| Objective:Dendritic cells(Dendritic cells,DCs)are the most potent antigen-presenting cells.The activation of DCs is considered to be the upstream of the pathogenesis of psoriasis.Cytokines secrete from dendritic cells(DCs)such as Interleukin-12(IL-12)and Interleukin-23(IL-23)can further promote the activation of Thl and Th17 cells,which secrete IL-17,IL-21,IL-22,TNF-alpha and other cytokines.Abundant cytokines,chemokines,and growth factors altogether constitute a "cytokine storm" which together lead to erythema in psoriasis.Studies have shown that,in psoriatic skin lesions,the number of DCs increases,treating with anti-IL-23 monoclonal antibody has obtained good curative effect.Therefore,controlling cytokine secretion from DCs may play a therapeutic role."From the blood to treat" is the main rule for the treatment of psoriasis,and "blood poison" is the core of its pathogenesis.Beijing Hospital of TCM and Beijing institute of TCM believe that the location of blood heat syndrome of psoriasis is blood system,heat toxin plays a crucial role on the prognosis and outcome of patients.The Liang xue jie du prescription will be accomplished by dosing Detoxification durgs further to cooling blood drugs,which is clinically effective.In this study we plan to observe the effects of Liang Xue jie du fomula and its main pharmaceutical composition β,β-dimethylacryloyl alkannin on mice psoriasis-like lesions induced by Imiquimod(IMQ).To study the effects of β,β-dimethylacryloyl alkannin on the function of human peripheral blood monocyte-derived dendritic cells and mouse bone marrow derived dendritic cells.To explore the exact immunological mechanisms of Liang Xue jie du fomula and β,β-dimethylacryloyl alkannin on the treatment of psoriasis,in order to provide theoretical basis for clinical applicationMethods:Balb/c male mice were randomly assigned into 6 groups:Control group(topical using vaseline on the back of mice and oral administration with NS once a day for 6 days),Model group(topical using 4%Imiquimod 42mg and oral administration with NS once a day for 6 days),Lang xue jie du group(oral administration with water decoction of Liang xue jie du fomula converted into mice dose,and topical Imiquimod as Model group),β,β-dimethylacryloyl alkannin High group(oral administration with 12mg/kg β,(3-dimethylacryloyl alkannin solution,and topical IMQ as Model group),β,β-dimethylacryloyl alkannin Median group(oral administration with 6mg/kg β,β-dimethylacryloyl alkannin solution,and topical IMQ as Model group),β,β-dimethylacryloyl alkannin Low group(oral administration with 3mg/kg alkannin solution,and topical IMQ as Model group),Methotrexate group(oral administration with lmg/kg methotrexate solution,and topical IMQ as Model group).The morphological changes of lesional skin were evaluated according to the psoriasis area and severity index(PASI)and HE staining.Proliferating cell nuclear antigen(PCNA),Ki67 and CD11c positive expression were investigated by immunohistochemical staining.The mRNA expression of cytokines was analyzed by RT-PCR,and the proteins of TLR7/8 pathway were analyzed by Western blotting assay.Moreover,the numbers of the CD11c positive celles were detected by flow cytometry.Human peripheral blood monocyte-derived dendritic cells and mouse bone marrow derived dendritic cells were cultured in vitro under GM-CSF and IL-4 condition,and cells were purified by magnetic beads.Cells were divided into Control,Model and drug administration groups(β,β-dimethylacryloyl alkannin 12.