The Study Of Anti-inflammatory Effects And Molecular Mechanisms Of Resovlin D1 In HaCaT Cells And Imiquimod-induced Mice Psoriasiform Dermatitis

Part Ⅰ SIRT1 inhibited the production of cytokines by decreasing LPS-induced acetylation of NF-κB p65 on HaCaT cellsObjective: To Study the roles and mechanism of resolvin D1(RvD1)on HaCaT cells proliferation induced by lipid polysaccharide(LPS).To investigate the effects of silent information regulator 1(SIRT1)inhibit cytokines and acetylation of NF-κB p65 on LPS-induced HaCaT cells.Methods:This part of the experiment had four groups: Control group,LPS(10ug/ml)group,LPS(10ug/ml)+ RvD1(100nmol/L)group and SIRT1 RNAi group.The effects of RvD1 and LPS on HaCaT cells viability were detected by CCK-8 assay firstly.The expression of LXA4 receptor(ALX),G protein-coupled receptor-32(GPR32)and toll-like receptor 4(TLR4)in HaCaT cells were detected by reverse transcription polymerase chain reaction(RT-PCR).HaCaT cells were induced by LPS(10ug/ml)with or without preincubation with RvD1(100nmol/L)for 30 min.After incubation for an appropriate time,m RNA levels and protein levels of interleukin-1β(IL-1β)and tumor necrosis factor-alpha(TNF-α)produced by HaCaT cells were determined by quantitative real-time polymerase chain reaction(QRT-PCR)and enzyme-linked immunosorbent assay(ELISA).protein levels of SIRT1 and acetylation of NF-κB p65 were determined by Western blot.Results: No significant effect on the cell viability of HaCaT cells by incubation with different concentrations of LPS and RvD1 for 24 h.HaCaT cells expressed ALX,GPR32 and TLR4.RvD1 up-regulate m RNA expression of SIRT1.RvD1 markedly inhibited m RNA and protein expressions of IL-1β and TNF-α induced by LPS in HaCaT cells.LPS obviously decreased the expression of SIRT1 in m RNA and protein levels,and increased acetylation levels of NF-κB p65.RvD1 reversed partly the effects of LPS on SIRT1 and acetylation of NF-κB p65.Conclusion: RvD1 decreased the production of IL-1β and TNF-α,the mechanisms might be related to RvD1 up-regulating expression of SIRT1 via inhibiting acetylation of NF-κB p65.PartⅡ LPS stimulates IL-1 beta production of HaCaT cells associated with the MAPK p38 signaling pathwayObjective: To investigate the effects of RvD1 and LPS on HaCaT cells apoptosis,and signaling pathway associated with the inflammatory cytokines production of HaCaT cells stimulated by LPS were determined.Methods:While HaCaT cell apoptosis was detected by flow cytometry,this part of the experiment had four groups: Control group,LPS group,LPS(10ug/ml)+ RvD1(100nmol/L)group and RvD1(100nmol/L)group.In follow-up study,divided the cells into five groups: LPS group,Control group,LPS+PD98059 group,LPS+ SP600125 group and LPS +SB203580 group,HaCaT cells were induced by LPS(10ug/ml)with preincubation with MAPK inhibitor for 30 min.The m RNA levels and protein levels of interleukin-1β(IL-1β)and tumor necrosis factor-alpha(TNF-α)were detected by real-time quantitative polymerase chain reaction(QRT-PCR)and enzyme-linked immunosorbent assay(ELISA)at different time point,the MAPKs phosphorylation and NF-κB p65 nuclear translocation were determined by Western blot.Electrophoretic mobility shift assay(EMSA)was used to check NF-κB p65 DNA binding activity.Results: RvD1 suppressed proliferation and apoptosis of both normal control and LPS-treated HaCaT cells.RvD1 inhibited the phosphorylation of MAPKs.An inhibitor of the MAPK p38,SB203580 decreased markedly the m RNA levels and protein levels of interleukin-1β(IL-1β).The NF-κB p65 nuclear translocation and DNA binding activity of LPS-induced HaCaT cells were both suppressed by RvD1.Conclusion: LPS-induced HaCaT cells secreted IL-1β through the MAPK p38 signaling pathway.PartⅢ Resolvin D1 attenuates imiquimod-induced mice psoriasiform dermatitis through MAPKs and NF-κB pathwaysObjective: The present study aimed to determine the protective effects and the underlying mechanisms of RvD1 on imiquimod(IMQ)-induced psoriasiform dermatitis,To explore the feasibility of RvD1 for psoriasis therapy.Methods: 30 BALB/c mice were randomly divided into six groups(n= 5 per group).(1)Control group(Control);(2)Imiquimod group(IMQ);(3)IMQ and 1μg/kg RvD1 group(RvD1,1+IMQ);(4)IMQ and 5μg/kg RvD1 group(RvD1,5+IMQ);(5)(RvD1,5+IMQ+Boc)group;(6)(IMQ+Boc)group.Mice were topically treated with IMQ to develop psoriasiform dermatitis on their shaved back,pretreated intraperitoneally(i.p.)with or without RvD1 or tert-butoxycarbonyl Met-Leu-Phe peptide(Boc),a lipoxin A4(ALX)receptor antagonist.The severity was monitored and graded using a modified human scoring system,the Psoriasis Area and Severity Index(PASI),histopathology,and the signature cytokines of psoriasis(IL-23,IL-17,TNF-α and IL-22).The m RNA and protein levels of these signature cytokines were detected using quantitative real-time PCR(QRT-PCR)and ELISA respectively.The expressions of signaling proteins MAPKs and NF-κB p65 were analyzed using western blotting.Electrophoretic mobility shift assay(EMSA)was used to check NF-κB p65 DNA binding activity.Results: Our study showed that RvD1 alleviated IMQ-induced psoriasiform dermatitis and improved skin pathological changes.RvD1 markedly inhibited IMQ-induced activation of ERK1/2,p38,JNK(c-Jun N-terminal protein kinase,a subfamily of MAPKs),and NF-κB.Furthermore,pretreatment with Boc,would not exacerbate skin inflammation of IMQ-induced mice,but significantly reversed the beneficial effects of RvD1 on IMQ-induced psoriasiform inflammation.Conclusion: RvD1 can obviously improve skin inflammation in IMQ-induced mice psoriasiform dermatitis.The protective mechanisms might be related to its selective reaction with lipoxin A4 receptor/ Formyl-peptide receptor 2(ALX/FPR2),by downregulating relevant cytokines of the IL-23/IL-17 axis expression,the inhibition of MAPKs and NF-κB signaling transduction pathways.Thus,these results show that RvD1 could be a possible candidate for psoriasis therapy.