Study On Tthe Mechanism Of Melatonin Inhibiting The Degradation Of Chondrocyte Matrix In OA Rats By Activating SIRT1/NF-κB/TGF-β1/Smad2

Osteoarthritis(OA)is a common degenerative joint disease with painful symptoms,characterized by chondrocyte apoptosis and an imbalance of extracellular matrix(ECM)catabolism.Melatonin(MT)is one of the hormones secreted by the pineal gland of the brain,which has antiinflammatory,antioxidant,anti-aging and circadian rhythm regulation functions.SIRT1(NADdependent deacetylasesirtuin-1,SIRT1)is a NAD-dependent nuclear histone deacetylase,involved in the regulation of aging,apoptosis,inflammation and other processes.Its deacetylation Capability can exert protective effects by modulating multiple pathways.In this study,an anterior cruciate ligament transection(ACLT)was used to establish a rat OA model in vivo,to observe the changes of melatonin on cartilage morphology,matrix and metabolic indicators,and to explore the effect of melatonin on SIRT1 expression in chondrocytes in vitro.The SIRT1 inhibitor EX527 was introduced to detect the expression of NF-κB and TGF-β1/Smad2 pathway-related proteins and chondrocyte matrix damage-related proteins.The purpose of this study was to investigate the effect of melatonin on rat OA cartilage and to explore its mechanism of action.In vivo experiments were divided into three groups,control group,model group,and melatonin treatment group(30 mg/kg),once every two days for 12 consecutive weeks,and then sacrificed after12 weeks.The tibial plateaus of each group were taken to make tissue sections.Safranin O fast green staining was used to observe the histopathological changes,and immunohistochemistry was used to detect the expressions of collagen type II,Aggrecan,MMP3,MMP13 and ADAMTS-4 in cartilage tissue.The changes of TNF-α,COX-2,PGE2 and i NOS in rat serum were detected by ELISA.In vitro experiments were divided into control group(Control),model group(IL-1β),melatonin group(IL-1β+100,200,400,800 ng/m L),negative control group(IL-1β+EX527),inhibitor group(IL-1β+melatonin+EX527).The rat knee articular chondrocytes were extracted and cultured to the second generation for processing.Protein and RNA were extracted,and Western blot was used to detect the changes of p-65,p-p65,IκBα,p-IκBα,TGF-β1,and Smad2 proteins.Changes in SIRT1,MMP3,MMP13,ADAMTS-4,COX-2,and COL2a1 genes were detected by q PCR.The nuclear translocation of SIRT1,p-p65 and p-Smad2 was detected by immunofluorescence.In vivo experiments found that the cartilage was severely worn,the cartilage was thinned,edema,and the matrix red staining became shallow and uneven in the modeling group,while the cartilage in the melatonin group was thickened,and the matrix red staining was relatively deep and uniform.Immunohistochemistry showed that the expressions of MMP3,MMP13 and ADAMTS-4in the modeling group increased,and the expressions of Aggrecan and type II collagen decreased.Melatonin significantly down-regulated the expressions of MMP3,MMP13 and ADAMTS-4,and increased the expressions of Aggrecan and type II collagen.Collagen expression.The expression levels of TNF-α,COX-2,PGE2 and i NOS in the serum of the modeling group were increased,and the melatonin group also significantly changed this trend.In vitro results found that the expression of SIRT1 and COL2a1 was decreased in the IL-1β group,and melatonin up-regulated their expression,but the addition of the inhibitor changed this situation.The expressions of p-p65,pIκBα,MMP3,MMP13,ADAMTS-4,COX-2 and i NOS were increased in the IL-1β group,and significantly decreased in the melatonin group,and the addition of the inhibitor reversed this trend.IL-1β inhibited the expression of TGF-β1 and Smad2,and the melatonin group restored their expression significantly,but the addition of the inhibitor EX527 did not change this trend.The experimental results show that: in vivo,melatonin can reduce the secretion of cartilage matrix degrading enzymes MMP3,MMP13,ADAMTS-4 and inflammatory factors,and increase the expression of type II collagen and Aggrecan.In vitro,melatonin can inhibit IL-1β-induced degradation of rat chondrocyte matrix by activating the SIRT1/NF-κB/TGF-β1/Smad2 pathway.This experiment provides new theoretical support for exploring the therapeutic effect and mechanism of melatonin in OA,and provides a new target for alleviating the progression of OA.