Effect Of Inhibited Chondrocyte Mitophagy On The Expression Of Osteoarthritis-related MMP-1 And MMP-13

The morbidity of osteoarthritis(OA)increased year by year in middle-aged and elderly,the pathogenesis of OA is very complicated and not yet fully understood.Cartilage degeneration is one of the most prominent features of OA joint degeneration.As the only cell in cartilage,the degeneration of chondrocytes becomes the core of the research.Mitochondria not only generate energy but also mediate other key biological behaviors in chondrocytes.They can affect the development of OA from many aspects such as cell autophagy,aging,cell death.Mitochondrial dysfunction seriously affects the survival of chondrocytes,so mitochondria damage become a key link in the pathogenesis of OA.Damaged mitochondria are mainly degraded by mitophagy.Therefore,mitophagy becomes an important regulatory mechanism for maintaining the quantity and quality of mitochondria,which maintains the homeostasis of chondrocytes and their survival.In addition mitophagy can reduce the production of reactive oxygen species(ROS)and the activation of inflammatory cytokines.However,the role of mitophagy in the pathogenesis of OA remains unclear.Objective To investigate the effect of mitophagy inhibited by cyclosporin A(Cs A)on the expression of osteoarthritis-related MMP-1 and MMP-13 in chondrocyte.Methods Patients who underwent knee arthroplasty were clinically diagnosed as osteoarthritis(OA).Knee arthroplasty required the interception of the cartilage-bearing surface bone.The tibial plateau and condyles of the femoral containing cartilage were sent to the laboratory under aseptic and low-temperature condition.they wereseparated on a clean bench to cultivate the chondrocytes.Toluidine blue staining and type II collagen immunofluorescence staining were applied to identify chondrocytes because type II collagen and proteoglycans are markers of chondrocytes.Interleukin1?(IL-1?)was used to stimulate chondrocytes in vitro as OA was a sterile inflammatory disease.In addition Cs A is a common tool to inhibit mitophagy.Cs A inhibited mitophagy under the condition of IL-1? stimulated chondrocytes.Chondrocytes was divided into control group,IL-1? group and IL-1? +Cs A group.Damaged mitochondria are mediated into autophagosomes by specific proteins,which fuse with lysosomes.The process is mitophagy.Mitochondria mass and mitochondrial proteins are reduced when mitophagy is increased.Tom20 is a mitochondrial outer membrane protein receptor.Expression of Tom20 protein and immunofluorescence localization was performed for mitochondria labeled with Mitotracker Deep Red(MTR),autophagosomes labeled with Lysotracker green(LTG)to observe mitophagy.MMP-1 and MMP-13 are markers of OA.Real-time PCR was used to measure the expression of m RNA of MMP-1 and MMP-13.Western blot was used to measure the expression of proteins of Tom20,MMP-1 and MMP-13.Results Compared with the normal group,the IL-1? group had a significant increase immunofluorescence colocalization of MTR and LTG,a signifcant reduction in the expression of the mitochondrial outermembrane protein Tom20(P<0.05),and a significant increase in the expression of the m RNA and proteins of MMP-1 and MMP-13(both P < 0.05).These results indicated that IL-1? could damage mitochondria to increase the expression of mitophagy,MMP-1 and MMP-13.Compared with the IL-1? group,the IL-1?+Cs A group had a significant reduction in the immunofluorescence colocalization of MTR and LTG,a significant increase in the expression of protein Tom20(P<0.05),and a significantly greater increase in the expression of the m RNA and proteins of MMP-1 and MMP-13(both P<0.05).These results indicated that Cs A could inhibit mitophagy of chondrocytes induced by IL-1?,and further increase the expression of MMP-1 and MMP-13.Conclusion Cs A can inhibit the degree of mitophagy in chondrocyte,and the inhibition of mitophagy can increase expression of osteoarthritis-related MMP-1 and MMP-13.