A Novel Inducible Nitric Oxide Synthase Inhibitor Alleviates The Progression Of Rat Osteoarthritis

Objective Osteoarthritis（OA）is a degenerative joint disease.Excessive nitric oxide（NO）mediates the chondrocyte inflammatory response,apoptosis,and extracellular matrix（ECM）degradation during the occurrence and development of OA.NO in chondrocytes is mainly produced by inducible nitric oxide synthase（i NOS）.The aim of this study was to design and synthesize an i NOS dimerization inhibitor and evaluate its effects on chondrocyte inflammation and articular cartilage injury in OA via in vitro and in vivo experiments.Methods Rat chondrocytes were extracted and cultured.The cytotoxicity of compound22 o at different concentrations（5,10,20 and 40μM）to chondrocytes was studied by MTT assay.The chondrocyte model of OA was constructed with IL-1β.The experiment was divided into 5 groups: control group,IL-1β group,IL-1β+5μM 22 o group,IL-1β+10μM 22 o group,and IL-1β+20μM 22 o group.Nitric oxide synthase assay kit was used to determine the effect of compound 22 o on the activity of i NOS in chondrocytes.The effect of compound 22 o on the content of NO in chondrocytes was determined by NO assay kit.Western Blot and RT-q PCR were used to detect the effect of compound 22 o on the expression of i NOS,ACAN,COL2A1,MMP3 and COX-2protein in chondrocytes of each group.Anterior cruciate ligament transection（ACLT）was used to construct the rat model of OA,and the rats were administered intragastrically for 6 weeks.The rats were divided into 5 groups: normal group,sham operation group,ACLT+ vector group,ACLT+40mg/kg 22 o group and ACLT+80mg/kg22 o group.The rats were sacrificed 6 weeks later.Removing the left knee joint,heart,liver,spleen,lung and kidney.Then proceed to histopathological analysis.Results Compound 22 o was not toxic to rat chondrocytes.It inhibited the formation of i NOS dimer in OA chondrocytes induced by IL-1β.It inhibited the IL-1β-induced expression of COX-2 and MMP3 in the chondrocytes,decreased NO production,and significantly increased the expression levels of the ECM anabolic markers,ACAN,and COL2A1.Gavage with compound 22 o was found to be effective in the rat OA model induced by ACLT,wherein it regulated the anabolism and catabolism and exerted a protective effect on the articular cartilage.The concentration of compound 22 o used in the experiment was not toxic to rats.Conclusions Compound 22 o showed effective i NOS inhibitory activity by inhibiting the dimerization of i NOS.It inhibited the inflammatory response and catabolism of the chondrocytes and reduced articular cartilage injury in the rat OA model,indicating its potential as a disease-modifying OA drug.