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| 补肾活血方调控血清外泌体miRNA表达改善骨质疏松的机制研究 |
| Mechanism of Bushen Huoxue Formula in ameliorating osteoporosis by regulation of the expression serum exosomal miRNA |
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| DOI：10.3969/j.issn.1006-7108.2026.09.002 |
| 中文关键词: 骨质疏松 补肾活血方 miRNAs表达谱 细胞周期 网络药理学 |
| 英文关键词:osteoporosis BSHX formula miRNAs expression profile cell cycle network pharmacology |
| 基金项目:国家自然科学基金（81460676）；苏州市卫生健康委临床重点疾病诊疗技术专项（LCZX202240）；
院管科学创新基金面上项目（SGY2021B05） |
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| 中文摘要: |
| 目的 为阐明补肾活血方（Bushen Huoxue Formula，BSHXF）改善骨质疏松（osteoporosis，OP）的分子机制，本研究整合外泌体miRNA组学与网络药理学，旨在鉴定其关键活性成分与调控的核心外泌体miRNA。方法 将SD大鼠随机分为假手术组（control）、去卵巢模型组（model）和BSHXF治疗组（BSHXF）、阳性处理组（ALN）。采用去卵巢法构建OP大鼠模型，BSHXF治疗组予以药物干预12周。通过Micro-CT检测各组大鼠股骨骨密度（bone mineral density，BMD）变化；比较各组大鼠体重变化；测定血清中碱性磷酸酶（alkaline phosphatase，ALP）、Ⅰ型胶原交联C-末端肽（C-terminal telopeptide of typeⅠcollagen，CTX）及Ⅰ型胶原N-末端前肽（procollagenⅠN-terminal propeptide，PINP）等指标。同时，提取血清外泌体，采用高通量测序技术筛选差异表达的miRNA，并整合网络药理学与分子对接技术，预测并分析BSHXF的活性成分、差异miRNA及其下游靶点与信号通路。结果 BMD和血清中OP相关指标检测显示，BSHXF干预显著改善了OP大鼠的BMD和骨质流失。高通量测序与RT-qPCR验证结果表明，BSHXF影响OP大鼠血清外泌体中miRNA的表达模式，其中rno-miR-30c-2-3p、rno-miR-181a-5p和rno-miR-145-5p是其发挥作用的核心miRNA。富集分析显示，其靶基因主要与细胞周期、p53、叉头框蛋白O亚家族（Forkhead box O，FoxO）和低氧诱导因子-1（hypoxia inducible factor-1，HIF-1）等信号通路有关。网络药理学进一步鉴定肉豆蔻酸（myristic acid）为BSHXF的关键活性成分，可直接与上述核心miRNA以及关键靶蛋白TP53和TNRC6B结合。体内药理学实验进一步证实，单独给予肉豆蔻酸可显著下调其靶向miRNA的表达，并有效调节血清骨转换标志物ALP、CTX-I、PINP水平，表明其具有直接改善OP的活性。结论 BSHXF的活性成分肉豆蔻酸可调控血清外泌体中以rno-miR-30c-2-3p等为核心的miRNA表达，进而影响p53、FoxO等关键信号通路，从而发挥改善OP的治疗作用。本研究通过揭示BSHXF“活性成分-核心miRNA-关键通路”的作用模式，不仅深化了对OP发病机制的认识，也为中药复方的现代化研究、临床精准应用及新药开发提供了重要科学支撑。 |
| 英文摘要: |
| Objective To elucidate the molecular mechanism by which Bushen Huoxue Formula (BSHXF) ameliorates osteoporosis (OP). This study integrated exosomal miRNA-omics with network pharmacology to identify its key active components and core regulatory exosomal miRNAs. Methods SD rats were randomly assigned to 4 groups: sham-operated (Control), ovariectomized model (Model), BSHXF-treated model (BSHXF), and positive drug group (ALN). The OP model was established using ovariectomy, followed by a 12-week BSHXF intervention. Changes in femoral bone mineral density (BMD) and body weight were monitored. The serum levels of ALP, CTX, and PINP and other indicators were determined. Serum exosomes were isolated, and high-throughput sequencing was employed to identify differentially expressed miRNAs (DE-miRNAs). An integrated approach of network pharmacology and molecular docking was utilized to predict and to analyze the active components of BSHXF, the DE-miRNAs, and their downstream target genes and signaling pathways. Results BSHXF intervention significantly improved BMD and mitigated bone loss in BSHXF-treated rats. High-throughput sequencing, validated by RT-qPCR, revealed that BSHXF modulated the expression profile of serum exosomal miRNAs, identifying rno-miR-30c-2-3p, rno-miR-181a-5p, and rno-miR-145-5p as core functional miRNAs. Enrichment analysis indicated that their target genes were predominantly involved in the cell cycle, p53, FoxO, and HIF-1 signaling pathways. Furthermore, network pharmacology identified myristic acid as a key active component of BSHXF, which predicted to directly bind to these core miRNAs and the key target proteins TP53 and TNRC6B. Furthermore, in vivo pharmacological validation demonstrated that administration of myristic acid alone significantly down-regulated the expressions of its target miRNAs and effectively regulated the levels of serum bone turnover markers (ALP, CTX-I, and PINP), confirming its direct anti-osteoporotic activity. Conclusion The active component of BSHXF, myristic acid, may exert its therapeutic effects by modulating the expressions of core exosomal miRNAs, including rno-miR-30c-2-3p, which in turn influences key signaling pathways such as p53 and FoxO to ameliorate osteoporosis. This study elucidates a potential active component-core miRNA-key pathway mechanism for BSHXF, providing a scientific basis for the modernization of traditional Chinese medicine formulas and offering new avenues for drug development in osteoporosis treatment. |
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