刺山柑异鼠李素通过抑制PI3K/AKT/mTOR信号通路保护软骨细胞外基质OA

Objective Osteoarthritis(OA),a highly prevalent degenerative joint disease worldwide,still lacks effective clinical treatments capable of delaying the progression of joint degeneration at present.Capparis spinosa L.is a commonly used herb in Uyghur medicine for the treatment of rheumatic arthralgia,has shown its therapeutic potential for OA in traditional clinical practice.However,the specific active substances responsible for its efficacy and the underlying molecular mechanisms remain unclear.Therefore,this study aims to systematically screen the key active components of Capparis spinosa L.against OA and elucidate their underlying molecular mechanisms.Methods In the network pharmacology section,① the known chemical components of Capparis spinosa L.were collected through literature retrieval,and the active components were screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and SwissADME web tools.② The potential targets of the active components were predicted using SwissTargetPrediction,and these targets were intersected with OA-related genes from the GeneCards,Online Mendelian Inheritance in Man(OMIM),Therapeutic Target Database(TTD)and DisGeNET databases to obtain common targets.③ Protein-protein interaction(PPI)network analysis and gene enrichment analysis were performed on the common targets to screen hub targets and key signaling pathways.④ Molecular docking was then applied to validate the binding affinity between the active components and hub targets,thereby locking the core component of Capparis spinosa L.,followed by a specific network pharmacology analysis of the core component.In the in vitro experimental section,the appropriate working concentration of the drug was determined by CCK-8 assay.A cellular model of OA was established by IL-1β-induced ATDC5 chondrocytes.Toluidine blue staining and Western blotting were used to evaluate the metabolism of the extracellular matrix(ECM).Finally,qPCR,Western blotting and immunofluorescence staining were performed to detect the expression levels of genes and proteins related to the signaling pathway and ECM metabolism,and a"rescue experiment"was conducted using Akt inhibitor MK-2206 and its activator SC-79 in combination with the target drug.Statistical analysis was performed using GraphPad Prism 10.1.2,with P<0.05 considered statistically significant.Results ① A total of 33 active components were screened from Capparis spinosa L.,and 174 OA-related common targets were identified.KEGG enrichment analysis revealed significant enrichment of the PI3K-AKT signaling pathway.PPI network analysis identified AKT1,TNF,IL6 and BCL2 as key hub targets.Molecular docking results indicated that isorhamnetin(ISO)exhibited the greatest average negative binding energy with the hub targets,suggesting that it may be the key component of Capparis spinosa L.in the treatment of OA.Network pharmacology analysis targeting ISO further confirmed that AKT1 was at the core node position,with the PI3K-AKT signaling pathway significantly enriched.Molecular docking simulation revealed that ISO achieved stable binding with AKT1 by forming hydrogen bonds with the THR195 and GLU191 residues of AKT1,as well as through hydrophobic interactions with its LEU295 and PHE161 residues.② In vitro experiments confirmed that ISO at a concentration of 60 μmol/L enhanced chondrocyte viability without exhibiting cytotoxicity.This concentration significantly reversed IL-1β-induced ECM degradation,markedly upregulated the expression of type Ⅱ collagen,and simultaneously reduced the expression level of matrix metalloproteinase 13(MMP13)(all P<0.01).At the mechanistic level,ISO treatment inhibited the overactivation of PI3K,AKT and mTOR induced by IL-1β.The rescue experiment ultimately established the core role of the PI3K/AKT/mTOR signaling pathway:the AKT inhibitor MK-2206 synergistically enhanced the inhibitory effect of ISO on the aforementioned pathway and its protective effect on the ECM;conversely,the AKT activator SC-79 antagonized the protective effect of ISO(all P<0.01).Conclusion Our results demonstrate that ISO is the key active component of Capparis spinosa L.showing anti-OA effects,and it may exert a therapeutic effect on OA by inhibiting the PI3K/AKT/mTOR signaling pathway.