何首乌提取物对失重小鼠骨质疏松和骨髓间充质干细胞成骨分化的影响

Effect of polygonum multiflorum extract on osteoporosis and differentiation of bone marrow mesenchymal stem cells in agravic mice

目的:探讨何首乌(PM)提取物对失重小鼠骨髓间充质干细胞(BMSCs)向成骨细胞分化和骨形成的促进作用及其机制,为治疗宇航员因失重导致的骨质疏松症(OP)提供潜在的治疗靶点和药物。方法:30只12周龄的雄性C57BL/6小鼠随机分为对照组、失重组和失重+PM(200 mg·kg^(-1)·d^(-1))组,每组10只。4周后,微型计算机断层成像(micro-CT)检测各组小鼠骨体积/组织体积比值、骨小梁数量、骨小梁厚度和骨小梁间隙。流式细胞术分选获得失重小鼠BMSCs,将细胞分为对照组(0μmol·L^(-1)PM)和10、20及40μmol·L^(-1)PM组,实时荧光定量PCR(RT-q PCR)法检测各组BMSCs中RUNX家族转录因子2(Runx-2)、碱性磷酸酶(ALP)、Osterix、过氧化物酶体增殖物激活受体γ(PPARγ)m RNA表达水平,Western blotting法检测各组细胞中Runx-2、ALP、Osterix、PPARγ、丝裂原活化蛋白激酶激酶(MEK)、磷酸化MEK 1/2(p-MEK1/2)、细胞外调节蛋白激酶(ERK)和磷酸化ERK1/2(p-ERK1/2)蛋白表达水平。结果:与对照组比较,失重组小鼠骨体积/组织体积比值降低(P<0.01),骨小梁数量减少(P<0.01),骨小梁的厚度降低(P<0.05),骨小梁间隙增加(P<0.01);与失重组比较,失重+PM组小鼠骨体积/组织体积比值升高(P<0.05),骨小梁数量增加(P<0.01),骨小梁厚度增加(P<0.05),骨小梁间隙降低(P<0.01);与失重组比较,失重+PM组小鼠BMSCs数量增加(P<0.01),成骨细胞数量增加(P<0.01),脂肪细胞数量减少(P<0.05),破骨细胞数量减少(P<0.05)。与对照组比较,10、20和40μmol·L^(-1)PM组小鼠BMSCs中Runx-2、ALP和Osterix m RNA和蛋白表达水平升高(P<0.05),PPARγm RNA和蛋白表达水平降低(P<0.05),与对照组比较,10μmol·L^(-1)PM组小鼠BMSCs中p-MEK1/2和p-ERK1/2蛋白表达水平升高(P<0.05);与10μmol·L^(-1)PM组比较,20μmol·L^(-1)PM组小鼠BMSCs中p-MEK1/2和p-ERK1/2蛋白表达水平升高(P<0.05);与20μmol·L^(-1)PM组比较,40μmol·L^(-1)PM组小鼠BMSCs中p-MEK1/2和p-ERK1/2蛋白表达水平升高(P<0.05)。结论:PM提取物能够激活MEK/ERK信号通路从而促进失重小鼠骨组织间中BMSCs向成骨细胞分化,提高小鼠骨量,改善骨小梁微结构,缓解因失重导致的OP。

Objective:To explore the promotion effect of polygonum multiflorum(PM)extract on the differentiation of bone marrow mesenchymal stem cells(BMSCs)into osteoblasts and bone formation in the mice and its mechanism,and to provide the potential therapeutic targets and drugs for the treatment of osteoporosis(OP)caused by weightlessness in the astronauts.Methods:Thirty 12-week-old male C57BL/6 mice were randomly divided into control group,weightlessness group and weightlessness+PM(200 mg·kg^(-1)·d^(-1))group,and there were 10 mice in each group.After 4 weeks,the ratio of bone volume/tissue volume,the trabecular number,trabecular thickness,and trabecular space of the mice in various groups was detected by micro CT;the BMSCs of the weightlessness mice were obtained by flow sorting.The BMSCs were divided into control group(0μmol·L^(-1)PM)and 10,20,and 40μmol·L^(-1)PM groups.The expression levels of Runx family transcription factor 2(Runx-2),alkaline phosphatase(ALP)and Osterix,peroxisome proliferator activated receptorγ(PPARγ)m RNA in BMSCs of the mice in various groups were detected by Real-time fluorescence quantitative PCR(RT-q PCR)method,and the expression levels of Runx-2,ALP,Osterix,PPARγ,mitogen activated protein kinase(MEK),phosphorylated MEK 1/2(p-MEK1/2),extracellular regulated protein kinase(ERK)and phosphorylated ERK 1/2(p-ERK1/2)proteins in BMSCs of the mice in various groups were detected by Western blotting method.Results:Compared with control group,the ratio of bone volume/tissue volume of the mice in weightlessness group was decreased(P<0.01),the trabecular number was decreased(P<0.01),the trabecular thickness was decreased(P<0.05),and the trabecular space was increased(P<0.01).Compared with weightlessness group,the ratio of bone volume/tissue volume of the mice in weightlessness+PM group was increased(P<0.05),the trabecular number was increased(P<0.01),the trabecular thickness was increased(P<0.05),and the trabecular space was decreased(P<0.01).Compared with weightlessness group,the number of BMSCs of the mice in weightlessness+PM group was increased(P<0.01),the number of osteoblasts was increased(P<0.01),the number of adipocytes was decreased(P<0.05),and the number of osteoclasts was decreased(P<0.05).Compared with control group,the expression levels of Runx-2,ALP,and Osterix m RNA in BMSCs of the mice in 10,20,and 40μmol·L^(-1)PM groups were increased(P<0.05),and the expression level of PPARγm RNA was decreased(P<0.05),the expression levels of Runx-2,ALP,and Osterix proteins were increased(P<0.05),and the expression level of PPARγprotein was decreased(P<0.05).Compared with control group,the expression levels of p-MEK1/2 and p-ERK1/2 proteins in BMSCs of the mice in10μmol·L^(-1)PM group were increased(P<0.05);compared with 10μmol·L^(-1)PM group,the expression levels of p-MEK1/2 and p-ERK1/2 proteins in BMSCs of the mice in 20μmol·L^(-1)PM group were increased(P<0.05);compared with 20μmol·L^(-1)PM group,the expression levels of p-MEK1/2 and p-ERK1/2 proteins in BMSCs of the mice in 40μmol·L^(-1)PM group were increased(P<0.05).Conclusion:The extract of PM can promote the differentiation of BMSCs into osteoblasts in the weightlessness mice by activating the MEK/ERK signaling pathway,and improve the bone mass and the micro-structure of trabeculae,relieve the OP caused by weightlessness.