目的:研究表明蛇床子素(osthole，OST)可以改善骨关节炎小鼠的软骨退化，但机制尚未充分阐明。本研究利用网络药理学和分子对接技术以及软骨细胞炎症损伤模型探讨OST改善骨关节炎软骨退化的机制。方法:从4个数据库中收集OST靶基因和膝骨关节炎(knee osteoarthritis，KOA)靶基因。通过UniProt数据库对获得的靶基因进行标准化转换，使用Venny工具获得了2个的交叉基因，并在STRING数据库中获得相应蛋白质的相互作用，随后通过Cytoscape软件筛选和可视化核心靶基因，并通过基因本体论(Gene Ontology，GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes，KEGG)分析通路富集交叉基因预测OST作用的靶基因。再通过分子对接模型验证其结合可能，并可视化其结合结果。通过体外培养兔原代软骨细胞，利用白细胞介素1β(interleukin-1β，IL-1β)诱导软骨细胞炎症损伤模型。将细胞分为对照组、IL-1β、IL-1β+OST低(IL-1β+OST-L)、中(IL-1β+OST-M)、高(IL-1β+OST-H)剂量处理组及IL-1β塞来昔布处理(IL-1β+celecoxib)组;CCK-8法检测软骨细胞活力;Western blot及免疫荧光检测肿瘤坏死因子α(tumor necrosis factor-α，TNF α)和基质金属蛋白酶 13(matrix metalloproteinase-13，MMP-13)蛋白表达;RT-qPCR法检测IL-6、TNF-α和MMP-13 mRNA的表达。结果:80个OST靶点被鉴定为治疗KOA的潜在靶点，并筛选了10个核心靶基因胱天蛋白酶3(caspase-3，CASP3)、TNF-α、缺氧诱导因子1A(hypoxia inducible factor 1 subunit alpha，HIF1A)、IL-1β、核因子KB1(nuclear factor kappa B subunit 1，NFKB1)、聚腺苷二磷酸-核糖聚合酶1[poly(ADP-ribose)polymerase 1，PARP1]、NFE2样bZIP转录因子2(NFE2 like bZIP transcription factor 2，NFE2L2)、Janus激酶2(Janus kinase 2，JAK2)、丝裂原活化蛋白激酶激酶1(mitogen-activated protein kinase 1，MAKP1)、糖原合酶激酶3b(glycogen synthase kinase 3 beta，GSK3B)。GO和KEGG分析进一步阐明了OST治疗KOA的分子机制，涉及多种信号通路。分子对接模拟证实了OST与IL-17信号通路中的TNF-α及IL-1β关键靶点稳定结合。兔软骨细胞实验证实，软骨细胞呈长梭型、铺路石样;2。5～40 µmol/L的OST增加软骨细胞存活。与IL-1β处理组比较，OST抑制L-1β处理的软骨细胞表达IL-6、TNF-α和MMP-13，且OST低、中、高剂量OST抑制IL-6和TNF-α表达的效应与塞来昔布处理组比较无统计学差异，但塞来昔布不能抑制IL-1β诱导的MMP-13的表达。结论:OST可以促进软骨细胞的存活，抑制IL-1β处理的软骨细胞表达IL-6、TNF-α和MMP-13，从而减轻软骨细胞损伤。这些结果提示OST抑制软骨细胞炎症反应和MMP的表达可能是其改善关节炎的分子机制。
Network pharmacology and molecular docking analysis on mechanism of osthole in treating knee osteoarthritis
AIM:This study aims to investigate the effects of Osthole(OST)on inflammation and cartilage-de-grading proteases in an interleukin 1β(IL-1β)-induced chondrocyte inflammation model.METHODS:Prediction:we collected target genes for OST and those related to knee osteoarthritis(KOA)from four databases.After standardizing the target genes obtained from the UniProt database,two overlapping genes were identified using the Venny tool.Additional-ly,protein interaction relationships were obtained from the STRING database,and core target genes were screened and vi-sualized using Cytoscape software.Enrichment analysis for the overlapping genes was performed through Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways.Finally,the active drug components and target proteins were validated and visualized through computational analysis.Validation:primary rabbit chondrocytes were iso-lated and cultured in vitro.Cell morphology and toluidine blue staining were employed to confirm chondrocyte identity.An inflammatory injury model was induced using IL-1β.The cells were divided into control,model,low-dose,medium-dose,high-dose OST,and celecoxib groups.Chondrocyte viability was assessed using the CCK-8 method.Western blot and im-munofluorescence techniques were utilized to detect the proteins IL-1β,tumor necrosis factor-alpha(TNF-α),matrix me-talloproteinase 13(MMP-13),and a disintegrin and metalloproteinase with thrombospondin motifs(ADAMTS-4).Gene expression levels of IL-1β,IL-6,TNF-α,and MMP-13 were measured via RT-qPCR.RESULTS:A total of 80 potential OST targets were identified for treating KOA,with 10 core target genes(CASP3,TNF,HIF1A,IL-1β,NFKB1,PARP1,NFE2L2,JAK2,MAPK1,and GSK3B)screened.GO and KEGG analyses further elucidated the molecular mechanisms of OST in KOA treatment,highlighting multiple biological processes and signaling pathways involved.Molecular docking simulations confirmed stable binding of OST to key targets TNF and IL-1β within the IL-17 signaling pathway.Chondro-cytes exhibited long spindle and pavement-like shapes.Based on the effects of varying OST concentrations on chondro-cytes,2.5,5,and 10 µmol/L OST were selected for pharmacodynamic testing.Compared to the model group,OST signif-icantly reduced inflammation in the chondrocyte inflammation model and inhibited the expression of cartilage matrix-de-grading enzymes,showing no statistically significant difference from the celecoxib group.CONCLUSION:OST promotes chondrocyte proliferation and differentiation.It effectively inhibits inflammation in the IL-1β-induced chondrocyte model and mitigates chondrocyte injury.The underlying mechanism may involve the degradation of chondroproteoglycans and the protection of the extracellular matrix.
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