Gubi Zhitong formula alleviates knee osteoarthritis induced by excessive mechanical loading in rats via regulating Piezo1 mediated Ca2+ signaling pathway and phosphoinositide 3-Kinase / protein kinase B / nuclear factor kappa-B axis

Ethnopharmacological relevance: Knee osteoarthritis (KOA) is a progressive chronic disease characterized by degenerative lesions of articular cartilage and secondary osteophyte formation. Gubi Zhitong Formula (GBZTF) is a Traditional Chinese Medicine prescription used for the treatment of KOA. Composed of Rehmannia glutinosa, Epimedium brevicornum, Cistanche deserticola, Pyrola calliantha, Drynaria roosii, Spatholobus suberectus, and Raphanus raphanistrum subsp. sativus, it has the effects of tonifying the kidney, promoting blood circulation, and relieving pain, but its mechanism of action remains unclear.

Aim of the study: To investigate the therapeutic effect of GBZTF and its potential mechanism in improving KOA induced by excessive mechanical loading in rats.

Method: Excessive mechanical loading was applied to rat right knee joints to establish KOA models using a static testing system in a dynamic cyclic loading mode. Behavioral and bone changes were evaluated using gait analysis, weight-bearing tests, Micro-CT scans, Western blotting, qRT-PCR, and histological staining across all experimental groups. The mechanism underlying the beneficial effects of GBZTF on KOA was elucidated through transcriptome Sequencing and subsequent validation.

Results: In this study, it was found that GBZTFM (2.42 g/kg/day) significantly improves exercise capacity of OA rats (P < 0.05), alleviates cartilage destruction (P < 0.05) and subchondral metabolic dysregulation (P < 0.05). It also downregulates key pro-inflammatory mediators in serum and joint fluid, including IL-1β (P < 0.01), IL-6 (P < 0.01) and TNF-α (P < 0.05), while enhancing anti-inflammatory factors TGF-β (P < 0.05) and IL-10 (P < 0.05). Transcriptomics analysis revealed 846 and 5125 differentially expressed genes (DEGs) between the Control and Model groups, and between the Model and GBZTF groups, respectively. Enrichment analysis revealed significant involvement of the CA²⁺ signaling, NF-κB signaling, and PI3K-AKT signaling pathways in both sets of DEGs. Western blot and immunofluorescence assays verified that GBZTF reduces articular cartilage damage and mitigates periarticular inflammation in OA rats by modulating the Piezo1-Ca²⁺/PI3K-AKT/NF-κB axis.

Conclusions: Through a series of experiments and transcriptome Sequencing, this study first reveals at the molecular level the considerable efficacy of GBZTF in ameliorating KOA induced by excessive mechanical loading. It also elucidates the underlying molecular mechanisms, providing a rationale to clinically apply GBZTF, this herbal compound, against KOA.

Articular cartilage; Gubi Zhitong formula; Knee osteoarthritis; Piezo1-Ca(2+)/PI3K-AKT/NF-κB signaling pathway.