中国骨质疏松杂志

五劳七损方对hBMSCs成骨分化影响研究

Research on the impact of Wulao Qisun Formula on osteogenic differentiation of hBMSCs

DOI：10.3969/j.issn.1006-7108.2026.05.003

中文关键词: 五劳七损方强直性脊柱炎 IL-17A-Wnt/β-catenin通路 hBMSCs成骨分化

英文关键词:WuLao QiSun Formula ankylosing spondylitis IL-17A-Wnt/β-catenin signaling pathway osteogenic differentiation of MSCs

基金项目:甘肃省科技计划资助（23JRRA1535）；第三批陇原青年英才培养项目（省委人才小组发[2024]11号）；国家中医药管理局高水平中医药重点学科建设项目[国中医药人教函(2022)226号]

作者	单位
陈平1 杨娟娟1 王海东1* 张智敏2 李浩林2 苏瑾2 金芳梅1 阚丽丽1 年芳红1	1.甘肃省中医院,甘肃兰州 730050 2.甘肃中医药大学中医临床学院,甘肃兰州 730000

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中文摘要:

目的基于强直性脊柱炎（ankylosing spondylitis，AS）的病理机制，探讨五劳七损方对人骨髓间充质干细胞（hBMSCs）成骨分化的影响及其作用机制。方法体外培养hBMSCs，将细胞分为对照组、IL-17A组、空白血清组（20 %空白血清）、20 %含药血清组、10 %含药血清组、5 %含药血清组和抑制剂组（15 μg/mL司库奇尤单抗），对照组选用无IL-17A的成骨诱导分化培养基，其余各组均在对照组的基础上加入质量浓度为50 ng/mL的IL-17A诱导hBMSCs并与各组血清共孵。采用CCK-8法检测实验所用五劳七损方含药血清干预体积分数。采用ALP活性检测和茜素红染色及定量检测细胞成骨矿化能力；Elisa法检测炎症因子水平变化；RT-qPCR法、Western Blot法和免疫荧光法等方法检测IL-17A-Wnt/β-catenin通路关键分子表达水平。结果 CCK-8法筛选用于实验的五劳七损方含药血清干预体积分数为5 %、10 %和20 %。与对照组比较，IL-17A组ALP活性升高，细胞颜色加深、钙结节数量增加，IL-17A、IL-6、IL-1β、TNF-α含量均升高，Act1、Wnt3a、β-catenin和Axin2蛋白水平均上升，GSK-3β蛋白水平下降，Runx2、OCN和Osx mRNA水平上升；与IL-17A组比较，空白血清组ALP活性、钙结节数量、IL-17A、IL-6、IL-1β及TNF-α含量、Act1、Wnt3a、β-catenin、Axin2、GSK-3β、Runx2、OCN和Osx的表达水平未见明显变化。而五劳七损方各浓度含药血清组及抑制剂组的ALP活性明显降低；细胞颜色、形态及钙结节数量存在较大差异，钙结节数量减少；IL-17A、IL-6、IL-1β、TNF-α含量均明显下降；IL-17RA、Act1、TRAF6、Wnt3a、β-catenin、Axin2、Runx2、OCN和Osx的表达水平降低，GSK-3β表达水平升高，同时IL-17RA、β-catenin和Runx2的平均荧光强度均降低。结论五劳七损方通过抑制IL-17A-Wnt/β-catenin信号通路活性进一步抑制成骨分化相关因子水平，从而发挥抑制hBMSCs的成骨分化能力。

英文摘要:

Objective To investigate the effect of Wulao Qisun Formula on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) and its underlying mechanism. Methods hBMSCs were cultured in vitro and divided into the following groups: Control, IL-17A, Blank Serum (20% blank serum), 20%, 10%, and 5% of Wulao Qisun Formula-containing serum, and Inhibitor (15 μg/mL Secukinumab). Cells in the control group were cultured in osteogenic induction medium without IL-17A, while cells in all other groups were treated with 50 ng/mL of IL-17A in addition to the respective serum interventions. The optimal concentration of Wulao Qisun Formula-containing serum was determined using CCK-8 assay. Alkaline phosphatase (ALP) activity and Alizarin red staining with quantitative analysis were used to assess osteogenic mineralization. Inflammatory cytokine levels were measured with ELISA. The expressions of key molecules in the IL-17A–Wnt/β-catenin pathway was evaluated via RT-qPCR, Western blotting, and immunofluorescence. Results CCK-8 results identified 5%, 10%, and 20% as suitable concentrations of Wulao Qisun Formula-containing serum for subsequent experiments. Compared with the control group, the IL-17A group showed increased ALP activity, darker staining, more mineralized nodules, elevated levels of IL-17A, IL-6, IL-1β, and TNF-α, upregulation of Act1, Wnt3a, β-catenin, and Axin2 protein expression, downregulation of GSK-3β, and increased mRNA levels of Runx2, OCN, and Osx. No significant differences were observed between the IL-17A and blank serum groups in any of these indicators. In contrast, all Wulao Qisun Formula-containing serum groups and the inhibitor group exhibited significantly reduced ALP activity, fewer mineralized nodules, decreased inflammatory cytokine levels, downregulated expression of IL-17RA, Act1, TRAF6, Wnt3a, β-catenin, Axin2, Runx2, OCN, and Osx, increased GSK-3β expression, and reduced mean fluorescence intensity of IL-17RA, β-catenin, and Runx2. Conclusion Wulao Qisun Formula inhibits the osteogenic differentiation of hBMSCs by suppressing the IL-17A–Wnt/β-catenin signaling pathway and downregulating osteogenesis-related factors.

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