Hyaluronan and proteoglycan link protein 1 (HAPLN1) mitigates the development of osteoarthritis by suppressing chondrocyte senescence mediated by Foxo3a

Background: Osteoarthritis (OA) is a prevalent degenerative joint disease marked by the continuous deterioration of cartilage and functional limitations, impacting millions, with few disease-modifying treatments available. The extracellular matrix protein hyaluronan and proteoglycan link protein 1 (HAPLN1) stabilises aggrecan-hyaluronan complexes in cartilage; nonetheless, its function in osteoarthritis pathogenesis is inadequately comprehended. This research examined HAPLN1 expression in osteoarthritis models and its impact on chondrocyte senescence. Methods: Primary articular mouse chondrocytes were extracted from neonatal C57BL/6 mice and grown in DMEM/F12 conditions. OA-like conditions were produced by TNF-α (10 ng/ml) or tert-butyl hydroperoxide (TBHP, 50 μM). HAPLN1 knockdown was accomplished using lentiviral shRNA transfection. Recombinant mouse HAPLN1 (rmHAPLN1, 2.5 ng/ml) was employed for rescue. SA-β-Gal staining, p16/p21 expression, and telomerase activity were used to measure senescence. Matrix breakdown indicators (MMP-13, Col2a1) and inflammatory cytokines (CXCL10, IL-1β, MCP-1) were evaluated by qPCR, Western blot, and ELISA. Oxidative stress was measured by ROS levels, γ-H2AX, SOD/CAT activity, and Foxo3a phosphorylation/localization. In vivo , post-traumatic OA was generated by destabilisation of the medial meniscus (DMM) in 8-week-old male mice, with intra-articular lentiviral HAPLN1 administration at weeks 1 and 4. Krenn’s approach was used to assess synovitis, whereas OARSI grading was used to score cartilage integrity. Results: TNF-α and TBHP-stimulated chondrocytes and cartilage from DMM-operated animals showed significantly lower levels of HAPLN1 mRNA and protein. Silencing HAPLN1 dramatically accelerated extracellular matrix breakdown, inflammatory cytokine release, cellular senescence, and oxidative stress, which was mediated by increased Foxo3a phosphorylation at Ser253/Thr32 and cytoplasmic retention. In contrast, supplementation with rmHAPLN1 or intra-articular lentiviral-mediated HAPLN1 overexpression improved these changes both in vitro and in vivo , considerably decreasing OARSI cartilage damage and synovitis scores when compared to untreated OA controls. Conclusion: Our results show that HAPLN1 protects against OA by reducing Foxo3a-induced chondrocyte senescence and oxidative stress, making it a suitable therapeutic target.