The Study Of Autophagy In The Treatment Of Osteoarthritis By Inhibiting TRAF3/NLRP3

Objective:Osteoarthritis(OA)is the most common joint disease.Its etiology is multifactorial,including the excessive production of reactive oxygen species(ROS).Autophagy,as an intracellular degradation system to maintain the stable state of intracellular energy metabolism,has been proved to restore the function of damaged chondrocytes and slow down the occurrence and development of OA.With the development of OA,the main negative regulator of autophagy,rapamycin target protein(mTOR),is up-regulated,which mediates the inhibition of autophagy signal transduction pathway in articular cartilage.Inflammasome such as NLRP3,induced by nuclear factor-κB(NF-κB)signal,can transform interleukin-1β(IL-1β)and IL-18 into mature pro-inflammatory cytokines,which is considered to be an important factor in inflammatory pathology.Although more and more evidence shows that NLRP3 and autophagy pathway are related through mutual regulation,the relevant regulatory mechanism in osteoarthritis has not been fully elucidated.TRAF3 is a member of the tumor necrosis factor receptor associated factor(TRAF)family.As a adaptor protein and E3 ubiquitin ligase,TRAF3 participates in a variety of immune pathways,eventually leading to the activation of transcription factors such as NF-kB and mitogen activated protein kinases.This study showed that TRAF3 was highly expressed in OA chondrocytes,but whether its existence in chondrocytes was regulated by autophagy pathway and whether it regulated NLRP3 were not clear.This study will clarify the regulation and influence of autophagy pathway on TRAF3/NLRP3 in rat chondrocytes through in vivo and in vitro experiments.Methods:1.In vivo experiment.Our previous experiments confirmed that moderate treadmill exercise can activate autophagy.After making OA model of knee joint,SD rats were divided into three groups:blank control group,OA group,OA+moderate intensity exercise group(OAM group).The expression of traf3 and other related proteins were detected by immunohistochemistry.2.In vitro experiment.The primary chondrocytes were cultured and then added with IL-13 and rapamycin.They were divided into four groups:blank control group,OA group,rapamycin group(Rapa group),OA+rapamycin group(OA+Rapa group).3.The expressions of MMP13,NLRP3,p62,LC3B and Beclinl in chondrocytes were detected by PCR and Western blotting.4.The changes of reactive oxygen species were detected by flow cytometry.5.The changes of cells were observed by transmission electron microscope.Results:1.Compared with normal rats,OA rats had less type Ⅱ collagen and more inflammatory factors such as TRAF3,MMP13 and NLRP3,while in moderate exercise group,OA rats had less autophagy related factors.2.The results of PCR and WB showed that IL-1 β had a certain degree of inflammatory effect on chondrocytes.After rapamycin activated autophagy,TRAF3 and inflammatory related factors decreased,autophagy related factors LC3 and Beclinl increased and p62 decreased,which confirmed that rapamycin activated autophagy had a protective effect on chondrocytes.3.Flow cytometry showed that reactive oxygen species accumulated in OA group and decreased after rapamycin treatment.4.Transmission electron microscopy showed that the cell morphology of inflammatory cells would be nuclear shrinkage,cell swelling and rupture.The activation of autophagy increases the number of autophagosomes and improves cell damage.Conclusion:1.TRAF3 and NLRP3 are closely related to the occurrence and development of osteoarthritis,and can be used as the indicator protein of osteoarthritis.2.Moderate intensity treadmill exercise and autophagy activation pathway induced by rapamycin can reduce the expression of TRAF3 and NLRP3 in Osteoarthritis Chondrocytes of OA rats,which has a certain degree of protective effect on cartilage damage.3.The activation of autophagy reduced the content of ROS in OA chondrocytes,increased autophagosomes in chondrocytes,and inhibited the changes of cell morphology,which may be due to the stress activation of mitochondria,which cleared the accumulated ROS.4.Transmission electron microscopy showed that the morphology of inflammatory cells changed,and the activation of autophagy increased autophagosome and improved cell damage.