Peptide as an Anti-Inflammatory Drug for the Treatment of Osteoarthritis

Osteoarthritis (OA) is a debilitating condition characterized by chronic inflammation and degradation of articular cartilage, affecting millions worldwide. Peptides have emerged as promising candidates for OA therapy due to their inherent bioactivity, target specificity, and reduced adverse effects compared to traditionally prescribed drugs. In this study, we investigated the therapeutic potential of the plasma transmembrane domain associated p65 (PTD-p65) peptide, which targets the nuclear factor-kappa B (NF-κB) pathway, a key regulator of inflammation in OA progression. This PTD-p65 peptide was combined with an additional peptide sequence tag to potentiate its anti-inflammatory properties. In this study, we evaluated the potency of PTD-p65 peptide and its derivatives: tag-PTD-p65, PTD-tag-p65, and PTD-p65-tag. Primary human OA-articular chondrocytes (OA-hACs) or a stably transduced NFκB response element driven Nano luciferase SW1353 reporter cell line were used. Either SW1353 reporter cells or OA-hACs were cultured overnight in 96-well plates with DMEM/F12 supplemented with 10% FCS. On Day 1, cells were pre-incubated with the peptide (50 μM) for 1 hour, followed by stimulation with TNF-α (1 ng/ml) for 5 hours. After 6 hours, the Nano luciferase signal was quantified using furimazine as a substrate with a Tristar2 plate reader. Expression of relevant cytokines, IL6, and CXCL8 was measured using ELISA. The expression of downstream NF-κB target genes COX2, IL6, and CXCL8 was analyzed by RT-qPCR. The potential cytotoxicity of the peptides was analyzed by LDH assay. OA-synovial fluid (OA-SF) was derived from total knee arthroplasty. All patient material received medical ethical approval. The PTD-tag-p65 peptide demonstrated potentiated inhibitory capacity of TNFα driven NF-κB activation compared to PTD-p65 and other peptide derivatives (tag-PTD-p65, PTD-p65-tag). This was evidenced by reduced luciferase activity, decreased expression of NF-κB target genes (COX2, CXCL8, IL6), and lower secreted IL6 and CXCL8 levels in the SW1353 reporter cell line. Apart from the tag-PTD-p65, the other peptides showed no cytotoxicity, and the tag alone did not show any significant effects on all measured parameters. The PTD-tag-p65 peptide decreased OA-SF-induced NF-κB activation in SW1353 cells. Effectivity of the PTD-tag-p65 peptide in lowering inflammatory cytokine expression could be validated in primary human OA chondrocytes. Our findings suggest that the PTD-tag-p65 peptide effectively suppresses NF-κB-mediated inflammatory responses in vitro, markedly better than the original PTD-p65 peptide. This novel peptide offers a promising therapeutic strategy for modulating OA-associated inflammatory processes, potentially addressing the critical need for disease-modifying treatments in OA.