Phenotypic screening identified polydatin alleviating cartilage degeneration by modulating SIRT3-dependent mitochondrial dysfunction.

BACKGROUND: Osteoarthritis (OA) is a prevalent degenerative disease, characterized by articular cartilage lesions, synovial inflammation, and osteophyte formation, which ultimately results in joint deformity and limited mobility. Polyphenols, a class of bioactive phytochemicals widely distributed in traditional Chinese medicine, have been extensively utilized for centuries in the management of OA.

PURPOSE: Despite extensive research efforts, effective pharmacological interventions for treating OA have yet to be fully realized. This study aimed to identify anti-inflammatory polyphenols as potential therapeutic alternatives for osteoarthritis treatment.

METHODS: The high-content screening (HCS) technique was utilized to identify active compounds from a polyphenol library. An interleukin-1β (IL-1β) stimulated inflammatory model, which significantly increased the expression of chondrocyte degradation markers such as matrix metallopeptidase 13 (MMP-13), was established in vitro. Polydatin (PD) was identified as a promising substance for attenuating MMP-13 expression and promoting type II collagen (COL2) expression. Subsequently, destabilization of the medial meniscus (DMM) and monosodium iodoacetate (MIA) mouse models of OA were utilized to evaluate the efficacy of PD in vivo. After model establishment and drug administration, knee joints were harvested at 4 and 12 weeks post-treatment and subjected to radiological and histological assessments. Finally, mitochondrial function assays were conducted to further explore the underlying therapeutic mechanisms.

RESULTS: Within a library of 16 polyphenolic compounds, HCS-based phenotypic screening identified six active compounds that potently inhibited the expression of MMP-13. PD exhibited promising anti-inflammatory effects in a dose-dependent manner. Western blot and immunofluorescence analyses revealed that PD markedly suppressed MMP-13 synthesis and COL2 degradation in a dose-dependent manner. The RNA sequencing results revealed that the anti-inflammatory efficacy of PD may be associated with the Wnt signaling pathway. Three-dimensional micro-CT reconstructions revealed a reduction in osteophyte formation in the PD treatment groups relative to the OA model groups. Safranin O and fast green (SO&FG) staining, hematoxylin and eosin staining, and COL2 immunofluorescence staining revealed that PD treatment alleviated cartilage matrix breakdown and reduced osteoarthritis scores. Furthermore, PD upregulated the expression of sirtuin 3 (SIRT3) and superoxide dismutase 2 (SOD2), thereby increasing mitochondrial membrane potential and reducing mitochondrial superoxide (mtROS) levels, suggesting antioxidant-driven anti-inflammatory effects.

CONCLUSION: This study successfully employed the HCS technique to identify polydatin from a polyphenol library as an active compound for treating OA. PD alleviates cartilage degradation and lesions by activating the SIRT3/SOD2/mtROS axis, increasing the mitochondrial membrane potential, and exerting antioxidant effects.