COX-2 and iNOS are critical in advanced glycation end product-activated chondrocytes in vitro

Background The advanced glycation end products (AGEs) accumulate in joints of osteoarthritis patients. This study aimed to investigate the roles of cyclooxygenase‐2 (COX‐2) and inducible nitric oxide synthase (iNOS) pathways in AGE‐mediated cartilage damage. Materials and methods Methylglyoxal‐modified albumin was used as the source of AGE. Porcine and human chondrocytes were prepared from the joint cartilage of pigs and osteoarthritis patients. The activation of COX‐2, iNOS, nuclear factor‐kappaB (NF‐κB), activator protein‐1 (AP‐1) and protein kinases was determined by Western blotting, kinase assay, electrophoretic mobility shift assay (EMSA) or transfection assay. Prostaglandin E₂ (PGE₂) and NO concentrations were determined by enzyme‐linked immunosorbent assay (ELISA) and Griess reaction respectively. The enzymatic activity of COX was determined by measuring the conversion of arachidonic acid to PGE₂. The release of sulphated glycosaminoglycan and the intensity of Safranin O staining were used to measure cartilage degradation. Results AGE potently induced COX‐2‐PGE₂ and iNOS‐NO activation in porcine and human chondrocytes. Meanwhile, the upstream molecules regulating COX‐2/iNOS activation, such as AP‐1, NF‐κB, extracellular signal regulated protein kinase (ERK) and c‐jun N‐terminal kinase (JNK), were activated by AGE. Although AGE could not activate p38 directly, by measuring COX enzyme activity, the inhibition of p38 resulted in suppressing AGE‐induced conversion of arachidonic acid to PGE₂. Furthermore, successful blockage of either COX‐2 or NOS activity significantly reduced AGE‐mediated proteoglycan release and cartilage degradation. Conclusions This study highlights the significance of COX‐2 and iNOS pathways in AGE‐mediated OA pathogenesis and their potential as therapeutic targets that are beyond pain killing for OA treatment.