Therapeutic Effect Of Neohesperidin On TNF-alpha-stimulated Human Rheumatoid Arthritis Fibroblast-like Synoviocytes

Background:Rheumatoid Arthritis(RA)is a chronic autoimmune clinical syndrome of unknown etiology,which is mainly manifested as chronic,systemic,and corrosive synovial tissue.Fibroblast-like Synovium(FLS)is the main cell of the synovium tissue.It is closely related to the pathogenesis of RA.In rheumatoid arthritis(RA)pathogenesis,activated RA fibroblast-like synoviocytes(RA-FLSs)combines similar proliferative features as tumour and inflammatory features as osteoarthritis,eventually lead to joint erosion.Therefore,it is imperative to search for compounds,which can effectively inhibit the abnormal activation of RA-FLSs and retard RA progression.Neohesperidin(Neo)is a major active component of flavonoid compounds with anti-inflammation and antioxidant properties.In this study,the anti-inflammation,anti-migration,anti-invasion,antioxidant and apoptosis-induced effects of Neo on RA-FLSs were explored to investigate the underlying mechanism.Since most mitogen-activated protein kinases are important signaling molecules in cell inflammatory response,proliferation,and apoptosis,we chose the MAPK pathway as the starting point of our study.Objective:To explore the therapeutic effect of neohesperidin on RA-FLSs and its underlying mechanism.Methods:Human synovium tissues were obtained from rheumatoid-arthritis patients.Firstly,Cell viability was determined by cell counting kit-8(CCK-8)assay.RA-FLSs(1×104 cells/well)were seeded into 96-well plates and treated with Neo at various concentrations(0.0,10.0,20.0,30.0,40.0,50.0,60.0 and 70.0 μM)for 48 h.The expression of MMP-3,MMP-9,MMP-13,p38,P-p38,JNK,P-JNK,ERK,P-ERK,Bcl-2,Bax and cleaved-caspase3 [Blank group 、 TNF-α group 、 TNF-α+NEO(5μM)group 、 TNF-α+NEO(10μM)group 、TNF-α+NEO(30μM)group 、 NEO(30μM)group] were detected using western blot.ELISA and q RT‐PCR assessed the levels of pro‐inflammatory cytokines [Blank group 、 TNF-α group 、 TNF-α+NEO(5μM)group 、 TNF-α+NEO(10μM)group 、TNF-α+NEO(30μM)group、NEO(30μM)group],including MMP9,MMP13,IL‐1β,IL‐6,IL-8,and TNF‐α、OPG、RANKL.The cell invasive ability was assessed by transwell chamber and the cell migration ability was measured by wound healing assay.Cell apoptosis was assessed by the Annexin V-FITC Apoptosis Kit.The reactive oxygen species(ROS)levels in RA-FLSs[Blank group 、TNF-α group 、 TNF-α+NEO(5μM)group 、 TNF-α+NEO(10μM)group 、TNF-α+NEO(30μM)group、NEO(30μM)group] were measured by the ROS assay kit.Results:(1)The CCK-8 assay showed that 10,20,and 30 μM Neo resulted in 98%,92% and86% cell viability.Since lower viability was observed on the RA-FLSs,the function of Neo in RA-FLSs at concentrations of 5–30 μM was studied;(2)Dramatic increases of MMP3,MMP9 and MMP13 were recorded in TNF-α-stimulated group using western blot.Nevertheless,pretreatment with Neo resulted in a reduction in them.ELISA and q RT‐PCR analyses showed that the levels of pro‐inflammatory cytokines,including IL‐1β,IL‐6,IL-8,and TNF‐α was decreased by Neo treatment in a concentration‐dependent manner;(3)The transwell chamber experiment showed that the number of cells passed through the Matrigel(blue-purple spots)was decreased as the drug concentration was increased.Scratch assays found that RA-FLSs treated with 5,10,or 30 μM Neo migrated less than the control cells(26.0%,16.2% and 9.2 vs.36.9%),and the difference became more evident after 36 h(41.0%,29.2% and 18.2 vs.52.9%).(4)Intracellular ROS detection indicated that,after exposure to TNF-α,the intensity of green fluorescence was higher than that in the blank group.However,this phenomenon was remarkably reversed with the treatment of Neo.(5)Flow cytometry assay found that the apoptosis rate of RA-FLSs was increased following the treatment with Neo.Moreover,with an increased concentration of Neo,the percentage of apoptotic cells was continuously elevated.A western blot assay showed that treatment with Neo substantially down-regulated the expression of Bcl-2 while up-regulated the expression of Bax and cleaved-caspase3.(6)The result of western blot assay showed that the administration of TNF-α remarkably activated the phosphorylation levels of p38,ERK and JNK,while the treatment with Neo resulted in a significant down-regulation in the expression of that compared with the blank group and TNF-α-treated group,in a concentration-dependent manner.Conclusions:(1)Neohesperidin exert its anti-inflammtory effect by downregulated the expression of cytokines like IL-1β,IL-6,IL-8,MMP-3,MMP-9,MMP-13 in RA-FLSs;(2)Different concentrations of Neo inhibited the migration and invasion of RA-FLSs,and had a certain dose-effect and time-effect relationship;(3)Treatment with Neo induced the apoptosis of FLSs;(4)It was also found that Neo could reduce the accumulation of reactive oxygen species(ROS)induced by TNF‐α;(5)Neo blocked the activation of MAPK pathways,suggesting that Neo plays a role in RA-FLSs,at least in part,by inhibiting MAPK signaling pathways;(6)Taken together,our results highlighted that Neo may be a potential and promising therapeutic drug for the management of RA..