摘要
目的探讨不同浓度虎杖苷(polydatin,PD)对地塞米松(dexamethasone,Dex)诱导的骨髓间充质干细胞(bone marrow stem cells,BMSCs)成骨成脂分化作用和机制。方法分离纯化和培养大鼠BMSCs,通过免疫荧光检测BMSCs表面阳性标志物,CCK-8检测不同浓度PD对BMSCs增殖作用影响。将BMSCs分为对照组(Control组)、地塞米松组(Dex组),虎杖苷低、中、高浓度组(PD-L组、PD-M组、PD-H组),虎杖苷高浓度+Wnt抑制剂组(PD-H+DKK1组)。通过碱性磷酸酶(ALP)染色、茜素红(ARS)染色观察成骨分化,油红O染色观察细胞脂滴形成情况,Western blot和RT-qPCR分别检测BMSCs内成骨、成脂特异性基因和Wnt/β-catenin通路相关蛋白及基因表达水平。结果BMSCs呈长梭状、形态均一、贴壁生长且胞质中阳性标志物高表达;CCK-8结果提示浓度为5~15μmol/L的PD对地塞米松诱导的BMSCs细胞增殖均无影响,选择PD浓度为5、10、15μmol/L进行后续实验;与Control组相比,Dex组ALP活性显著降低,矿化结节减少,脂滴明显增多,ALP、RUNX-2、OCN、Wnt10b、β-catenin mRNA表达水平显著降低,PPAR-γ、FABP4 mRNA表达水平显著升高;与Dex组比较,PD-L组、PD-M组、PD-H组ALP活性、矿化结节数依次升高、脂滴数量依次减少,ALP、RUNX-2、Wnt10b、β-catenin mRNA表达水平依次升高、PPAR-γ、FABP4 mRNA表达水平依次降低;与PD-H组比较,PD-H+DKK1组ALP活性与矿化结节显著降低,脂滴数量下降RUNX-2、OCN、ALP、Wnt10b、β-catenin mRNA表达水平显著降低,PPAR-γ、FABP4 mRNA表达水平升高,Western blot结果与RT-qPCR结果一致。结论不同浓度的PD通过激活Wnt/β-catenin信号通路促进RUNX-2、ALP和OCN的表达,抑制PPAR-γ、C/EBP-α和FABP4的表达从而促进大鼠BMSCs成骨分化,抑制其成脂分化作用。
Objective To investigate the effects and mechanisms of different concentrations of Polydatin(PD)on the osteogenic and adipogenic differentiation of dexamethasone(Dex)-induced bone marrow mesenchymal stem cells(BMSCs).Methods Rat bone marrow mesenchymal stem cells(BMSCs)were isolated,purified and cultured,with their surface markers identified by immunofluorescence.The proliferative effects of different concentrations of polydatin(PD)on BMSCs were determined using CCK-8 assay.The cells were divided into control group(Control),dexamethasone group(Dex),low-,medium-and high-dose polydatin groups(PD-L,PD-M,PD-H),and high-dose polydatin plus Wnt inhibitor group(PD-H+DKK1).Osteogenic differentiation was evaluated by alkaline phosphatase(ALP)staining and alizarin red S(ARS)staining,while adipogenic differentiation was assessed by Oil Red O staining for lipid droplet formation.The expression levels of osteogenic-and adipogenic-specific genes as well as Wnt/β-catenin pathway-related proteins and genes were detected by Western blot and RT-qPCR,respectively.Results BMSCs were spindle-shaped,uniform in morphology,adhered to the surface,and showed high expression of positive markers in the cytoplasm.CCK-8 result indicated that PD concentrations of 5-15μmol/L had no effect on BMSCs induced by dexamethasone;therefore,PD concentrations of 5,10,and 15μmol/L were selected for subsequent experiments.Compared with the Control group,the Dex group showed significantly reduced ALP activity,fewer mineralized nodules,and increased lipid droplets.Additionally,ALP,RUNX-2,OCN,Wnt10b,andβ-catenin mRNA expression levels were significantly reduced,while PPAR-γand FABP4 mRNA levels were significantly increased.Compared with the Dex group,the PD-L,PD-M,and PD-H groups showed progressively increased ALP activity and mineralized nodule numbers,and decreased lipid droplet counts.Furthermore,ALP,RUNX-2,Wnt10b,andβ-catenin mRNA levels increased progressively,while PPAR-γand FABP4 mRNA levels decreased.Compared with the PD-H group,the PD-H+DKK1 group exhibited significantly reduced ALP activity,mineralized nodule numbers,and lipid droplet counts.RUNX-2,OCN,ALP,Wnt10b,andβ-catenin mRNA levels were significantly reduced,while PPAR-γand FABP4 mRNA levels increased.Western blot result were consistent with RT-qPCR findings.Conclusion Different concentrations of PD promote osteogenic differentiation of rat BMSCs and inhibit adipogenic differentiation by activating the Wnt/β-catenin signaling pathway,enhancing the expression of RUNX-2,ALP,and OCN,and suppressing the expression of PPAR-γ,C/EBP-α,and FABP4.
作者
张俊娇
何敏聪
肖方骏
侯文渊
杨晓强
林锟
廖家如
韩龙飞
方伟华
田佳庆
彭鹏
陆舜
何宪顺
刘振鑫
庄旭锐
杨帆
梁祖建
魏秋实
ZHANG Junjiao;HE Mincong;XIAO Fangjun;HOU Wenyuan;YANG Xiaoqiang;LIN Kun;LIAO Jiaru;HAN Longfei;FANG Weihua;TIAN Jiaqing;PENG Peng;LU Shun;HE Xianshun;LIU Zhenxin;ZHUANG Xurui;YANG Fan;LIANG Zujian;WEI Qiushi(The Third Clinical Medical School,Guangzhou University of Chinese Medicine,Guangzhou 510378,China;Guangdong Research Institute for Orthopedics and Traumatology of Chinese Medicine,Guangzhou 510378,China;The Third Affiliated Hospital,Guangzhou University of Chinese Medicine,Guangzhou 510378,China)
出处
《中国骨质疏松杂志》
北大核心
2025年第8期1128-1136,共9页
Chinese Journal of Osteoporosis
基金
2022年广东省中医骨伤研究院开放课题青年基金项目(GYH202201-01)
2023年广州中医药大学青年拔尖人才(团队)揭榜挂帅项目。
