Study Of Molecular Mechanism Of Indoleamine 2,3 Dioxygenase 1 Promoting Chondrocyte Apoptosis

Objective: Osteoarthritis(OA)is the most common chronic degenerative disease affecting joint activity and causing joint pain.It is more common in the lesions of hip and knee joints.However,the exact pathogenesis of OA is still unclear,but it is known that chondrocyte apoptosis is one of the important reasons for the occurrence and development of OA.Targeting the pathological factors that mediate chondrocyte apoptosis is helpful to alleviate cartilage degeneration and joint pain.Indoleamine 2,3 dioxygenase 1(IDO1)is highly expressed in the synovial fluid of OA patients and can be used as a pathological marker of cartilage degeneration.However,whether the high expression of IDO1 in chondrocytes can promote chondrocyte apoptosis,and the molecular mechanism of IDO1 promoting chondrocyte apoptosis is still unclear.Therefore,this study aimed to explore the role and molecular mechanism of IDO1 in chondrocyte apoptosis.Methods:(1)In this study,human normal chondrocytes were isolated from the articular cartilage of patients with femoral neck fracture requiring joint replacement,and the chondrocytes were identified after passage to P2 generation.(2)The chondrocytes were co-cultured with different concentrations of IDO1(0ng/ml,10ng/m L,50ng/m L,100ng/m L),and then the CCK8 experiment was performed,and the apoptotic activity was detected after co-culture with 50ng/m L IDO1 for72 hours to establish a microenvironment model of chondrocyte apoptosis.(3)The total RNA of chondrocytes in the IDO1 group and the Cont group was collected and the differential changes of m RNA were screened by high-throughput sequencing technology.RT-q PCR and immunofluorescence were used to explore the molecular mechanism of IDO1 regulating chondrocyte apoptosis through m RNA.(4)The differential expression of miRNA in chondrocytes of IDO1 group and Ctrl group was screened by high-throughput sequencing technology,and Target Scan analysis and RT-q PCR experiment were used to explore the molecular mechanism of IDO1 regulating m RNA expression through miRNA.Results:(1)Primary chondrocytes were isolated and identified by the expression of type II collagen and proteoglycan in chondrocytes.(2)To explore the role of IDO1 in chondrocyte apoptosis,IDO1 and chondrocyte co-culture found that the chondrocyte was apoptotic state.The chondrocytes apoptosis significantly increased after 50ng/m L IDO1 for 72h,and the apoptosis rate was the fastest.(3)To explore the mechanism of IDO1 promoting chondrocyte apoptosis,the RNA of chondrocytes under the action of IDO1 was subjected to m RNA high-throughput sequencing,and it was found that the expression of m RNA-Fibronectin1(FN1)was significantly down-regulated.(4)The expression of FN1 and COL2A1 in chondrocytes under the action of IDO1 was decreased by RT-q PCR and immunofluorescence assay,and the expression of caspase3 was increased,indicating that IDO1 promotes chondrocyte apoptosis by down-regulating the expression of FN1.(5)To explore that IDO1 regulates the expression of FN1 by miRNA,the RNA of chondrocytes under the action of IDO1 was subjected to miRNA high-throughput sequencing,and it was found that the expression of miRNA-146a-5p in chondrocytes under the action of IDO1 was significantly up-regulated.(6)Bioinformatics analysis in Target Scan indicated that FN1 was an ideal downstream target of miRNA-146a-5p,and the RT-q PCR experiment found that the expression of miRNA-146a-5p was up-regulated,thereby inhibiting the expression of FN1.Conclusion:(1)As the concentration and time of IDO1co-cultured with chondrocytes increased,the ratio of apoptosis also increased,and the apoptosis rate of IDO1 and chondrocytes co-cultured at50ng/ml for 72 h was the fastest.(2)In chondrocytes,IDO1 promotes chondrocyte apoptosis by down-regulating FN1.(3)In chondrocytes,IDO1 inhibits the expression of FN1 by up-regulating miRNA-146a-5p.Therefore,the molecular mechanism of IDO1 in chondrocyte apoptosis in this experiment is that IDO1 upregulates miRNA-146a-5p,inhibits the expression of FN1,and promotes chondrocyte apoptosis.