KLK8 promotes cartilage degradation and pyroptosis in osteoarthritis by activating the NF-κB and NLRP3 inflammasome pathways.

Cartilage degradation, inflammation, and pyroptosis are key drivers of Osteoarthritis (OA) pathogenesis, contributing to chondrocyte impairment. However, the therapeutic potential of KLK8, a cartilage-associated protease, remains unexplored in OA. KLK8 expression was detected in meniscal/ligamentous injury (MLI)-induced OA mouse models and human OA cartilage tissues. KLK8 expression in chondrocytes stimulated with interleukin-1β (IL-1β) was also detected and quantified. Small interfering RNA (siRNA) was used to inhibit KLK8 in IL-1β-stimulated chondrocytes and its influence on the expression of cartilage catabolic factor and inflammatory factors expression were examined. The mechanism through which KLK8 regulates NF-κB activity was investigated. Chondrocyte pyroptosis were also measured. MLI-induced OA in mice were used to investigate KLK8's in vivo therapeutic potential. KLK8 expression was significantly upregulated in articular cartilage from OA patients and mice models. IL-1β stimulation increased KLK8 levels in chondrocytes in vitro. Functional assays showed that KLK8 knockdown attenuated IL-1β-induced inflammatory responses and extracellular matrix degradation in chondrocytes. KLK8 directly interacts with IκBα to promote its cytoplasmic degradation, which leads to p65 release, nuclear translocation and extended nuclear retention, thus enhancing NF-κB activity in IL-1β-stimulated chondrocytes. Furthermore, the levels of NLRP3, ASC and cleaved Caspase-1 were decreased after IL-1β stimulation in KLK8-deficient chondrocyte, indicating a stimulatory effect on NLRP3 inflammasome activation. Flow cytometry showed reduced chondrocyte pyroptosis after KLK8 knockdown, and ELISA detected decreased TNF-α, IL-6, and IL-18 levels, indicating KLK8 regulates chondrocyte pyroptosis. In vivo, KLK8 knockdown mitigated OA progression and reduced the expression of NLRP3, MMP13, COL2A1, and IL-18 in OA mice. These findings indicate that KLK8 promotes cartilage degradation, inflammation and pyroptosis via activating the NF-κB and NLRP3 inflammasome pathways. In vivo, KLK8 inhibition significantly alleviates OA progression, highlighting KLK8 as a potential therapeutic target for OA.