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.

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.

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).

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.

The immuno-inflammatory mechanisms underlying acute and chronic Brucella osteoarthritis (BOA) remain unclear, and the dynamic changes in T follicular helper (Tfh) cells and follicular regulatory T (Tfr) cells in this disease, as well as their associations with immune-inflammatory responses, have not been systematically reported. This study aimed to characterize the stage-specific changes in Tfh and Tfr cells in peripheral blood and synovial tissue of BOA patients, and to analyze their relationships with related cytokines, transcription factors, and immunoglobulins, thereby providing the immunological evidence for pathogenesis and clinical management of the disease.

A cross-sectional study design was adopted in this research. A total of 13 patients with acute BOA and 21 patients with chronic BOA, who were diagnosed in the Department of Orthopedics at the First Affiliated Hospital of Shihezi University and its medical consortium hospitals from January 2024 to August 2025, were enrolled, and another 20 healthy individuals were subjected and served as the control group. Peripheral blood samples were collected from all participants, while synovial tissue specimens were obtained from 3 chronic BOA patients and 3 patients with non-infectious joint injury undergoing surgery for local tissue-level validation, which were not included in the overall clinical statistical analysis. Flow cytometry was performed to determine the proportions of Tfh cells (CD4⁺CXCR5⁺CD25^-CD127⁺) and Tfr cells (CD4⁺CXCR5⁺CD25⁺CD127^-) in peripheral blood, as well as the Tfh/Tfr ratio. Serum levels of IL-4, IL-21, IL-10, TGF-β, and immunoglobulins (IgA, IgG, IgE and IgM) were measured by ELISA. The mRNA expression of Tfh-related transcription factor BCL-6 and cytokine IL-21, as well as Tfr-related transcription factor FOXP3 and cytokine IL-10, was assessed in peripheral blood mononuclear cells (PBMCs) using qRT-PCR. Western blotting was used to detect the expression of IL-21, FOXP3, and joint matrix metabolism-related proteins MMP-13, type Ⅱ collagen (COLⅡ), and ADAMTS5) in synovial tissues.

① In the synovial tissues, the chronic BOA group showed significantly higher protein levels of FOXP3 (P=0.0201), MMP-13 (P=0.0048), and ADAMTS5 (P=0.0023), while lower levels of IL-21 (P < 0.0001) and COLⅡ (P=0.0092) compared with the control group. ② Flow cytometric analysis of peripheral blood revealed that the proportion of Tfh cells in acute-phase patients was significantly higher than that in the control group (P=0.0014) and the chronic-phase group (P < 0.0001); so was the Tfh/Tfr ratio when compared with that in the control group (P=0.0204) and the chronic-phase group (P < 0.0001); whereas the proportion of Tfr cells was lower than that in the control group (P=0.0155) and the chronic-phase group (P < 0.0001). ③ qRT-PCR in PBMCs showed that the mRNA levels of the Tfh-related cytokine IL-21 and transcription factor BCL-6 were upregulated in acute-phase patients than the control group (P=0.0008, P < 0.0001); those of the cytokine IL-10 and transcription factor FOXP3 were also enhanced in chronic-phase patients compared with the control group (both P < 0.0001). ④ ELISA results showed that serum levels of IL-21, IL-4, and IgG were significantly elevated in acute-phase patients, all higher than those in the control group (all P < 0.0001); these 3 indicators showed a decreasing trend in the chronic phase compared with the acute group (P < 0.05). In contrast, serum IL-10 level was significantly increased in the chronic phase, higher than that in the control group (P < 0.0001) and the acute-phase patients (P=0.0343). Serum IgA and IgM levels in patients in both acute and chronic phases were significantly higher than those in the control group (P < 0.05), but no significant difference was observed between the 2 phase groups. ⑤ Spearman correlation analysis indicated that, in the acute phase, the proportion of Tfh cells was positively correlated with IL-21, IgA, and IgM (r=0.26, 0.34, and 0.37, respectively), and negatively with Tfr cells (r=-0.54). IL-21 showed a strong positive correlation with IgG and IgM (r=0.75 and 0.58). In the chronic phase, the above correlations overall weakened, while correlations with Tfr-related indexes exhibited stronger associations compared with the other indicators.

This study preliminarily revealed the changes in Tfh/Tfr cell imbalance in BOA, with the acute phase characterized by Tfh cells and high expression of related factors, and the chronic phase by larger proportion of Tfr cells and high expression of related factors, accompanied by pathological joint damage.