| BackgroudRheumatoid arthritis(RA)is a chronic disease mainly caused by synovium damage due to immune dysfunction.Based on big data analysis,the proportion of Rheumatoid arthritis is about 100/1 in the middle-aged and elderly population in the world,and the proportion of the damage caused by this disease is on the rise.Rheumatoid arthritis,characterized by synovitis,joint bone destruction,and systemic complications,is one of the common causes of disability,reduced quality of life,and reduced life expectancy.In addition to easily leading to joint deformity and dysfunction,RA often involves extrarticulatory organs such as heart,lung and kidney,causing great perplexity to patients' life and work.An analysis of U.S.medical statistics shows that annual direct medical expenses for patients suffering from RA are,on average,$2,000 more than those for patients without RA.It can be seen that RA not only reduces the quality of life and work of patients,but also significantly increases the economic burden of the families affected,leading to huge social burden.During the pathogenesis of rheumatoid arthritis,various types of immune-related cells and their secreted autoantibodies interact with cytokines and fibroblast synovial cells.Among them,fibroblast-like synoviocytes(FLSs)are considered to be the most abundant and play a key role in the process of inflammation.Current treatment for RA relies on hormone,conventional and biologic disease-modifying antirheumatic drugs,which often lead to toxicity and only partial responses.Therefore,it is essential to explore a safe and effective treatments for RA.Epimedium is a kind of Chinese herbal medicine that can be used to prevent and treat bone and joint dysfunction.Icariin(ICA),a therapeutic monomer extracted from Epimedium,has been shown to have beneficial therapeutic and preventive effects on bone healing and osteoporosis.The protective effect of icariin on RA has been reported in recent years,but its mechanism remains unclear.RA is featured by synovial inflammation,NLRP3 inflammasome is critical in inflammation and RA pathogenesis.Recent study has found that lupus nephritis was suppressed by icariin through NF-?B pathway inhibition and NLRP3 inflammasome in MRL/lpr mice and osteoarthritis was also alleviated by icariin via inhibiting NLRP3-mediated pyroptosis.Whether and how icariin affects RA still needs further investigation.MicroRNAs(miRNAs)belong to small non-coding RNAs which may regulate gene expression post-transcriptionally.Increasing evidence has shown that abnormal expression level of miRNA exists in synovial tissue and synovial fibroblasts during the process of RA.Among them,miR-223-3p is considered to be one of the possible biomarkers of RA,and miR-223-3p overexpression suppresses osteoclastogenesis in vitro.It has not been reported whether icariin regulates miR-223 expression,thus affects the progress of RA.At the early stage,we predicted that there might be targeted binding sites between miR-233-3p and NLRP3 through Starbase software.In summary,we speculated that icariin may inhibit the expression of NLRP3 in RA by upregulation of miR-223-3p,thereby inhibiting the proliferation and inflammatory response of fibroblast synovium cells in RA,promoting cell apoptosis,and then alleviating the occurrence of RA.In order to verify this hypothesis,this study conducted the following three explorations:In the first part,we investigated the effect of Icariin on the function of Rheumatoid arthritis fibroblast-like synoviocyte(RA-FLS).In the second part,we investigated whether icariin affected the function of fibroblastoid synovial cells(RA-FLs)in rheumatoid arthritis by regulating miR-223-3p.In the third part,the targeting binding relationship between miR-223-3p and NLRP3 was investigated.To verify whether icariin affects the function of RA-FLS cells through miR-223-3p/NLRP3 signal.Part I: Effect of icariin on fibroblast synovial cells in rheumatoid arthritisObjective:To observe the effects of icariin on the proliferation,inflammatory cytokine secretion and apoptosis induction of rheumatoid arthritis fibroblast synovial cells(RA-FLS).Methods:Collection of synovial tissue from patients with rheumatoid arthritis after joint replacement or synoviectomy.Synovial tissue samples were prepared and FLSs was obtained from synovial tissues by digesting with 2.5 g/L trypsin with gentle agitation for 1h at 37?.Cells were cultured in Dulbecco's Modified Eagle's medium(DMEM)containing 10% heat-inactivated FBS,penicillin,and streptomycin.After the end of culture,P3 RA-FLS in the culture bottle with good growth and fusion degree up to80%-90% were identified and centrifuged for flow cytometry detection.Positive CD90 and negative CD14 and CD68 were