putative kinase 1(PINK1),Parkin,p62,and microtubule-associated protein 1A/1B-light chain 3(LC3-Ⅱ)proteins in VSMC.The results showed that Tianma Gouteng Decoction-containing serum at a concentration of 8%could significantly inhibit VSMC growth after 48 hours of intervention.Compared with the normal group,the model group showed significantly increased cell proliferation activity and migration,significantly decreased levels of SOD and CAT,significantly increased levels of MDA,significantly enhanced ROS fluorescence intensity,significantly decreased expression of PINK1,Parkin,and LC3-Ⅱproteins,and significantly increased expression of p62 protein.Compared with the model group,the Chinese medicine group showed significantly reduced cell proliferation activity and migration,significantly increased levels of SOD and CAT,significantly decreased levels of MDA,significantly weakened ROS fluorescence intensity,
作者
时拥月
符雁榕
秦钦
陈雨丝
姚佳梅
钟广伟
SHI Yong-yue;FU Yan-rong;QIN Qin;CHEN Yu-si;YAO Jia-mei;ZHONG Guang-wei(Department of Geriatric,Xiangya Hospital,Central South University,Changsha 410008,China;School of Medicine,Hunan Normal University,Changsha 410006,China;International Medical Center,Xiangya Hospital,Central South University,Changsha 410008,China;National Clinical Medical Research Center for Geriatric Diseases,Xiangya Hospital,Central South University,Changsha 410008,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2024年第14期3894-3900,共7页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(81673858)
湖南省自然科学基金项目(2023JJ30979)
湖南省中医药科研计划项目(C2022032)
湖南省生态环境科研项目(HBKYXM-2023017)。
