人参皂苷Rh1对MC3T3-E1细胞增殖和分化的促进作用及其机制  

Promotion effect of ginsenoside Rh1 on proliferation and differentiation of MC3T3-E1 cells and its mechanism

作　　者：毛天娇 孙铎 高幸[1] 魏溦 李熙恒 姜可新 姜秋[2] 李江[1,3] MAO Tianjiao;SUN Duo;GAO Xing;WEI Wei;LI Xiheng;JIANG Kexin;JIANG Qiu;LI Jiang(Department of Prosthodontics,Stomatology Hospital,Jilin University,Changchun 130021,China;Department of Pediatric Dentistry,Stomatology Hospital,Jilin University,Changchun 130021,China;Department of Prosthodontics,Affiliated Stomatology Hospital,Guangzhou Medical University,Guangzhou 510150,China)

机构地区：[1]吉林大学口腔医院口腔修复科,吉林长春130021 [2]吉林大学口腔医院儿童口腔科,吉林长春130021 [3]广州医科大学附属口腔医院口腔修复科,广东广州510150

出　　处：《吉林大学学报（医学版）》2021年第4期896-903,共8页Journal of Jilin University：Medicine Edition

基　　金：科技部国家重点研发计划项目(2020YFE010636);吉林省科技厅科研项目(20200201025JC);吉林省科技厅科技发展计划项目(20190201082JC);吉林省财政厅科研项目(jsz2018170-3);吉林省长春市科技局科技计划项目(17YJ001)。

摘　　要：目的:筛选靶向上调雌激素受体β(ERβ)转录和表达的人参皂苷单体,研究其对MC3T3-E1细胞增殖和分化的影响及其机制。方法:采用PGL2-ERβ和内参Renilla荧光素酶质粒Prep7-Rluc共同转染HEK293T细胞,细胞分为对照组,雌二醇组(1×10^(-6)mmol·L^(-1)),人参皂苷Rb1组、Rb2组、Rd组、Rg1组、Rg2组和Rh1组(1×10^(-5)mmol·L^(-1)),通过双荧光素酶报告基因实验检测各组细胞双荧光素酶活性。进一步将MC3T3-E1细胞分为对照组、雌二醇组和不同浓度(1×10^(-6)、1×10^(-5)及1×10^(-4)mmol·L^(-1))人参皂苷Rh1组,采用Western blotting法检测各组MC3T3-E1细胞中ERβ蛋白表达水平。通过Auto Dock分子对接实验,模拟人参皂苷Rh1与ERβ蛋白分子的结合情况。MC3T3-E1细胞分为对照组、雌二醇组和不同浓度(5×10^(-6)、1×10^(-5)、5×10^(-5)、1×10^(-4)、1×10^(-3)及5×10^(-3)mmol·L^(-1))人参皂苷Rh1组,分别作用24、48和72 h后,采用CCK-8法检测各组细胞增殖率。MC3T3-E1细胞分为对照组、雌二醇组(1×10^(-6)mmol·L^(-1))和不同浓度(1×10^(-5)及1×10^(-4)mmol·L^(-1))人参皂苷Rh1组,配制成骨诱导液,分别诱导MC3T3-E1细胞7、14和21 d,采用碱性磷酸酶(ALP)染色和茜素红染色检测细胞中ALP和钙化结节染色面积,观察人参皂苷Rh1对MC3T3-E1细胞成骨分化的影响。结果:双荧光素酶报告基因实验,各组HEK293T细胞中转染ERβ-PGL2质粒后,与对照组比较,1×10^(-5)mmol·L^(-1)人参皂苷Rh1组细胞荧光素酶活性明显升高(P<0.05)。Western blotting法检测,与对照组比较,不同浓度人参皂苷Rh1组细胞中ERβ蛋白表达水平明显升高(P<0.05),且1×10^(-4)mmol·L^(-1)人参皂苷Rh1组细胞中ERβ蛋白表达水平最高。Auto Dock分析,人参皂苷Rh1可以结合在ERβ蛋白的配体结合口袋内。CCK-8实验,培养24、48和72 h后,与对照组比较,1×10^(-5)、5×10^(-5)、1×10^(-4)和1×10^(-3)mmol·L^(-1)人参皂苷Rh1组MC3T3-E1细胞增殖率均明显升高(P<0.01),Objective:To screen the ginsenoside monomer targetedly up-regulating the transcription and expression of estrogen receptorβ(estrogen receptorβ,ERβ),and to explore its effect on the proliferation and differentiation of MC3T3-E1 cells and its possible mechanism.Methods:PGL2-ERβwas cotransfected into the HEK293T cells with Renilla luciferase plasmid Prep7-Rluc,then the HEK293T cells were divided into control group,estradiol group(1×10^(-6)mmol·L^(-1)),ginsenoside Rb1,Rb2,Rd,Rg1,Rg2 and Rh1 groups(1×10^(-5)mmol·L^(-1)).The dual luciferase activities in various groups were detected by dual luciferase reporter gene assay.The MC3T3-E1 cells were further divided into control group,estradiol group(1×10^(-6)mmol·L^(-1)),ginsenoside Rh1 group(1×10^(-6)mmol·L^(-1),1×10^(-5)mmol·L^(-1)and 1×10^(-4)mmol·L^(-1)).The expression levels of ERβprotein in the MC3T3-E1 cells in various groups were detected by Western blotting method.The binding of ginsenoside Rh1 to ERβprotein was simulated by Auto Dock expreriment.The MC3T3-E1 cells were divided into control group,estradiol group(1×10^(-6)mmol·L^(-1)),ginsenoside Rh1 group(5×10^(-6)mmol·L^(-1),1×10^(-5)mmol·L^(-1),5×10^(-5)mmol·L^(-1),1×10^(-4)mmol·L^(-1),1×10^(-3)mmol·L^(-1)and 5×10^(-3)mmol·L^(-1)),after treated for 24,48 and 72 h,the cell proliferation rates in various groups were detected by CCK-8 method.The MC3T3-E1 cells were divided into control group,estradiol group(1×10^(-6)mmol·L^(-1)),and ginsenoside Rh1 groups(1×10^(-5)mmol·L^(-1)and 1×10^(-4)mmol·L^(-1)).The MC3T3-E1 cells were induced with osteogenic induction solution for 7,14 and 21 d,respectively;alkaline phosphatase(ALP)staining and alizarin red staining were used to detect the staining areas of ALP and calcified nodules in the MC3T3-E1 cells and observe the effect of ginsenoside Rh1 on the osteogenic differentiation of the MC3T3-E1 cells.Results:In double luciferase reporter gene experiment,compared with control group,the luciferase activity of cells in ginsenoside Rh1 group(1

关 键 词：人参皂苷RH1 骨质疏松症 MC3T3-E1细胞 雌激素受体Β 

分 类 号：R285[医药卫生—中药学]