RA-FLS cells.The RA-FLS cells were treated with icariin of different concentrations(0,10,20,40,80,160,320?M)for24h,and divided into Control group and icariin group.The state of cells in each group was observed under microscope.The proliferation of cells was detected by CCK8 and Brd U methods.Flow cytometry was used to detect apoptosis.The inflammatory cytokines TNF-??IL-6?IL-1? were detected by ELISA.Western Blot detected Bcl-2,Bax,cleaved Caspase-3,and cleaved Caspase-9;The expression of miR-223-3p was detected by q RT-PCR.Through the above experiments,the alleviating effect of icariin on fibroblast synovial cell injury in rheumatoid arthritis was determined,and the optimal drug concentration was selected.Results:1.Icariin inhibited the activity of RA-FLS cells in a dose-dependent manner.When the concentration of icariin was 40?M,the survival rate of RA-FLS cells decreased by about 50%,which was the best concentration.2.Brdu detection of cell proliferation found that the proliferation of RA-FLS cells was inhibited after icariin treatment.3.Icariin could significantly increase the apoptosis of RA-FLS cells.4.Western blot results showed that bcl-2 expression was significantly reduced in RA-FLS cells after icariin treatment,while protein levels of Bax,cleaved Caspase-3and cleaved caspase-9 increased.5.Icariin reduces the production of inflammatory factors TNF-??IL-6?IL-1?.6.The expression level of miR-223-3p was significantly up-regulated after icariin treatment.Conclusion:Icariin can inhibit the proliferation of RA-FLS cells,inhibit the secretion of inflammatory factors,and promote the apoptosis of RA-FLS cells.Moreover,after icariin treatment,miR-223-3p level was higher than that of the control group.Part II: Whether icariin affects the function of fibroblast synovium cells(RA-FLS)in rheumatoid arthritis by regulating miR-223-3pObjective:To observe whether icariin affects the proliferation,inflammatory response and apoptosis of RA-FLS cells by regulating miR-223-3p.Methods:Synovial tissue samples of patients with rheumatoid arthritis after joint replacement or synoviectomy were collected,and peripheral blood of the patients and healthy controls were collected,and the expression difference of miR-223-3p in serum was detected by q RT-PCR.Then primary isolation,culture and identification of rheumatoid arthritis synovial-like fibroblasts(FLS)were performed.Screening CD90 positive,CD14,CD68 negative rheumatoid arthritis synovial fibroblasts.First study the function of miR-223-3p high expression in RA cell lines,We divided RA-FLS cells into miR-223-3p mimics and NC mimics group.The relative expression of miR-223-3p in RA-FLS cells transfected with miR-223-3p mimics and NC mimics group was detected by q RT-PCR,cell viability of miR-223-3p mimics and NC mimics group was detected by CCK-8 assay,apoptosis of the two groups was detected by flow cytometry,and TNF-?,IL-1? and IL-6 concentrations were detected by ELISA.Then,the effect of Icariin on RA-FLS cell function was studied,which was divided into four groups: Control group,Icariin group,Icariin+ NC inhibitor group,and Icariin+ miR-223-3p inhibitor group.The optimal concentration of icariin was 40?M.The activity,proliferation,apoptosis,apoptotic-related proteins,inflammatory cytokines and miR-223-3p levels of RA-FLS cells in each group were detected by the same method as in the first part of the experiment.Results:1.The expression of miR-223-3p in serum of RA patients was significantly higher than that in serum samples of healthy people.The expression of miR-223-3p in RA-FLS cells was significantly higher than that in FLS cells.2.Function of high expression of miR-223-3p in RA cell lines.2.1 After transfection with miR-223-3p mimics,the expression level of miR-223-3p in RA-FLS cells was significantly increased.2.2 CCK8 assay showed that miR-223-3p upregulation significantly reduced the proliferation of RA-FLS cells.2.3 Overexpression of miR-223-3p significantly increased the rate of apoptosis.2.4 The overexpression of miR-223-3p significantly decreased the secretion of TNF-?,IL-1? and IL-6.3.Icariin significantly increased the expression of miR-223-3p,and the effect of Icariin on the function of RA-FLS cells was detected.3.1 miR-223-3p inhibitor down-regulated the expression of miR-223-3p in RA-FLS cells,and significantly inhibited the upregulation of miR-223-3p in icariin-induced RA-FLS cells(P <.01).3.2 CCK-8 assay showed that knockdown of miR-223-3p reversed the inhibitory effect of icariin on the proliferation of RA-FLS cells.3.3 BrdU test showed that cell proliferation ability was significantly reduced after Icarin treatment;the knockdown of miR-223-3p significantly restored the cell proliferation inhibited by Icariin.3.4 