Research On The Mechanism Of ATF4-modified Serum Exosomes In The Treatment Of Osteoarthritis By Inducing Autophagy

ObjectiveOsteoarthritis(Osteoarthritis,OA)is one of the most common chronic crippling diseases involving the whole joint in the world.The main pathogenesis of OA is the degeneration of articular cartilage,that is,the apoptosis and aging of chondrocytes.Therefore,the treatment of OA should focus on the protection of chondrocytes.There is evidence that autophagy is closely related to the occurrence and development of OA,and autophagy enhancement has a protective effect on OA articular cartilage.At present,it has been proved by animal experiments that genetically modified exosomes can also promote cartilage tissue regeneration and prevent the progress of OA.However,the current research is mainly focused on the exosomes derived from bone marrow mesenchymal stem cells,and it is impossible to determine whether the edited exosomes extracted from autologous serum samples have an effect on the occurrence of OA.Activating transcription factor 4(ATF4)is responsible for regulating the proliferation and differentiation of chondrocytes during endochondral osteogenesis.ATF4 also plays an important role in the regulation of autophagy,suggesting that ATF4 may have potential in the treatment of OA by regulating the mechanism of autophagy.In this study,the exosome derived from the serum of osteoarthritis mice was constructed as the gene vector for overexpression of ATF4 gene,and the ATF4 gene was integrated into the exosome of serum samples of experimental OA mice to explore its therapeutic effect and mechanism on osteoarthritis.MethodsThe OA animal model of meniscus injury was established by excising the anterior horn of the medial meniscus of the right knee.Exosomes were isolated from serum samples of mice in sham group and OA group,which were called sham-Exo and OAExo,respectively.ATF4 mRNA was introduced into OA-Exo,by electroporation to overexpress the ATF4 of OA-Exo(that is,to construct ATF4-OA-Exo).Four weeks after operation,OA mice received intra-articular injection of Exo,OA-Exo and ATF4OA-Exo in sham group.Twelve weeks after operation,the animals in each group were sacrificed,and the cartilage tissue of knee joint was dissected.The structural changes of joint tissue after exocrine injection were evaluated by H&E staining and safranin-O fast green staining,and the progress of OA was evaluated by Mankin’s grading system.The expression of autophagy-associated protein and ATF4 was evaluated by western blot analysis.Autophagy was evaluated by transmission electron microscope.QRTPCR analysis was used to detect the level of ATF4 mRNA in serum samples or serum exosomes of mice.Serum inflammatory cytokines were quantitatively determined by ELISA.Chondrocytes were extracted from suckling mice.Cell proliferation and apoptosis were detected by endoplasmic reticulum stress and inflammation induced by tunicamycin(TM)and TNF-α.ResultsIn this study,the ATF4-modified serum exosomes derived from OA mice inhibited the apoptosis of chondrocytes induced by tunicamycin/TNF-α increased the level of COL-Ⅱ and the expression of autophagy-related proteins(ATG5 and LC3Ⅱ/LC3-Ⅰ)in cartilage,decreased the levels of MMP-13 and inflammatory factors(TNFα,IL-1 β,IL-6),reduced the injury of articular cartilage of OA mice,improved the condition of articular osteophytes of OA mice,and promoted autophagy of chondrocytes.The ATF4-modified serum exosomes alleviated OA progression by inducing autophagy.ConclusionATF4-modified serum exosomes derived from OA mice protect cartilage by inducing autophagy and alleviate the progression of OA.Induced autophagy may be the molecular mechanism of ATF4-OA-Exo in the treatment of osteoarthritis.