Anti-rheumatoid Arthritis Effects And Mechanism Of Bioactive Lignans VN5 And VN2 From Vitex Negundo Fruits

Background and objective: Rheumatoid arthritis(RA)is a chronic and debilitating autoimmune disease characterized by extensive and aggressive synovitis.With persistent inflammatory cells infiltration and proliferation of synovial cells,RA can affect the health of patients by directly destroying the structure and function of joints,cartilages and bones,thus resulting in bone erosive destruction,irreversible joint damage,and even a high degree of disability.Natural compounds represent an extraordinary resource of structural scaffolds with high diversity that can offer promising chemical drugs for making RA less devastating and more curable.Thus,it is urgently needed to pursue anti-RA drugs with high efficacy and low adverse effects from the natural sources.Vitex negundo L.(VN)is a Vitex medicinal plant of Verbenaceae family.Its fruits have been traditinally used in folk medicine as a traditional Chinese medicine for the treatment of RA,with efficacy of expelling wind,removing damn,regulating qi-flowing and relieving pain.Modern pharmacological researches have revealed that these medicinal fruits possessed anti-inflammatory,antioxidant,antitumour,and anti-osteoporotic activites.Our previous studies revealed that the phenylnaphthalene lignans(vitexdoins)were the major type of bioactive components in VN fruits.The bioactive fraction,total vitexdoins(TOV),was prepared by our group and displayed significant anti-RA effects.TOV mainly included four phenylnaphthalene-type lignans,including VNL(VN1),vitedoin A(VN2),vitexdoin A(VN12)and vitedoamine A(VN5).However,the detailed mechanisms involved in the anti-RA acitivies of these TOV lignans had not been clarified yet.Therefore,our study was conducted to investigate the in vitro anti-RA effects and underlying mechanisms of two representative lignans,VN5 and VN2,using several inflammatory and osteoclastic cell models,to lay the foundation for preclinical research of TOV.Methods:(1)The effects of VN5 and VN2 on the cell viability of 293 T cells,RAW 264.7 cells,osteoclasts and fibroblast-like synoviosytes was assessed using CCK8 method.(2)The effects of VN5 and VN2 on the NF-?B transcriptional activity in 293 T cells were evaluated using Dual-Glo ? Luciferase Assay System.(3)After LPS-stimulated RAW 264.7 cells were co-incubated with different doges of VN2 or VN5 for 24 h,the level of NO in the supernatants was measured by using Griess reagent.(4)The osteoclasts stimulated by MCSF(50 ng/mL)and RANKL(100 ng/mL)were co-incubated with different concentrations of VN2 and VN5 for 4 days until osteoclasts were formed and the total TRAP activity was determined using p-nitrophenyl phosphate method.(5)The mature osteoclasts were stained with a TRAP assay and the number of TRAP positive cells(more than three nuclei)was observed using microscopy.The mature osteoclasts were stained with rhodamine-conjugated phalloidin and the area of actin ring were visualized and photographed with Fluorescent Microscope.(6)The effects of VN5 and VN2 on the inflammatory signaling pathways of NF-?B and MAPK were studied in LPS/IL-1?/TNF?-stimulated RAW 264.7 cells,RANKL-stimulated osteoclasts,and in IL-1?-stimulated fibroblast-like synoviosytes by Western-blot.The related inflammatory factors of IL-1?,IL-6,IL-8,TNF-? and the characteristic osteoclastic genes NFATc1,TRAP,CTSK,were detected by Real-time RT-PCR.(7)The IKK activity of RAW 264.7 cells treated with VN5 was detected by IKK? kinase activity kit.The molecular docking technique was applied to deduce and identify the potential binding mode for VN5 and its potential target IKK?.Result:(1)Anti-inflammatory effects and mechanism of VN5 and VN2 in LPS/IL-1?/TNF-?-stimulated RAW 264.7 cells.Our results indicated that VN5 could dose-dependently inhibit the production of NO in LPS-stimulated RAW 264.7 cells and down-regulate the activity of NF-?B in TNF-?