淫羊藿苷调控LAP自噬促进酒精抑制的MC3T3-E1细胞成骨分化  

Icariin promotes alcohol-inhibited osteogenic differentiation of MC3T3-1-E1 cells by regulating LAP autophagy

作　　者：曾麒 陈跃平[2] 宋世雷 赖渝 吴华华 ZENG Qi;CHEN Yue-ping;SONG Shi-lei;LAI Yu;WU Hua-hua(Graduate School,Guangxi University of Chinese Medicine,Nanning 530000,China;Traumatic Orthopedics and Hand Surgery,Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine,Nanning 530000,China)

机构地区：[1]广西中医药大学研究生院,广西南宁530000 [2]广西中医药大学附属瑞康医院创伤骨科与手外科,广西南宁530000

出　　处：《中国中药杂志》2025年第3期590-599,共10页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81960803);广西中医药大学研究生教育创新计划项目(YCBXJ2023025)。

摘　　要：研究细胞自噬在MC3T3-E1细胞成骨诱导(生理)、酒精(alcohol,AL)干预(病理)分化过程中的影响机制,以及淫羊藿苷(icariin,ICA)对AL干预病理状态下MC3T3-E1细胞成骨分化的作用机制。成骨矿化结节染色鉴定该细胞可分化为成骨细胞。CCK-8实验筛选合适的AL和ICA浓度后,实验分为7组:完全培养基(complete medium,CM)组,成骨诱导培养基(osteogenic induction medium,OIM)组,OIM+0.25 mol·L^(-1)AL组,OIM+0.25 mol·L^(-1)AL+1×10^(-8)mol·L^(-1)ICA组,OIM+0.25 mol·L^(-1)AL+1×10^(-7)mol·L^(-1)ICA组,OIM+0.25 mol·L^(-1)AL+1×10^(-6)mol·L^(-1)ICA组,OIM+0.25 mol·L^(-1)AL+1×10^(-5)mol·L^(-1)ICA组,培养7 d。碱性磷酸酶(alkaline phosphatase,ALP)染色检测ALP相对面积,Western blot、RT-qPCR检测成骨、自噬相关蛋白及mRNA表达情况,活性氧(reactive oxygen species,ROS)染色检测ROS水平,细胞凋亡线粒体膜电位实验检测细胞凋亡情况。结果显示,ICA可上调AL干预后下调的ALP相对面积;AL下调成骨相关蛋白Wnt家族成员1(Wnt family member 1 gene,Wnt1)及成骨相关基因Wnt1、β-连环蛋白(β-catenin)、核心结合因子2(Runt-related transcription factor 2,Runx2)、骨保护素(osteoprotegerin,OPG)、ALP的表达水平,抑制细胞成骨分化,ICA可上调AL抑制的成骨相关蛋白及mRNA的表达水平,促进细胞成骨分化;AL抑制典型自噬,ICA调控Rubicon抑制LC3相关吞噬作用(LC3-associated phagocytosis,LAP)促进典型自噬;ICA下调AL诱导后上升的ROS水平;ICA下调AL干预后导致的成骨细胞凋亡。综上,ICA可调控Rubicon抑制LAP促进典型自噬,清除ROS减少细胞凋亡,最终通过调控Wnt/β-catenin信号通路促进AL干预病理状态下MC3T3-E1细胞的成骨分化。This study investigated the mechanism of autophagy in the differentiation processes of MC3T3-E1 cells under osteogenic induction(physiological) and alcohol(AL) intervention(pathological), as well as the mechanism by which icariin(ICA) affected osteogenic differentiation of MC3T3-E1 cells under the pathological condition of AL intervention. Osteogenic mineralized nodule staining confirmed that the cells could differentiate into osteoblasts. After determining the appropriate concentrations of AL and ICA using the CCK-8 assay, seven groups were set up in this study: complete medium(CM) group, osteogenic induction medium(OIM) group, OIM+0.25 mol·L^(-1) AL group, OIM+0.25 mol·L^(-1) AL+1×10^(-8) mol·L^(-1) ICA group, OIM+0.25 mol·L^(-1) AL+1×10^(-7) mol·L^(-1) ICA group, OIM+0.25 mol·L^(-1) AL+1×10^(-6) mol·L^(-1) ICA group, and OIM+0.25 mol·L^(-1) AL+1×10^(-5) mol·L^(-1) ICA group, with a culture period of 7 days. Alkaline phosphatase(ALP) staining was used to detect the relative ALP area. Western blot and RT-qPCR were employed to analyze the expression of osteogenesis-and autophagy-related proteins and mRNAs. Reactive oxygen species(ROS) staining was used to detect ROS levels, and apoptosis was assessed through mitochondrial membrane potential assays. The results showed that ICA increased the relative ALP area that had been reduced by AL intervention. AL down-regulated the expression levels of Wnt family member 1(Wnt1), along with the osteogenesis-related mRNAs Wnt1, β-catenin, Runt-related transcription factor 2(Runx2), osteoprotegerin(OPG), and ALP, thereby inhibiting osteogenic differentiation. ICA up-regulated the expression levels of the osteogenesis-related proteins and mRNAs that had been inhibited by AL, promoting osteogenic differentiation. AL inhibited typical autophagy, while ICA regulated Rubicon to suppress LC3-associated phagocytosis(LAP) and promote typical autophagy. ICA also reduced the ROS levels that were elevated by AL and decreased the apoptosis of osteoblasts induced by AL interve

关 键 词：淫羊藿苷 酒精 成骨细胞 MC3T3-E1细胞 Wnt1/β-catenin通路 成骨分化 骨代谢失衡 自噬 LAP 

分 类 号：R285[医药卫生—中药学]