Melatonin Protects Osteoarthritis-induced Cartilage Degradation Via Upregulating Mir-140-5p

Objective:Osteoarthritis(OA)is a common degenerative disease which manifests as destruction of articular cartilage,sclerosis of subchondral bone,and synovium hyperplasia and ligament laxity.Most OA patients complain about painful symptoms,while ankylosis,disability,and malformation may occur at the end stage.For early OA,basal therapy and medicine treatment are recommended and arthroplasty is applied for end-staged patients.However,how to avoid degradation of cartilage is still a pivotal problem in OA treatment.It is known that deterioration of Extracellular Matrix(ECM)including Collagen II and ACAN is important during the OA progress.Meanwhile,activation of matrix degrading enzymes such as MMPs and AD AMTS are also involved in this process.Recently,miRNAs are reported to participate in the progression of OA and miR-140-5p is considered as a crucial role.Melatonin(MT)is a kind of hormone that secreted from pineal gland,which has multiple biological functions,for example anti-oxidant,anti-inflammation,and anti-aging.Studies have revealed that MT enhances the chondrogenic differentiation of mesenchymal stem cells(MSCs)and regulates expression of several miRNAs,In brief,the present study is designed to investigate:1.the protective effect of MT on chondrocytes in OA environment;2.the regulation of MT on miR-140-5p;3.the amelioration of MT on OA in vivo.Methods:In vitro experiment:we cultured human chondrocytes in normal and in OA environment(mimicked by IL-1?)and observed the cellular state under different concentrations of MT.CCK-8 was used to detect the cell proliferation and immunofluorescence was performed to detect the expression of Collagen ?.RT-PCR and Western Blot were conducted to verify the alteration of mRNA and protein levels of cartilage related anabolic and catabolic enzymes,Moreover,miRNA inhibitor was added to examine the regulation function of MT on OA.In vivo experiment;we built mice OA model using DMM surgery and injected MT via intra-articular method.Histological staining and immunohistochemical analysis were used to assess the degrading score of articular cartilage.In addition,miRNA antagomir was injected to further verify the regulation of MT on OA.Results:In vitro experiment,our results indicated that:1.MT promoted the proliferation of chondrocytes in normal environment;2.MT increased the expression of anabolic enzymes(Collagen ?,ACAN,and SOX-9)in normal environment;3.MT enhanced the expression of anabolic enzymes(Collagen ?,ACAN,and SOX-9)as well as suppress the expression of catabolic enzymes(MMP-9,MMP-13,ADAMTS-4,and ADAMTS-5)in OA environment;4.MT increased the expression of miRNA-140-5p via RT-PCR;5.miRNA-140-5p inhibitor decreased the expression of anabolic enzymes and increased the expression of catabolic enzymes.In vivo experiment,our results indicated that:1.Intra-articular injection of MT ameliorated the OA progress by decreasing loss of glycosaminoglycan,increasing expression of Collagen ?,and decreasing expression of Collagen I;2.miRNA-140-5p antagomir weaken the protective effect of MT on OA.Conclusion:Our experiment demonstrates that melatonin protects articular cartilage from OA-induced degradation by targeting miR-140-5p.Intra-articular administration of melatonin may benefit OA patients.