-stimulated 293 T cells(p<0.05),without causing obvious cytoxicity in tested concentrations.However,VN2 did not show significant effect on either the trascriptional activity of NF-?B or NO production.Further mechanism studies revealed that VN5 significantly inhibited the expression of p-IKK,and subsequently decreased the expressions of p-p65 and p-ERK and suppressed the LPS/IL-1?/TNF-?-induced degradation of I?B,thus inhibiting the activation of NF-?B and MAPK pathways(p<0.05).Also,the expressions of NF-?B downstream inflammatory cytokines such as IL-1?,IL-6,and TNF-? were markedly reduced after treatment with VN5 in LPS-stimulated RAW 264.7 cells(p<0.05).(2)The function and mechanism of VN5 and VN2 on osteoclastogenesis.Our results showed that the total TRAP activity was increased immediately after stimulated with RANKL,while administration of VN2 and VN5 significantly inhibited the osteoclast formation and TRAP activity in a concentration-dependent manner(p<0.05),without causing evident cytoxicity in tested concentrations.In addition,further studies revealed that TRAP-positive multinucleated cells could be obviously observed in the presence of RANKL during osteoclastogenesis.However,we found that the number of TRAP-positive multinucleated cells was significantly declined treated with VN2 and VN5 at the different doses(p<0.05),especially at high concentrations compared with the model group.The staining images of actin ring displayed that compared with the the entire and normal periphery of control cells,VN2 or VN5-treated cells had thinner actin rings and the individual podosomes were dose-dependently diminished or absent.VN2 displayed much better activity and thus selected to be furter studied in term of its underlying mechanism.Western-blot analysis indicated that the phosphorylation of ERK was strongly inhibited in RANKL-induced osteoclastogenesis by VN2 in a concentration dependent manner(p<0.05),without affecting the expressions of I?B and p65 in the NF-?B pathway.Also,the expressions of phosphorylated NFATc1,CTSK and TRAP were significantly decreased.However,VN5 significantly inhibited the expression of p-IKK,and subsequently decreased the expressions of p-p65 and p-ERK,and suppressed the RANKL-induced degradation of I?B,thus inhibiting the actication of NF-?B and MAPK pathway(p<0.05).Furthermore,the mRNA expressions of NFATc1 and its regulated genes CTSK,TRAP and OSCAR were all inhibited considerably in RANKL-reduced osteoclasts differentiation by treatment with VN2 and VN5(p<0.05).(3)Anti-inflammatory effects,mechanism and target of VN5 in IL-1? stimulated fibroblast-like synoviosytes.VN5 significantly inhibited the expression of p-IKK,and subsequently decreased the expressions of p-p65 and p-ERK,and suppressed the degradation of I?B? in IL-1?-induced fibroblast-like synoviosytes,thus inhibiting the activationof NF-?B and MAPK pathways.Also,the expressions of NF-?B downstream inflammatory cytokines such as IL-1?,IL-6,TNF-?,and IL-8 were markedly reduced.Furthermore,the IKK? activity was significantly inhibited by VN5 at the consentration of 40?? with the inhibitory rate of 48%.Molecular docking results displayed that VN5 could bind with Cys99 and Asp103 with hydrogen.Cys99 is located at the center of the ATP binding pocket,which indicated that VN5 could prevents ATP from binding with IKK?,thereby inhibiting the activity of IKK?.Conclusion:(1)VN5 could exert anti-RA activity mainly through its significant anti-inflammatory effects,which inhibited both the expression and activity of p-IKK and subsequently suppressed the activation of NF-?B and MAPK signaling pathways.And IKK? may be the potential binding target of VN5.(2)VN2 could exert anti-RA activity mainly through its effects in attenuating osteoclastic bone destruction,which effectively inhibited osteoclastogenesis mainly by suppressing the ERK-NFATc1 signaling and the podosomes formation of actin rings.Our results indicated that different lignans in TOV of VN fruits could synergistically exert anti-RA effects.