[关键词]
[摘要]
目的 基于生物信息学、分子对接技术结合实验验证,探究川续断皂苷 VI (Asperosaponin VI,ASD) 改 善激素性股骨头坏死 (SONFH) 后骨相关细胞损伤的作用机制。方法 生物信息学与网络药理学研究:通过 GEO 数据库筛选 SONFH 的差异表达基因 (DEGs) 并进行富集分析,探究其发病相关的作用机制;将预测得到 的 ASD 靶点与疾病的 DEGs 取交集,构建蛋白互作网络 (PPI) ,筛选药物发挥治疗作用的关键靶点进行富集分 析并通过分子对接进行验证,以明确 ASD 治疗 SONFH 的潜在分子机制。临床研究:收集 SONFH 患者股骨头 标本,HE 染色及免疫荧光观察股骨头坏死区域的炎性病理表现。体外实验:地塞米松 (Dex) 干预 MC3T3-E1 和 MLOY4 细胞进行体外 SONFH 模型复制,CCK-8、Calcein/PI 细胞活性与毒性实验、qPCR 法结合体外共培 养体系验证实验结果。结果 生物信息学与网络药理学结果:基于生物信息学蛋白互作分析,筛选出 TNF-α、 IL-1β、IL-6 等 7 个关键靶点,富集分析发现与炎症反应与调控的生物过程及 TNF、NF-κB 信号通路关系密 切。临床研究:HE 结果示,与非坏死组比较,坏死组可见炎症细胞浸润 (P<0.05) ;免疫荧光结果示,与非坏 死组比较,坏死组的 iNOS、CD86 表达升高 (P<0.01) ,而 CD206 表达降低 (P<0.05) 。体外实验:第 1 轮的 CCK-8 结果示,与对照组比较,地塞米松可抑制 MC3T3-E1 和 MLOY4 细胞增殖率 (P<0.01,P<0.05) ;qPCR 结果示,与对照组比较,受损的 MC3T3-E1 和 MLOY4 细胞可引起 RAW264.7 细胞 TNF-α、IL-1β、IL-6、 iNOS 和 CD86 mRNA 表达升高 (P<0.05,P<0.01,P<0.001) ,CD206 mRNA 表达降低 (P<0.05,P<0.001) ; CCK-8 及 Calcein/PI 结果显示,与对照组比较,M1 极化后的 RAW264.7 可引起 MC3T3-E1 和 MLOY4 细胞增 殖率 (P<0.05,P<0.001) 及活性 (P<0.05) 降低;第 2 轮的 CCK-8 及 Calcein/PI 结果示,与地塞米松组比较, 地塞米松+ASD 组的 MC3T3-E1 和 MLOY4 细胞增殖率 (P<0.01,P<0.05) 及细胞活性 (P<0.05) 升高;qPCR 结果示,与地塞米松组比较,活性恢复的 MC3T3-E1 和 MLOY4 细胞可引起 RAW264.7 细胞的 TNF-α、IL-1β、 IL-6、iNOS 和 CD86 mRNA 表达降低 (P<0.05,P<0.01,P<0.001) ,而 CD206 mRNA 表达升高 (P<0.05) 。 结论 ASD 可缓解 SONFH 的进程,其作用可能与抑制受损骨相关细胞 MC3T3-E1 和 MLOY4 诱导的巨噬细胞 M1 型极化相关。
[Key word]
[Abstract]
Objective To investigate the mechanism of Asperosaponin VI (ASD) in improving bone-related cell damage in steroid-induced osteonecrosis of the femoral head (SONFH) based on bioinformatics,molecular docking technology and experimental validation. Methods Bioinformatics and network pharmacology study: Differentially expressed genes (DEGs) of SONFH were screened from the GEO database for enrichment analysis to explore disease- related mechanisms. Predicted ASD targets were intersected with disease DEGs to construct a protein-protein interaction (PPI) network. Key therapeutic targets were identified for enrichment analysis and validated via molecular docking to elucidate ASD's potential molecular mechanisms in SONFH treatment. Clinical study:Femoral head specimens from SONFH patients were collected,and inflammatory pathology in necrotic areas was observed using HE staining and immunofluorescence. In vitro experiments:Dexamethasone (Dex)-treated MC3T3-E1 and MLOY4 cells were used to establish an in vitro SONFH model. Results were validated using CCK-8,Calcein/PI cell viability and toxicity assays, qPCR with a co-culture system. Results Bioinformatics and network pharmacology:PPI analysis identified seven key targets (TNF- α,IL-1β,IL-6,etc.),with enrichment analysis revealing strong associations with inflammatory responses and TNF,NF- κB signaling pathways. Clinical study:HE staining showed increased inflammatory cell infiltration in necrotic versus non-necrotic groups (P<0.05) . Immunofluorescence demonstrated elevated iNOS and CD86 (P<0.01) but reduced CD206 (P<0.05) in necrotic areas. In vitro experiments:First-round CCK-8 assays confirmed Dex suppressed MC3T3-E1 and MLOY4 proliferation (P<0.01,P<0.05) . qPCR showed damaged bone cells upregulated RAW264.7 mRNA expression of TNF-α,IL-1β,IL-6,iNOS,and CD86 (P<0.05,P<0.01, P<0.001) while downregulating CD206 mRNA (P<0.05,P<0.001) . M1-polarized RAW264.7 reduced bone cell proliferation (P<0.05,P<0.001) and viability (P<0.05) . Second-round CCK-8 and Calcein/PI assays revealed ASD+Dex improved bone cell proliferation (P<0.01,P<0.05) and viability (P<0.05) versus Dex alone. qPCR showed recovered bone cells downregulated RAW 264.7 mRNA of TNF-α、IL-1β,IL-6,iNOS and CD86 (P<0.05, P<0.01,P<0.001) and upregulated CD206 mRNA (P<0.05) in macrophages. Conclusion ASD alleviates SONFH progression,potentially by inhibiting M1 polarization of macrophages induced by damaged bone-related cells.
[中图分类号]
R285.5
[基金项目]
2024年度国家级中医药继续教育项目 (T20241520005)。