摘要
探讨银杏内酯B(ginkgolide B,GB)对MH7A人成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS)的增殖抑制作用及其潜在机制。采用20μg/L肿瘤坏死因子-α(tumor necrosis factor-a,TNF-α)刺激MH7A构建关节炎细胞模型。经不同浓度GB作用于MH7A细胞后,CCK-8法检测细胞活力;Transwell实验检测细胞侵袭力;流式细胞术检测细胞凋亡率和细胞周期;实时荧光定量PCR(Real-time quantitative PCR,RT-qPCR)和蛋白免疫印迹分别检测基因转录和蛋白表达量。与对照组相比,GB对细胞活力的抑制作用呈现出一定的浓度和时间依赖性;GB显著抑制细胞侵袭力、增加细胞凋亡率和G_(0)/G_(1)期比例;GB显著上调细胞Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)和p21 mRNA和下降Bcl-2、髓系白血病1(myeloid cell leukemia 1,Mcl-1)、蛋白激酶B(protein kinase B,PKB;又称AKT)、磷脂酰肌醇-3激酶(phosphati-dylinositol 3-kinase,PI3K)、Cyclin D1和细胞周期调节蛋白激酶4(cyclin-dependent kinase 4,CDK4)mRNA转录水平;同时,GB显著上调Bax、p21和Cleaved-caspase 3蛋白和下调Bcl-2、Mcl-1、p-AKT、p-PI3K、Cyclin D1和CDK4蛋白表达量,且伴有p-PI3K/PI3K、p-AKT/AKT和Bcl-2/Bax比值的降低。综上,GB通过抑制PI3K/AKT信号通路,阻滞MH7A细胞G_(1)期向S期转化、抑制细胞活力和侵袭力,并诱导MH7A人成纤维样滑膜细胞凋亡。
To explore the inhibitory effect of ginkgolide B(GB)on MH7A human fibroblast-like synoviocytes(FLS)and its potential mechanism.Firstly,20μg/L tumor necrosis factor-α(TNF-α)was pretreated with MH7A to establish a cell model of arthritis.After incubation of MH7A cells with various concentrations of GB,CCK-8 assay,Transwell assay,and flow cytometry(FCM)were separately used to detect cell viability,cell invasion,and cell apoptosis rate and cell cycle;Real-time quantitative PCR and Western blot assay were performed to detect the apoptosis-and cycle-related gene transcriptions and protein expressions,respectively.The results showed that compared with the control group,GB dose-and time-dependently suppressed cell viability to a greater extent;GB significantly reduced cell invasive ability and increased cell apoptosis rate and proportion of G_(0)/G_(1) phase in MH7A cells,along with increased transcription levels of Bcl-2-associated X protein(Bax)and p21 mRNA and decreased transcription levels of Bcl-2,myeloid cell leukemia 1(Mcl-1),protein kinase B(PKB;AKT),IP3K,Cyclin D1 and cyclin-dependent kinase 4(CDK4)mRNA;GB remarkably increased expression levels of Bax,p21,and cleaved-Caspase 3 protein and decreased expression levels of Bcl-2,Mcl-1,p-AKT,p-PI3K,Cyclin D1,and CDK4 protein,with decreased ratios of p-PI3K/PI3K,p-AKT/AKT,and Bcl-2/Bax.In conclusion,GB blocks the G_(1)-to-S cell cycle transition,suppresses cell viability and cell invasion and induces cell apoptosis of MH7A human RA-FLS via suppressing the PI3K/AKT signaling pathway.
作者
刘璘琛
徐晓龑
孙春萌
俞济荣
施青
孙君君
逄丹丹
卫斐然
刘兴
LIU Linchen;XU Xiaoyan;SUN Chunmeng;YU Jirong;SHI Qing;SUN Junjun;PANG Dandan;WEIFeiran;LIU Xing(Department of Rheumatology,Southeast University,Zhongda Hospital,Nanjing 210009;Department of Pharmaceutics,School of Pharmacy,China Pharmaceutical University,Nanjing 210009;School of Medicine,Southeast University,Nanjing 210009,China)
出处
《中国药科大学学报》
北大核心
2025年第2期216-224,共9页
Journal of China Pharmaceutical University
基金
国家自然科学基金项目(No.22278442)。
作者简介
通信作者:刘兴,Tel:18066068585,E-mail:llc2430782663@163.com。
