摘要
目的探讨薯蓣皂苷(Dioscin)通过调控腺苷酸活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)自噬信号通路对人骨肉瘤MG63细胞增殖及侵袭的影响。方法取对数期生长人骨肉瘤MG63细胞分为空白对照组、Dioscin不同剂量组(20、40、80、100μmol/L)。CCK-8法检测细胞增殖能力,划痕实验检测细胞迁移能力,细胞迁移实验检测细胞侵袭能力,丹酰尸胺法观察细胞自噬情况,蛋白质印迹法检测磷酸化腺苷酸活化蛋白激酶(p-AMPK)/AMPK、磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)/mTOR、转录因子EB(TFEB)、人微管相关蛋白轻链3Ⅱ(LC3-Ⅱ)、自噬效应蛋白beclin-1表达水平。结果与空白对照组比较,Dioscin20、40、80、100μmol/L组MG63细胞增殖率[(65.21±4.09)%、(51.94±3.95)%、(46.57±3.72)%、(37.31±4.23)%比100.00%]、划痕愈合率[(85.43±3.84)%、(70.62±3.17)%、(55.83±2.92)%、(44.23±3.27)%比(98.25±1.23)%]、侵袭细胞比率[(72.43±2.83)%、(63.76±3.34)%、(51.92±3.13)%、(42.44±3.09)%比(91.14±2.91)%]依次降低,呈浓度依赖性(P<0.05);空白对照组中仅有少量细胞自噬泡,作用48 h后Dioscin各组自噬泡依次增多;与空白对照组比较,Dioscin20、40、80、100μmol/L组MG63细胞中p-AMPK/AMPK[(0.35±0.04)、(0.50±0.02)、(0.60±0.03)、(0.88±0.03)比(0.20±0.02)]、TFEB[(0.42±0.02)、(0.68±0.03)、(0.92±0.04)、(1.36±0.10)比(0.30±0.02)]、LC3-Ⅱ[(0.41±0.03)、(0.70±0.04)、(0.88±0.03)、(1.17±0.07)比(0.26±0.02)]、beclin-1[(0.32±0.03)、(0.50±0.04)、(0.68±0.05)、(0.75±0.06)比(0.16±0.02)]蛋白表达依次增加,p-mTOR/mTOR[(0.75±0.02)、(0.52±0.03)、(0.40±0.04)、(0.31±0.02)比(0.86±0.05)]蛋白表达依次降低,差异均具有统计学意义(P<0.05);与Dioscin(100μmol/L)组比较,Dioscin(100μmol/L)+AMPK抑制剂(Compound C 10μmol/L)组p-AMPK/AMPK[(0.49±0.03)比(0.63±0.04)]、TFEB[(0.16±0.03)比(0.24±0.02)]、LC3-Ⅱ[(0.35±0.02)比(0.44±0.04)]、beclin-1[(0.44±0.03)比(0.56±0.04)]蛋白表达�
Objective To investigate the effect of dioscin on the proliferation and invasion of human osteosarcoma MG63 cells by modulating the adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)autophagy signaling pathway.Methods Osteosarcoma MG63 cells in the logarithmic growth phase were divided into a blank control group and different doses of the dioscin groups(20,40,80,100μmol/L).Cell proliferation ability was assessed using CCK-8 assay,cell migration ability was evaluated using scratch assay,cell invasion ability was examined using cell migration assay,cell autophagy was observed using monodansylcadaverine staining,and protein expression levels of phosphorylated AMPK(p-AMPK)/AMPK,phosphorylated mTOR(p-mTOR)/mTOR,transcription factor EB(TFEB),the microtubule-associated protein light chain 3Ⅱ(LC3-Ⅱ),and the autophagy-related protein beclin-1 were detected using Western blot analysis.Results Compared to the blank control group,the proliferation rates of MG63 cells in 20,40,60,80μmol/L groups of dioscin[(65.21±4.09)%,(51.94±3.95)%,(46.57±3.72)%,(37.31±4.23)%vs.100.00%],the scratch healing rates[(85.43±3.84)%,(70.62±3.17)%,(55.83±2.92)%,(44.23±3.27)%vs.(98.25±1.23)%],and the ratios of invading cells[(72.43±2.83)%,(63.76±3.34)%,(51.92±3.13)%,(42.44±3.09)%vs.(91.14±2.91)%]were decreased in a dose-dependent manner(P<0.05).In the blank control group,only a small number of autophagic vacuoles were observed.After 48 h of treatment,the number of autophagic vacuoles in each dioscin group increased sequentially.Compared to the blank control group,the protein expression levels of p-AMPK/AMPK[(0.35±0.04),(0.50±0.02),(0.60±0.03),(0.88±0.03)vs.(0.20±0.02)],TFEB[(0.42±0.02),(0.68±0.03),(0.92±0.04),(1.36±0.10)vs.(0.30±0.02)],LC3-Ⅱ[(0.41±0.03),(0.70±0.04),(0.88±0.03),(1.17±0.07)vs.(0.26±0.02)],and beclin-1[(0.32±0.03),(0.50±0.04),(0.68±0.05),(0.75±0.06)vs.(0.16±0.02)]of MG63 cells in 20,40,60,80μmol/L groups of dioscin were increased sequentially,while the p
作者
吴良邦
葛云林
WU Liang-bang;GE Yun-lin(The Third Department of Orthopedics,903 Hospital,Joint Logistics Support Force of Chinese People's Liberation Army,Hangzhou,Zhejiang 311243,China)
出处
《浙江中西医结合杂志》
2024年第4期304-309,316,共7页
Zhejiang Journal of Integrated Traditional Chinese and Western Medicine
基金
浙江省教育厅一般科研项目(No.Y202249274)。
