摘要
目的探究薯蓣皂苷元(diosgenin,DG)对风湿性关节炎(rheumatoid arthritis,RA)滑膜成纤维细胞(fibroblast-like synoviocytes,FLS)凋亡及细胞周期的影响及机制。方法将FLS细胞分为对照组,10mg/L、20mg/L和40mg/LDG组,分别用0、10、20和40mg/L的DG处理细胞。MTT检测细胞活性,Hoechst染色检测细胞凋亡,流式细胞术检测细胞周期分布,免疫印迹检测Ki67、cleaved Caspase-3、Bcl-2、Bax、细胞周期蛋白B(cyclinB)、Cdc-2和p27的表达及通路蛋白磷酸化磷脂酰肌醇-3-激酶(phosphorylase-phosphatidyl inositol 3 kinase,p-PI3K)、PI3K、p-蛋白激酶B(p-Akt)、Akt和雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)的表达;IGF-1处理细胞,Hoechst染色检测细胞凋亡,流式细胞术检测细胞周期分布情况。结果与Control组比较,DG(10、20、40mg/L)组细胞活性和Ki67、Bcl-2表达水平显著降低,细胞凋亡率和cleavedCaspase-3、Bax的表达水平明显升高;同时,DG(10、20、40mg/L)组细胞G2/M期与Control组比较显著延长、CyclinB和Cdc2蛋白表达水平明显升高,p27表达明显减少;此外,DG(20、40mg/L)组p-PI3K/PI3K和p-Akt/Akt的比值及mTOR表达水平与Control组比较显著降低。IGF-1能显著减弱DG(40mg/L)对细胞凋亡及细胞G2/M周期阻滞的诱导作用。结论DG可通过抑制PI3K/Akt信号通路激活诱导人FLS细胞凋亡和G2/M周期阻滞。
This study was performed to investigate the effects of diosgenin (DG) on cell apoptosis and cell cycle of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA).FLS cells were divided into four groups: Control,DG (10 mg/L,20 mg/L,40 mg/L) groups and treated by 0,10,20 and 40 mg/L DG,respectively.MTT assay was performed for cell viability;Hoechst staining was used to detect the cell apoptosis,while flow cytometry was used for cell cycle analysis.The proteins of cell growth/cycle,apoptosis and pathway related were determined by Western blotting.Finally,cells were treated with IGF-1,then Hoechst staining and flow cytometry were performed for cell apoptosis and distribution of cell cycle.Compared with control group,the DG (10,20,40 mg/L) groups demonstrated lower levels of cell viability,Ki67 expression and Bcl-2 expression,while higher levels of cell apoptosis,cleaved Caspase-3 expression,and Bax expression.Meanwhile,the G2 / M phase of cells was prolonged,the expression levels of Cyclin B and Cdc2 were up-regulated,while the p27 was down-regulated in DG (10,20,40 mg/L) groups;the ratio of p-PI3K/ PI3K to p-Akt/Akt,and mTOR expression were decreased notably in DG (20,40 mg/L) groups.Furthermore,IGF-1 alleviated the effects of DG (40 mg/L) on cell apoptosis and cell cycle arrest.Taken together,DG can induce apoptosis and G2/M phase arrest in FLS cells through inhibiting activation of PI3K/Akt signaling pathway.
作者
冯要菊
王中晓
杨晶
FENG Yaoju;WANG Zhongxiao;YANG Jing(Department of Nephropathy and Rheumatology,First Affiliated Hospital of Nanyang Medical College,Nanyang 473000,China;Department of Basic Medicine,Nanyang Medical College,Nanyang 473000,China;Departmentof Pharmacy,First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)
出处
《免疫学杂志》
CAS
CSCD
北大核心
2019年第9期752-759,共8页
Immunological Journal
基金
河南省医学科技攻关计划(201404163)
关键词
滑膜成纤维细胞
细胞凋亡
周期阻滞
Fibroblast-like synoviocytes
Apoptosis
Cell cycle arrest