连翘苷调节相关信号通路对地塞米松诱导的成骨细胞自噬和凋亡的影响  

Effects of phillyrin on dexamethasone-induced autophagy and apoptosis of osteoblasts by regulating PI3K/AKT/mTOR signal pathway

作　　者：周凡[1] 高扬[1] 胡艳平[1] 向超 熊和然 周茹 ZHOU Fan;GAO Yang;HU Yanping;XIANG Chao;XIONG He′ran;ZHOU Ru(Department of Massage,Wuhan Hospital of Traditional Chinese Medicine,Wuhan 430000;Department of Encephalopathy,Wuhan Traditional Chinese Medicine Hospital,Wuhan 430000,China)

机构地区：[1]武汉市中医医院推拿科,湖北武汉430000 [2]武汉市中医医院脑病科,湖北武汉430000

出　　处：《中国骨质疏松杂志》2023年第7期976-981,共6页Chinese Journal of Osteoporosis

基　　金：武汉市医学科研项目(WZ21C09)。

摘　　要：目的 探讨连翘苷(phillyrin, PHN)调节磷脂酰肌醇3-激酶(PI3K)/丝氨酸苏氨酸激酶(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对地塞米松(dexamethasone, DEX)诱导的成骨细胞自噬和凋亡的影响。方法 将MC3T3-E1细胞分为对照组(NOR组)、DEX组(10μmol/L DEX处理MC3T3-E1细胞)、L-PHN组(5μmol/L PHN处理MC3T3-E1细胞)、M-PHN组(10μmol/L PHN处理MC3T3-E1细胞)、H-PHN组(20μmol/L PHN处理MC3T3-E1细胞)、ZSTK474组(用20μmol/L PHN和2μmol/L的PI3K/AKT/mTOR信号通路抑制剂ZSTK474处理MC3T3-E1细胞);MTT法检测细胞毒性和细胞活力;流式细胞术检测MC3T3-E1细胞凋亡;透射电子显微镜(TEM)观察自噬小体;Western blot法检测MC3T3-E1细胞中自噬、凋亡和PI3K/AKT/mTOR通路相关蛋白表达;ALP活性及ALP染色检测MC3T3-E1细胞分化能力。结果 0~80μmol/L PHN对MC3T3-E1细胞无明显毒性影响。与NOR组相比,DEX组OD570值、Bcl-2蛋白水平、p-PI3K/PI3K、p-AKT/AKT、mTOR蛋白水平、ALP活性、Beclin1、LC3-Ⅱ/Ⅰ蛋白水平以及自噬小体数目显著降低(P<0.05),凋亡率、Bax、cleaved-Caspase-3显著升高(P<0.05),与DEX组相比,L-PHN组、M-PHN组、H-PHN组凋亡水平显著降低(P<0.05),增殖活性、自噬水平、成骨分化能力、通路蛋白水平显著升高(P<0.05);而ZSTK474消除了PHN对MC3T3-E1细胞的有利作用。结论 PHN可能通过激活PI3K/AKT/mTOR信号通路促进成骨细胞自噬,进而抑制其凋亡。Objective To investigate the effects of phillyrin(PHN)on dexamethasone(DEX)-induced autophagy and apoptosis of osteoblasts by regulating phosphatidylinositol 3-kinase(PI3K)/serine threonine kinase(AKT)/mammalian target of rapamycin(mTOR)signaling pathway.Methods MC3T3-E1 cells were divided into control group(NOR group),DEX group(10μmol/L DEX treated MC3T3-E1 cells),L-PHN group(5μmol/L PHN treated MC3T3-E1 cells),M-PHN group(10μmol/L PHN treated MC3T3-E1 cells),H-PHN group(20μmol/L PHN treated MC3T3-E1 cells),and ZSTK474 group(treated with 20μmol/L PHN and 2μmol/L ZSTK474,an inhibitor of PI3K/AKT/mTOR signaling pathway).MTT assay was used to detect cytotoxicity and cell viability.The apoptosis of MC3T3-E1 cells was detected with flow cytometry.The autophagosomes were observed with transmission electron microscopy(TEM).Western blotting was used to detect the expression of autophagy,apoptosis,and PI3K/AKT/mTOR pathway related proteins in MC3T3-E1 cells.ALP activity and ALP staining were used to detect the differentiation ability of MC3T3-E1 cells.Results 0-80μmol/L PHN had no obvious toxic effect on MC3T3-E1 cells.Compared to those in NOR group,the OD 570 value,Bcl-2 protein level,p-PI3K/PI3K,p-AKT/AKT,mTOR protein levels,ALP activity,Beclin1,LC3-II/I protein levels,and the number of autophagosomes in DEX group decreased significantly(P<0.05),and the apoptosis rate,Bax,and cleaved Casase-3 increased significantly(P<0.05).Compared to that in DEX group,the level of apoptosis in L-PHN group,M-PHN group,and H-PHN group decreased significantly(P<0.05),and the proliferation activity,autophagy level,osteogenic differentiation ability and pathway protein levels increased significantly(P<0.05).ZSTK474 eliminated the beneficial effects of PHN on MC3T3-E1 cells.Conclusion PHN promotes autophagy of osteoblasts by activating PI3K/AKT/mTOR signaling pathway,and then inhibits apoptosis of osteoblasts.

关 键 词：连翘苷 PI3K/AKT/mTOR信号通路 地塞米松 成骨细胞 自噬 凋亡 

分 类 号：R-332[医药卫生] R965