The Anti-Inflammatory Effect And Its Molecule Mechanisms Of Euphorbia Factor L2 On Rheumatoid Arthritis

Objective::In this study,we investigated the potential anti-arthritic mechanisms of EFL2（Euphorbia factor L2）identified both in vivo and in vitro.Meanwhile,explored EFL2’s pharmacokinetic features.Methods:In animal experiments:EFL2’s anti-arthritic effects were evaluated with STA（K/BxN serum transfer arthritis）murine model.The changes of joint inflammation were observed by arthritis score after mice were gavage or intraperitoneal injection of EFL2.HE（Hematoxylin-eosin）staining was used to observe the infiltration of inflammatory cells,synovial hyperplasia and degree of joint damage.in mice.RT-PCR（Reverse Transcription-Polymerase Chain Reaction）were used to detect the expression of inflammatory cytokines in mouse joints.CBA（Cytometric Bead Array）were used to detect the expression of inflammatory cytokines in serum of mice.C57BL/6 mice were treated by intraperitoneal injection of EFL2,then we applied UPLC-MS（Ultra-Performance Liquid Chromatography/Tandem mass spectrometry）method to measure the concentrations of EFL2 in plasma at various time points.In vitro cell experiments:The effects of EFL2 on RAW264.7（Mouse leukemia cells of monocyte macrophage）,BMDMs（Bone marrow-derived macrophages）and PBMCs（Human Peripheral Blood Mononuclear Cells）viability was evaluated by CCK8（Cell counting kit-8）assay.In addition,RT-PCR were used to test the anti-inflammatory effect of EFL2 on TLR7（Toll-like receptor 7）ligand agonist induced the m RNA of IL-1βand IL-6 in RAW264.7 cells.ELISA（Enzyme linked immunosorbent assay）were used to test the concentration of IL-1βand IL-6 in supernatant of RAW264.7 cells,primary murine bone marrow-derived macrophages（BMDMs）and human peripheral blood molecular cells（PBMCs）after R837（Imiquimod）induced.Western blotting were used to confirm if IL-1βand IL-6 reduction induced by EFL2 is via inhibiting signaling pathway of MAPKs（Mitogen-activated protein kinase）（p38,JNK,p-p38,p-JNK）,NF-κB（IKKαβ,IκB-α,p65,p-IKKαβ,p-IκBα,p-p65）and TLR7-IKKβ-IRF5（IRAK4,IKKβ,IRF5,p-IRAK4,p-IKKβ,p-IRF5）activation.The inhibiting effect of EFL2 on R837 induced IRF5 translocation in RAW264.7 cells was analyzed by immunofluorescence staining and confocal microscopy.Results:As shown,the clinic scores in STA mice group rapidly increased and reached the peak at day 5,following with slow remission between day 5 and13.In contrast,the arthritis scores in STA mice receiving DEX（Dexamethasone）were significantly lower during the effector phase of arthritis.this ameliorating effect was only observed in STA mice i.p.injected with EFL2.Gavage mice with EFL2 did not affect the arthritis severity of STA mice neither at the acute phase nor the remission phase of arthritis.As the HE results shown,there were abundant infiltrated inflammatory cells in the knee of STA mice,accompanied with obvious synovial hyperplasia.Both DEX gavage and STA（i.p.administration）significantly suppressed the inflammatory cells infiltration and synovium hyperplasia.As RT-PCR shown that there have no significant changes in TNF-αlevels were observed among na（?）ve,STA,and STA mice administrated with EFL2 or DEX on day 13.Whereas,high levels of IL-1βand IL-6 were detected in the sera of STA mice,which were significantly reduced to normal level in mice administrated with EFL2 or DEX.As CBA shown,the gene expressions of pro-inflammatory cytokine IL-1βand IL-6 were also up-regulated in the ankles of STA mice on day 13.EFL2 i.p.treatment robustly decreased these two cytokines expression at the gene transcriptional level.after intraperitoneal injection of EFL2,the concentration of EFL2 reached a maximum plasm concentration(C_max)of 4525.12±1630.01 ng/ml,and the time to reach the maximum concentration(T_max)was 2.50 ± 1.73 h.The T max is shortly.The area under the plasma concentration versus time curve from zero to time t(AUC_0–t)was（374.10±163.96）h·ng/ml for EFL2.Besides,the value of elimination half time(t_1/2)were 21.17± 7.11 h,which suggested EFL2 was slowly eliminated.As the CCK8 assay results shown,EFL2 did not display significant cytotoxicity in RAW264.7 cells until the concentration reached 25μM.Similar to RAW264.7cells,EFL2,at the concentrations from 0.1 to 10μM,did not show cytotoxicity in BMDMs neither.Surprisingly,EFL2 did not affect PBMCs viability until its concentration reached 100μM.As RT-PCR results shown that the m RNA of IL-1βand IL-6 in R837-stimulated RAW264.7 were significantly blocked by different concentrations of EFL2.ELISA results showed that EFL2 effectively inhibited the concentrations of IL-1βand IL-6 release in the supernatant of RAW264.7 cells,BMDMs and PBMCs induced by R837.ELISA results showed JNK,p38 and NF-κB reactive R837-induced IL-1βand IL-6 secretion in RAW264.7 cells.All those results indicating that TLR7 is implicated into activation of MAPKs and NF-κB signaling pathway.Western blot results shown that R837 can activated RAW264.7 cells and rapidly caused phosphorylation of p38,JNK,p65,IKKαβ,IκB-α,IRAK4,IKKβ,IRF5 of MAPKs,NF-κB and IRF5 signaling pathway.EFL2 could effectively inhibit IKKαβ,IκB-α,p65,IRAK4,IKKβ,IRF5phosphorylation expression,but had little effect on R837 induced p38 and JNK phorsphorylation expression in RAW264.7 cells.EFL2 did not exert its intensity anti-inflammatory effect via inhibiting p38 and JNK activation.How,interesting in contrast,this compound robustly decreased IL-1βand IL-6 production through downregulating TLR7 mediated NF-κB and IRAK4-IKKβ-IRF5signaling pathway.Immunofluorescence staining further confirmed that R837could dramatically induce p65 and IRF5 phosphorylation enhanced p65 and IRF5translocation from cytoplasm to nucleus.Meanwhile,p65 and IRF5 translocation was also effectively blocked by EFL2 at different concentrations.All these data indicate that EFL2 can block NF-κB and signaling pathway and alter the phosphorylation of p65 and IRF5 to suppressed their translocational activity.Conclusion:EFL2 ameliorates arthritis severity mainly by suppressing IL-1βand IL-6 pro-inflammatory cytokines production.The anti-inflammatory effect of EFL2 are mediated by blocking NF-κB and IRAK4-IKKβ-IRF5signaling pathway.Therefore.