Therapeutic Effect And Mechanism Of MiRNA-146a-5p On Temporomandibular Joint Osteoarthritis In Rats

Objectives:Osteoarthritis is the most common musculoskeletal disease,mainly manifested as joint pain,stiffness,limited activity,occasional effusion,different degrees of local inflammation,etc.,which seriously affect the quality of life of patients.Osteoarthritis has a high incidence rate worldwide,and the governments of various countries spend a lot of money on bone and joint treatment every year.For decades,many scholars have made a lot of research on the etiology,pathogenesis and treatment of osteoarthritis.As the most important joint in the maxillofacial region,the temporomandibular joint plays an important role in speech,swallowing,expression and facial development.Like other osteoarthritis,temporomandibular joint osteoarthritis is also a degenerative disease,which is characterized by progressive cartilage degeneration,soft bone remodeling,local inflammation,chronic pain and dysfunction.MicroRNA(miRNA)is a kind of endogenous non-coding RNA with a length of about 20-24 nucleotides.It can interact with homologous mRNA to regulate gene transcription by enhancing degradation,inhibiting translation or other mechanisms.Some miRNAs are closely related to the occurrence and development of arthritis.These miRNAs can regulate the genes related to cartilage development,cartilage homeostasis and pathological changes of osteoarthritis.miRNAs have broad application prospects for the prediction,diagnosis and treatment of osteoarthritis.There is a lack of research on the relationship between miRNAs and temporomandibular joint osteoarthritis.In this study,on the basis of establishing the rat model of temporomandibular joint osteoarthritis,we screened the differentially expressed miRNAs,and studied the therapeutic effect and mechanism of related miRNAs in temporomandibular joint osteoarthritis.This study provides an experimental basis for the clinical application of miRNAs in temporomandibular joint osteoarthritis or other arthritis in the future.Methods:In this study,IL-1 β and TNF-α were used to stimulate rat condylar chondrocytes to establish an inflammatory microenvironment model,the expression of related inflammatory factors(MMP9,MMP13,COX2,adamts5)and cartilage phenotype(COL2A1,Sox9)were detected by qRT-PCR and WB;Then the differentially expressed miRNAs in the inflammatory microenvironment of condylar chondrocytes were screened by high-throughput sequencing technology;Then the function of mir-146a-5p(up-regulated)was verified and its mechanism was studied by target gene screening,cell transfection,qRT-PCR.WB,dual luciferase and other experimental methods;Finally,the animal model of TMJ osteoarthritis was established by injecting IL-1β(10000ng/ml)and TNF-α(10000ng/ml)into TMJ cavity of rats.After injecting mir-146a-5p agomir into TMJ,the therapeutic effect of mir-146a-5p on TMJ osteoarthritis was investigated by HE staining,safranin o-solid green staining and immunohistochemistry.Results:The inflammatory microenvironment model of condylar chondrocytes was established by IL-1β and TNF-α,qRT-PCR and WB results showed that the expression of related inflammatory factors was significantly increased,while the expression of cartilage phenotype factors was decreased;High throughput sequencing results showed that the expression of 49 miRNAs changed(P<0.05),of which 23 were up-regulated and 26 were down regulated,and 13 of the 49 miRNAs had significant differences(P<0.01),there were 7 up-regulated miRNAs and 6 down regulated miRNAs,and the most significant change of these miRNAs was rno-mir-146a-5p(up-regulated);Condylar chondrocytes in inflammatory microenvironment were transfected with mirna-146a-5p mimic,the results of qRT-PCR and WB showed that the expression of related inflammatory factors(MMP9,MMP13,COX2,adamts5)was significantly decreased;Dual luciferase assay showed that rno-mir-146a-5p could directly regulate the expression of tnfsf4 through the predicted binding site(AAGUCAAGAG);After injection of IL-1β and TNF-αinto TMJ,cartilage phenotype protein type Ⅱ collagen and Sox9 were significantly decreased,and inflammatory factors MMP9 and COX2 were significantly decreased.The expression of MMP-9,COX-2,TNFSF4 and CD3 were decreased after mir-146a-5p agomir was injected into TMJ,while the expression of COL2al was increased and the Mankin’s score was decreased.Conclusion:The combined stimulation of IL-1β and TNF-α can establish an effective and stable model of temporomandibular joint osteoarthritis;MiRNA-146a-5p,as an inflammatory protective factor in temporomandibular joint osteoarthritis,plays an anti-inflammatory role by inhibiting the secretion of related inflammatory factors;MiRNA-146a-5p may regulate T cell-mediated immunity by targeting tnfsf4(ox401),thus inhibiting the occurrence of inflammation In inflammatory environment;Condylar chondrocytes may act as a non professional antigen presenting cell,and affect T cell-mediated immune function through the interaction of TNFSF4(OX40L)-OX40.