Flow cytometry showed that the apoptosis rate was significantly increased after Icarin treatment;however,knockdown of miR-223-3p significantly inhibited the cell apoptosis induced by Icariin.3.5 Icarin treatment significantly down-regulated Bcl-2 expression,and up-regulated Bax,cleaved caspase-3,and cleaved caspase-9 expression;The knockdown of miR-223-3p was significantly reversed.3.6 Icarin treatment significantly reduced the levels of inflammatory factors TNF??,IL?1?,and IL?6;the knockdown of miR-223-3p was significantly reversed.Conclusion:Icariin inhibits the proliferation and inflammatory response of RA-FLS cells and promotes the apoptosis of RA-FLS cells by upregulating the expression of miR-233-3p.Part III: Whether icariin affects the function of RA-FLS cells through miR-223-3P /NLRP3 signalingObjective:The targeted binding relationship between miR-223-3p and NLRP3 was investigated.To verify whether icariin affects the function of RA-FLS cells through miR-223-3p /NLRP3 signaling.Methods:To further investigate the regulatory mechanisms of miR-223-3p in RA,Star Base virtual online screening was performed(http://starbase.sysu.edu.cn/).By using Star Base software to predict the binding sites between miR-233-3p and NLRP3,we found that miR-223-3p may bind to the 3'-UTR of NLRP3 m RNA.CD90 positive and CD14 and CD68 negative rheumatoid arthritis fibroblast(RA-FLS)cell lines were screened as above,and divided into 4 groups:NC mimic group(negative control simulation group),miR-223-3p mimic group(miR-223-3p simulation group),NC inhibitor group(negative control inhibitor group),and miR-223-3p inhibitor group(miR-223-3p inhibitor group).The binding relationship between miR-223-3p and NLRP3 was detected by dual luciferase reporter genes.The expression of NLRP3 was detected by q RT-PCR and WB.Then it was further verified whether icariin affected the function of RA-FLS cells through miR-223-3p/NLRP3 signal.First,the RA-FLS cell lines were selected and divided into 4 groups:Control group,Icariin group,Icariin+ miR-223-3p inhibitor group,Icariin+ miR-223-3p inhibitor+MCC950 group,MCC950 is a specific inhibitor of NLRP3.The activity,proliferation,apoptosis,apoptotic-related proteins,inflammatory cytokines and miR-223-3p levels of RA-FLS cells in each group were detected by the same method as in the first part of the experiment.Meanwhile,the expression of NLRP3 in RA and normal synovial tissues was detected by immunohistochemistry.Results:1.Targeting binding relationship between miR-223-3p and NLRP3.1.1 StarBase software predicted potential binding sites between miR-223-3p and NLRP3.1.2 Dual-luciferase reporter gene assay proved that there was an interaction between miR-223-3p and NLRP3.1.3 miR-223-3p mimics reduced the m RNA and protein levels of NLRP3 in RA-FLS cells.However,the expression of NLRP3 was significantly increased after transfection with miR-223-3p inhibitor,and miR-223-3p negatively regulated the expression of NLRP3.2.Icarin can inhibit the proliferation and inflammatory response of RA-FLS cells by regulating miR-223-3p/NLRP3,and promote cell apoptosis.2.1 The expression of NLRP3 in synovial tissue isolated from RA patients was significantly higher than that in healthy synovial tissue.2.2 Western blot results showed that miR-223-3p inhibitor significantly up-regulated the expression of NLRP3 inhibited by icariin,while in the presence of miR-223-3p inhibitor and icariin,MCC950 treatment down-regulated the expression of NLRP3.2.3 miR-223-3p inhibitor can significantly improve the cell viability and proliferation ability inhibited by icariin.However,the cell viability and proliferation were decreased after the administration of miR-223-3p inhibitor and icariin at the same time with MCC950 inhibitor.2.4 Inhibition of miR-223-3p can significantly reduce icariin-induced apoptosis;However,in the presence of miR-223-3p inhibitor and MCC950,the apoptosis rate of RA-FLS cells was significantly increased.2.5 Icarin treatment significantly down-regulated Bcl-2 expression,and up-regulated Bax,cleaved caspase-3,and cleaved caspase-9 expression;Knockdown of miR-223-3p showed the opposite effect,while the addition of MCC950 significantly reversed the expression of these apoptosis-related proteins.2.6 After knockdown of miR-223-3p,the level of inflammatory factors was significantly increased,and this effect could be significantly inhibited by miR-223-3p and NLRP3 inhibitors.Conclusion:miR-223-3p targets the m RNA of NLRP3 and inhibits the expression of NLRP3.Icariin inhibits the proliferation and inflammatory response of RA-FLS cells and promotes the apoptosis of RA-FLS cells by upregulating the expression of miR-233-3p. |
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