淫羊藿苷通过PI3K/AKT信号通路促进MC3T3-E1细胞的增殖分化  

Effect of icariin on the proliferation and differentiation of MC3T3-E1 cells via PI3K/AKT signalling pathway

作　　者：范凯健 钮艾雯 吴辉辉[2] FAN Kai-jian;NIU Ai-wen;WU Hui-hui(Department of Pharmacy,Mental Health Center,Chongming District,Shanghai 202150;Department of Rheumatology,Shuguang Hospital Attachedto Shanghai University of Traditional Chinese Medical,Shanghai 201203)

机构地区：[1]上海市崇明区精神卫生中心药剂科,上海202150 [2]上海中医药大学附属曙光医院风湿科,上海201203

出　　处：《中南药学》2025年第2期417-422,共6页Central South Pharmacy

基　　金：上海市崇明区科委科技项目(No.CKY2020-15,No.CKY2024-63)。

摘　　要：目的探讨淫羊藿苷对成骨细胞增殖分化的影响。方法采用CCK-8法检测淫羊藿苷对MC3T3-E1细胞增殖活性的影响,筛选合适浓度的淫羊藿苷用于后续实验。将MC3T3-E1细胞随机分为对照组和淫羊藿苷组,于成骨细胞诱导的第7日,采用碱性磷酸酶染色法检测细胞碱性磷酸酶活性;成骨细胞诱导第21日,采用茜素红染色检测各组MC3T3-E1细胞中钙化结节形成情况;划痕愈合和Transwell细胞迁移实验检测MC3T3-E1细胞的迁移能力;Western blot技术检测PI3K/AKT信号通路相关蛋白与成骨相关蛋白的表达情况,以验证淫羊藿苷对MC3T3-E1细胞分化的作用机制。并结合分子对接方法计算淫羊藿苷与PI3K、AKT的结合能力。结果淫羊藿苷能显著升高MC3T3-E1细胞内碱性磷酸酶的表达水平,增加钙盐沉积和钙化结节的数量,促进MC3T3-E1细胞的钙化;与对照组比较,0.1、0.2μg·mL^(-1)淫羊藿苷处理组促进细胞迁移。与对照组比较,0.1、0.2μg·mL^(-1)淫羊藿苷组的SMAD4、BMP-2、TGF-β的蛋白表达水平明显上调,PI3K、AKT、SMAD2磷酸化水平显著升高(P<0.01)。分子对接结果显示,淫羊藿苷与PI3K、AKT1蛋白结合稳定。结论淫羊藿苷可能是通过激活PI3K/AKT信号通路,增强PI3K、AKT1蛋白的表达从而发挥促进成骨分化作用。Objective To determine the effect of icariin on the proliferation and differentiation of osteoblasts.Methods The proliferation of MC3T3-E1 cells in each group was detected by CCK-8 assay,and the appropriate concentration of icariin was screened for later use in the experiments.MC3T3-E1 cells were randomly divided into a control group and an icariin group.The alkaline phosphatase activity of the cells was detected by alkaline phosphatase staining on the 7th day of osteoblast induction.On the 21st day of osteoblast induction,alizarin red staining was used to detect the formation of mineralised nodules in the MC3T3-E1 cells in each group.The migration capacity of MC3T3-E1 cells was detected by scratch healing and Transwell cell migration assay.Western blot was used to detect the expression of PI3K/AKT signalling pathway related proteins and osteogenesis related proteins to verify the mechanism of action of icariin on the differentiation of MC3T3-E1 cells.The binding capacity of icariin with PI3K and AKT was measured by molecular docking.Results Icariin significantly increased the expression level of alkaline phosphatase within MC3T3-E1,increased the number of calcium salt deposition and calcified nodules,and promoted the calcification of MC3T3-E1.Compared with the control group,the 0.1 and 0.2μg·mL^(-1) icariin-treated groups promoted cell migration.Compared with the control group,the protein expression levels of SMAD4,BMP-2,and TGF-βwere obviously up-regulated in the 0.1 and 0.2μg·mL^(-1) of icariin;the levels of phosphorylation of PI3K,AKT,and SMAD2 were significantly elevated(P<0.01).The molecular docking showed that the binding of icariin with PI3K and AKT1 proteins was stable.Conclusion Icariin plays a role in promoting osteogenic differentiation by activating the PI3K/AKT signalling pathway and enhancing the expression of PI3K and AKT1 proteins.

关 键 词：淫羊藿苷 成骨细胞 增殖 分化 分子对接 

分 类 号：R285[医药卫生—中药学]