中国骨质疏松杂志

虎杖苷对地塞米松诱导的BMSCs成骨成脂分化影响机制

Mechanisms underlying the effects of polydatin on dexamethasone-induced osteogenic and adipogenic differentiation of BMSCs

DOI：10.3969/j.issn.1006-7108.2025.08.006

中文关键词: 虎杖苷骨髓间充质干细胞成脂分化成骨分化 Wnt/β-catenin信号通路

英文关键词:polydatin BMSCs adipogenic differentiation osteogenic differentiation Wnt/β-catenin signaling pathway

基金项目: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

作者	单位
张俊娇1 何敏聪2, 3* 肖方骏1 侯文渊1 杨晓强1 林锟1 廖家如1 韩龙飞1 方伟华1 田佳庆1 彭鹏1 陆舜1 何宪顺1 刘振鑫1 庄旭锐1 杨帆2, 3 梁祖建3 魏秋实2, 3	1.广州中医药大学第三临床医学院，广东广州 510378 2.广东省中医骨伤研究院，广东广州 510378 3.广州中医药大学第三附属医院，广东广州 510378

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中文摘要:

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5~15 μmol/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、15 μmol/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。

英文摘要:

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 results indicated that PD concentrations of 5-15μmol/L had no effect on BMSCs induced by dexamethasone; therefore, PD concentrations of 5, 10, and 15 μmol/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, and β-catenin mRNA expression levels were significantly reduced, while PPAR-γ and 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, and β-catenin mRNA levels increased progressively, while PPAR-γ and 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, and β-catenin mRNA levels were significantly reduced, while PPAR-γ and FABP4 mRNA levels increased. Western blot results 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.

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