Metrnl Regulated Proliferation,apoptosis And Function Of RA-FLS By PPARγ

Objective: Rheumatoid arthritis(RA)is an autoimmune disease with synovitis of the joints as the main pathological change.It can cause synovial hyperplasia,blood vessel formation,bone and cartilage destruction,and eventually lead to disability.The exact cause is unknown,but it is widely believed that decreased immune tolerance and activation of autoreactive T and B lymphocytes can lead to the development of RA.Studies have shown that the secretion of various inflammatory cytokines and angiogenesis factors in the peripheral blood and synovial fluid of RA patients is increased,such as interleukin-6(IL-6),interleukin-17(IL-17,IL-17),tumor necrosis factor-α alpha(TNF-α),vascular endothelial growth factor Growth factor(VEGF),platelet-derived growth factor(PDGF),etc.,eventually lead to the proliferation of RA fibroblast-like Synoviopolitos(FLS),angiogenesis and joint destruction,participating in the pathogenesis of RA.At present,biotargeted therapy(such as anti-TNFα,anti-IL-6,etc.)for RA patients has achieved good clinical efficacy,but some patients still do not respond to treatment.This suggests that there are other cytokines involved in the regulation of RA,which prompts us to further explore the pathogenesis of RA and find new targets for its treatment.Glial cell differentiation regulator-like factors(Meteorin-like,Metrnl),also known as Meteorin-β,Subfatin and Cometin,are located in the q25.3 region of human chromosome 17,the q E2 region of mouse chromosome 11,and the q25.3 region of human chromosome 17.Metrnl is a newly discovered secreted protein involved in inflammation and immune regulation in recent years.Metrnl is mainly secreted by adipocytes,activated macrophages,and is highly expressed in mucosal barrier tissue,adipose tissue,skin and muscle.At present,the research on Metrnl at home and abroad mainly focuses on diabetes,asthma,inflammatory bowel disease and myocardial infarction.Metrnl’s initial exploration in the field of immunity originated from a scientific team using their own synovial gene expression database of rheumatoid arthritis to find that Metrnl is highly expressed in the synovial membrane of RA,and subsequent researchers found that Metrnl is highly expressed in tissue samples of synovial fluid and tendon attachment sites of patients with psoriatic arthritis,and TNF-α and IL-17 can induce Metrnl secretion.Our previous studies found that serum Metrnl levels in RA patients were significantly elevated and positively correlated with RA disease activity and antibody levels.Therefore,we speculate that Metrnl may be involved in the regulation of RA pathogenesis.To confirm this hypothesis,we intend to study the effect of exogenous Metrnl on proliferation and apoptosis of RA-FLS cells.To study the effect of Metrnl on RA-FLS inflammatory cytokines and angiogenesis-related cytokines.At the same time,the effect of exogenous Metrnl on the pathogenesis of collagen-induced arthritis(CIA),an animal model of RA,was observed.Research methods:In order to further explore the signaling pathway and mechanism of Metrnl in the regulation of RA pathogenesis,we selected RA-related datasets in GEO database through Bioinformation analysis and found that Peroxisome Proliferator Activated γ Receptor γ(PPARγ)was poorly expressed in RA synovium.PPARγ belongs to a subtype of PPAR,which can be transcribed and translated into the participation of various subpopulations in physiological processes,and has important roles such as inhibiting inflammation and inhibiting angiogenesis.In diabetes studies,it has been found that Metrnl can improve insulin sensitivity and reduce insulin resistance by upregulating the expression of PPARγ.Therefore,we suspect that Metrnl may regulate the proliferation,apoptosis,and function of RA-FLS cells in RA patients by upregulating the expression of PPARγ.To confirm this hypothesis,we intend to examine the effect of exogenous Metrnl on the expression of PPARγ in RA-FLS.We performed PPARγsi RNA targeted silencing on RA-FLS,blocked the role of PPARγ in RA-FLS,and observed whether Metrnl affected the proliferation,apoptosis and function of RA-FLS cells by regulating PPARγ.Through this study,a new theoretical basis is provided for the pathogenesis of RA and a new target is further provided for the treatment of RA.1.The human RA synovial membrane related dataset in the public database was selected to analyze the PPARγ expression level in the model.2.Different concentrations of Metrnl stimulated LPS-induced RA-FLS inflammatory cell models,and RT-PCR detected the expression of inflammatory factor PPAR-γm RNA;Western blot detects the expression of PPAR-γ protein;3.Different concentrations of Metrnl stimulated LPS-induced RA-FLS inflammatory cell model,CCK8 method and flow cytometry to detect FLS proliferation and