Paeoniflorin Ameliorates Osteoarthritis via Ring Finger Protein 216/Optineurin‐Mediated Autophagy Activation in Chondrocytes

ABSTRACT Osteoarthritis (OA) is a prevalent degenerative joint disorder. Paeoniflorin (PF), a primary bioactive compound from Paeonia lactiflora roots, demonstrates therapeutic potential for OA. This study investigated the effects and mechanisms of PF in OA. In vitro, an OA model was established using interleukin‐1β (IL‐1β)‐treated chondrocytes. In vivo, an OA mouse model was generated via destabilization of the medial meniscus (DMM). Molecular docking, bioinformatics analysis, and experimental validation identified ring finger protein 216 (RNF216) as a PF target. RNF216 overexpression and optineurin (OPTN) overexpression were achieved via genetic manipulation. Autophagy was evaluated by Western blot, transmission electron microscopy, and monomeric red fluorescent protein (mRFP)‐green fluorescent protein (GFP)‐microtubule‐associated protein 1A/1B‐light chain 3 (LC3) reporter. PF attenuated chondrocyte injury in vitro and alleviated OA progression in vivo. Through molecular docking and cellular thermal shift assay (CETSA), we identified carbonic anhydrase IX (CAIX, CA9) as a direct target of PF. RNF216 overexpression abolished PF's chondroprotective effects, which were rescued by OPTN overexpression. The autophagy inhibitor 3‐methyladenine (3‐MA) blocked PF‐mediated chondroprotection via the RNF216/OPTN axis. Mechanistically, PF inhibited RNF216‐dependent ubiquitination and degradation of OPTN, enhancing autophagic flux to mitigate cartilage damage. PF alleviates OA by suppressing RNF216‐mediated OPTN ubiquitination, stabilizing OPTN to activate autophagy and protect chondrocytes. These findings highlight PF as a promising therapeutic agent for OA.