RLX-2 ameliorates post-traumatic joint contracture by inhibiting the cGAS-STING signaling pathway.

OBJECTIVES: Post-traumatic joint contracture (PTJC) is a debilitating fibrotic disorder whose underlying molecular drivers, particularly the involvement of cellular senescence, remain poorly elucidated. This study aimed to evaluate the therapeutic potential of Relaxin-2 (RLX-2) in attenuating knee joint fibrosis and to explore its regulatory effects on the cGAS-STING signaling axis. METHODS: In vitro fibrotic phenotypes were recapitulated using TGF-β1-stimulated rat synovial fibroblasts. An in vivo PTJC model was established via surgical trauma combined with internal fixation-induced immobilization in rats. The anti-fibrotic and anti-senescent properties of RLX-2 were characterized by quantifying markers such as Collagen I, α-SMA, p16, and p53. Transcriptomic profiling via RNA-sequencing was employed to identify potential signaling hubs. The mechanistic involvement of the cGAS-STING pathway was further interrogated using the pharmacological inhibitor H-151 in both experimental settings. RESULTS: In vitro, RLX-2 treatment exerted a concentration-dependent inhibitory effect on TGF-β1-induced fibrogenic transition and cellular senescence. In the rat model, intra-articular administration of RLX-2 resulted in a significant reduction in synovial hyperplasia and extracellular matrix deposition. These phenotypic improvements were associated with the downregulation of senescence markers in the synovium. Mechanistically, RNA-seq analysis pointed toward the cGAS-STING pathway as a primary target of RLX-2. RLX-2 administration was associated with decreased phosphorylation of STING and its downstream effector TBK1. Notably, pharmacological blockade of STING by H-151 partially phenocopied the protective effects of RLX-2, suggesting a coordinated regulation of fibrosis and senescence. CONCLUSION: Our findings demonstrate that RLX-2 attenuates post-traumatic knee joint fibrosis in rats, a process associated with the modulation of the cGAS-STING-senescence axis. These results suggest that targeting the cGAS-STING pathway may represent a viable strategy for managing PTJC, with RLX-2 serving as a promising pharmacological candidate for further clinical investigation.