apoptosis,and further detected the effect of Metrnl on the proliferation and apoptosis of RA-FLS under si RNA-mediated PPARγ inhibition.4.Different concentrations of Metrnl stimulated LPS-induced RA-FLS inflammatory cell models,and RT-PCR detected the expression of inflammatory factors TNF-α,IL-6,IL-17 and angiogenesis factors VEGF and PDGF m RNA;Western blot detected the expression of inflammatory factors TNF-α,IL-6,IL-17 and angiogenesis factors VEGF and PDGF proteins.ELISA detected the secretion levels of inflammatory cytokines TNF-α,IL-6,IL-17,angiogenesis factors VEGF and PDGF in the supernatant.5.si RNA-mediated PPARγ-targeted silencing of LPS-induced RA-FLS inflammatory cell models: RT-PCR detected the expression of inflammatory factors TNF-α,IL-6,IL-17,angiogenesis factors VEGF and PGDF m RNA;Western blot detected the expression of inflammatory factors TNF-α,IL-6,IL-17 and angiogenesis factors VEGF and PDGF proteins.ELISA detected the secretion levels of inflammatory factors TNF-α,IL-6,IL-17 and angiogenesis factors VEGF and PDGF in the supernatant.6.DBA/1J mice were used to induce the establishment of CIA models,and Metrnl and Phosphate Buffer Saline(PBS)were given intraperitoneal injection in the experimental group and the model group to evaluate the general state and arthritis of mice,and the joint bone tissue was stained with crobus O and Hematoxylin-eosin(HE)staining to evaluate the role of Metrnl in cartilage and bone destruction in CIA mouse models.Results:1.GEO database bioinformation analysis showed that the expression of PPARγ in synovial tissue of RA patients was downregulated by-2.28 times.2.After different concentrations of Metrnl(0,50,100,200 and 300ng/m L)stimulated the LPS-induced RA-FLS inflammatory cell model,the m RNA expression of PPARγ in RA-FLS cells was increased by RT-PCR,and the protein expression of PPARγ in RA-FLS cells was increased by Western blot.3.Different concentrations of Metrnl(0,50,100,200 and 300ng/m L)stimulated LPS-induced RA-FLS inflammatory cell models:(1)The proliferation of RA-FLS was detected by CCK8 method and found that Metrnl could inhibit the proliferation of RA-FLS and was concentration-dependent.PPARγsi RNA-targeted silencing cannot antagonize the inhibitory effect of Metrnl on RA-FLS proliferation.(2)Flow cytometry to detect the apoptosis of RA-FLS found that Metrnl could promote apoptosis of RA-FLS and there was a concentration-dependent.PPARγsi RNA-targeted silencing can antagonize the promotion of Metrnl on apoptosis in RA-FLS cells4.After different concentrations of Metrnl stimulated LPS-induced RA-FLS inflammatory cell models:(1)The expression of m RNA in RA-FLS cells detected by RT-PCR to detect TNF-α,IL-6,IL-17,VEGF and PDGF decreased significantly,and there was a concentration-dependent.PPARγ si RNA-targeted silencing can antagonize Metrnl’s inhibition of IL-17 and PDGF m RNA expression,but cannot antagonize Metrnl’s inhibition of m RNA expression of TNF-α,IL-16 and VEGF.(2)Western blot detected that the protein expression of TNF-α,IL-6,IL-17,VEGF and PDGF in RA-FLS cells decreased significantly,and there was a concentration-dependent.PPARγ si RNA-targeted silencing can antagonize the inhibitory effect of Metrnl on the expression of inflammatory cytokines and angiogenesis-related factor proteins in RA-FLS cells.(3)ELISA detected that the secretion levels of TNF-α,IL-6 and IL-17 in the supernatant of RA-FLS cells decreased significantly and were concentration-dependent.PPARγsi RNA targeted silence antagonizes the inhibitory effect of Metrnl on the secretion of RA-FLS inflammatory cytokines and angiogenesis-related factors.5.Compared with the control group,the body weight of mice in CIA+Metrnl group and CIA group showed a downward trend,and the overall weight of CIA+Metrnl group was higher than that of CIA group.The joint swelling and arthritis index of the CIA+Metrnl group were significantly lower than those in the CIA group.HE staining showed that Metrnl significantly reduced synovial hyperplasia and inflammatory cell infiltration,aggregation,and bone tissue destruction.Croca staining suggests that Metrnl can significantly reduce the destruction of joint cartilage in CIA.Metrnl affects RA-FLS proliferation,apoptosis,and function by upregulating the expression of PPARγ,and Metrnl can alleviate the pathogenesis in CIA mouse models.It provides a new theoretical basis for the study of the pathogenesis of RA and a new target for the treatment of RA.Conclusion:Metrnl targeted regulation of PPARγ promoted LPS-induced apoptosis and inhibited proliferation of RA-FLS cells,reduced the expression of inflammatory cytokines and angiogenesis factors,and slowed down the inflammatory reaction and cartilage injury in CIA mouse model.Metrnl provides a new theoretical basis for the study of the pathogenesis of RA,or becomes a new drug target.