Anti-inflammatory Mechanism Of Iguratimod In Rheumatoid Arthritis By Regulating Autophagy And Mitophagy

Background:Rheumatoid arthritis is a chronic and systemic features of autoimmune disease,the disease is the main clinical features of aggressive symmetrical polyarthritis RA is the basic pathological change is characterized by chronic inflammation of the synovial joints pannus formation,the occurrence of articular cartilage and bone destruction,the above process ultimately caused the joint deformity and loss of function.Fibroblast-like synovial cells play a role not only as damaged target cells but also as immune cells in the pathogenesis of RA.RA-FLS can secrete inflammatory factors,leading to the continuous development of synovitis;RA-FLS also secretes vascular endothelial growth factor,which is related to the production of pannus;the high expression of cell adhesion factor on the surface of RA-FLS enables it to adhere to extra-articular cartilage;RA-FLS also secretes matrix proteases,which leads to cartilage destruction;RA-FLS also promotes osteoclast activity by regulating the RANKL/OPG system,which leads to bone destruction.NF-?B is a widely influential transcription factor,which is a dimer protein composed of Rel family.It can promote the transcription and expression of a variety of genes,which is closely related to inflammatory response,immune response,cell proliferation,differentiation and apoptosis and other important physiological and pathological processes.In general,the dimer NF-?B and I?B combine to form a trimer,which exists in the cytoplasm in an inactivated state.When cells are stimulated by extracellular signals such as TNF-?,IL-1? and LPS,activation of IKK,results in phosphorylation and degradation of I?B,dissociation of NF-?B and I?B,then rapid translocation of NF-?B from cytoplasm to nucleus,specific binding to target gene ?B site,promotion of target gene transcription and activation of NF-?B pathway.The excessive activation of NF-?B promotes the immune disorder and inflammatory storm of RA,which leads to the continuous progress of RA. As a new type of small molecule DMARDs,Iguratimod is widely used in the treatment of RA and other autoimmune diseases.Iguratimod has anti-inflammatory effects,inhibits the synthesis of immunoglobulins and inflammatory factors,and has a protective effect on destruction and pulmonary fibrosis.Iguratimod can inhibit the activation of NF-?B pathway in RA,but the specific mechanism is not clear.Autophagy is a process in which cells wrap some proteins or organelles in specific membrane structures,send them to lysosomes for degradation,and produce energy and small molecules for cell reuse.It widely exists in the cells of higher vertebrates.Autophagy not only provides energy for cells and maintains cell homeostasis,but also is closely related to the immune system.If autophagy regulation is abnormal,it will lead to autoimmune diseases,such as rheumatoid arthritis.Autophagy plays a pathological role in rheumatoid arthritis by regulating inflammatory factors and bone destruction.Studies have shown that the increased level of autophagy in RA-FLS is related to disease activity.In addition,mitophagy is a special form of selective autophagy,which refers to the process in which redundant or damaged mitochondria can be selectively cleared by cells through autophagy mechanism.However,the role of mitophagy in RA is not clear and needs further study.Therefore,we infer that Iguratimod can not only regulate the level of global autophagy in RA-FLS,but also play an anti-inflammatory role by regulating the level of autophagy of NF-?B pathway,especially the level of autophagy of IKK kinase.We will also study the level of mitophagy in RA-FLS and the effect of Iguratimod on it.Objective:To explore the effect of Iguratimod on autophagy and mitophagy in RA-FLS,and the specific mechanism of anti-inflammatory effect of Iguratimod by regulating autophagy of IKK in NF-?B pathway,and to further reveal the mechanism of Iguratimod in the treatment of RA.Methods:MH7A cells(human rheumatoid arthritis fibroblast line)were cultured in vitro,and the time and concentration of LPS and Iguratimod were determined by MTT method.Western blotting was used to detect the expression of related proteins in NF-?B pathway in MH7 A before and after treatment with LPS.After the autophagy inhibitor Baf A1 was added,the protein expression in MH7 A was detected by Western blotting and compared with that of LPS+Iguratimod group.The selective autophagy receptors that can bind to IKK were identified by co-immunoprecipitation and immunofluorescence techniques.CBA technique was used to detect the effect of Iguratimod on the secretion of inflammatory factors in MH7 A cells.Western blotting was used to detect the content of mitochondrial outer membrane protein TOMM20 and intimal protein TIM23 in MH7 A before and after Iguratimod treatment under LPS induction.After adding Baf A1,the expression levels of the two proteins were detected again.ROS,was labeled with probe and immunofluorescence was used to detect the effect of Iguratimod on ROS in MH7 A and its relationship with mitochondrial autophagy.Results:MTT method determined 20?g/ml and 40?M as the best action time of LPS and Iguratimod.Western blotting results showed that NF-?B protein in MH7 A induced by LPS increased,I?B? decreased,p-I?B? increased,IKK?,IKK? and p-IKK?+?increased,indicating that NF-?B pathway in MH7 A was activated.After the addition of Iguratimod,NF-?B protein decreased,I?B? increased,p-I?B? decreased,IKK?,IKK? and p-IKK?+? decreased,indicating that Iguratimod inhibited the activation of NF-?B in MH7 A.After the addition of LPS and Iguratimod,the expression of LC3 B protein in MH7 A was increased,indicating that Iguratimod enhanced the overall autophagy level of MH7 A.On the basis of adding LPS and Baf A1,the results showed that IKK?,IKK? and p-IKK?+? increased,which proved that the effect of Iguratimod on the content of IKK was realized by regulating its autophagy.The results of co-immunoprecipitation and immunofluorescence showed that the quantitative and spatial connection between IKK and p62 increased,indicating that p62 is a selective autophagy receptor binding to IKK under the action of Iguratimod.CBA results showed that TNF in MH7 A cells induced by LPS increased,TNF decreased after Iguratimod was added,and TNF increased after Iguratimod and Baf A1 were added,which proved that Iguratimod could inhibit the secretion of inflammatory cytokines by increasing autophagy of IKK.The results of Western blotting showed that TOMM20 and TIM23 in MH7 A induced by LPS increased,decreased after the addition of Iguratimod,and increased after the addition of Iguratimod and Baf A1.The results showed that the level of mitophagy in MH7 A decreased,Iguratimod increased mitophagy,and Baf A1 blocked this effect.After labeling ROS with probe,immunofluorescence results showed that ROS increased in MH7 A induced by LPS,while ROS decreased in Iguratimod group.CBA results showed that TNF decreased after the addition of mitophagy inducers Oligomycin and Antimycin A,indicating that Iguratimod reduced the secretion of inflammatory cytokines by enhancing mitophagy.Conclusions:1.Iguratimod inhibits the activation of NF-?B pathway in MH7 A cells induced by LPS.2.Iguratimod inhibits the activation of NF-?B and reduces the release of pro-inflammatory factors by increasing autophagy of IKK and the selective autophagy receptor associated with IKK is p62.3.Iguratimod enhanced mitophagy in MH7 A cells,and enhanced mitophagy could reduce the release of pro-inflammatory factor TNF.4.Iguratimod reduced ROS production by enhancing mitophagy.