μg/ml,10μg/ml,5μg/ml,2.5μg/ml).Cells viability was investigated by CCK-8 assay,surface molecules markers were detected by flow cytometry,the mRNA expression and cytokines secretion were analyzed by RT-PCR,CBA and ELISA respectively,and protein expression of TLR7/8 pathway were analyzed by Western blotting assay.Results:1.Compared with model group,the psoriasis-like lesions of LXJD-H,LXJD-M,LXJD-L,MTX groups were alleviated,with decreased PASI scores,epidermal parakeratosis,and reduced expression of Ki67 and CD11c positive cells.Among them,the curative effect of LXJD-L group was the most obvious.2.Compared with model group,the lesions symptoms of the DMA groups were alleviated,with decreased PASI scores,epidermal parakeratosis and reduced expression of PCNA.Among them,the curative effect of DMA-L group was the most obvious.The CD11c+ dendritic cells were decreased in the lesion and spleen.The expression of IL-23,IL-12,IL-1β mRNA and TLR8,MyD88 protein expression were inhibited significantly.3.Human peripheral blood monocyte-derived dendritic cells were identified by fluorescence immunoassay,the rate of CD la positive cells reached 94.3%,after stimulation with 10 ng/ml R848,the expression of surface marker molecules such as CD80 and CD83 were increased(P<0.01,P<0.05),promoted lymphocyte proliferation(P<0.05),and the mRNA expression of IL-6,IL-12 p40,IL-1β,TNF-a,IL-23 were significantly increased(P<0.01),the mRNA expression of IL-10 was decreased,IL-6,IL10,IL-1β,TNF-a and IL-23 cytokine secretion were increased(P<0.05,P<0.01,P<0.05,P<0.01,P<0.01);after treated with 12.5μg/ml DMA,the mRNA expression of IL-12p40,IL-1β,TNF-α,IL-23 were suppressed(P<0.01,P<0.01,P<0.05,P<0.01),IL-10 were increased(P<0.01);IL-1β,TNF-α,IL-23,IL-10 secretion were significantly suppressed after alkannin treatment.4.Mouse bone marrow derived dendritic cells cells were identified by fluorescence immunoassay,the rate of CDllc positive cells reached 91.93%,after stimulation of DCs with 10 ng/ml R848,the expression of surface marker molecules such as I-E/I-A,CD80 and CD83 increased(P<0.01),the mRNA expression of IL-1β,IL-23,IL-12 increased(P<0.01,P<0.05,P<0.001),IL-1β and IL-23 cytokine secretion increased(P<0.001,P<0.01),DCs promoted T-bet,RORyT,Foxp3 mRNA expression CD4+T cells(P<0.01,P<0.05,P<0.05),the expression of TLR7,TLR8,MyD88,IRAK,p-IRF7,p-p65,p-stat3,p-stat6 increased(P<0.01,P<0.01,P<0.05,P<0.05,P<0.001,P<0.001,P<0.001,P<0.01),treatment of R848-treated cells with DMA showed that the I-E/I-A expression was inhibited by 10,5,and 2.5 μg/mL of DMA(P<0.05,P<0.01,P<0.01),CD80 expression was inhibited by 5 and 2.5 μg/mL of DMA(P<0.01,P<0.05),and CD86 expression was only inhibited by 5 μg/mL of DMA.It was found that IL-12p40 mRNA expression was inhibited by 5、2.5μg/ml DMA(P<0.01,P<0.001),IL-1βmRNA expression was inhibitied by 10μg/ml DMA(P<0.05).IL-1(3 cytokine secretion was decreased after DMA 10μg/ml、5μg/ml、2.5μg/ml treatment(P<0.001,P<0.001,P<0.01),IL-23 cytokine secretion was inhibited(P<0.01),the expression of the TLR7,TLR8,MyD88,P-p65,P-stat3,P-stat6 proteins were inhibited(P<0.01,P<0.01,P<0.01,P<0.05,P<0.001,P<0.05),10μg/ml DMA suppressed the expression of IRAKM(P<0.05).Conclusion:1.Liang Xue jie du fomula significantly improved the psoriasis-like lesions of imiquimod-induced models.2.The main pharmaceutical composition β,β-dimethylacryloyl alkannin significantly improved the psoriasis-like lesions of imiquimod-induced models.The anti-inflammatory effect of DMA is related to inhibiting the activation of dendritic cells.And it may be one of the mechanisms of Liang Xue jie du fomula in treating psoriasis.3.β,β-dimethylacryloyl alkannin showed inhibition effect on the activation of dendritic cells by inhibiting the TLR7/8 pathway and NF-κB pathway.4.β,β-dimethylacryloyl alkannin can be used as an antagonist of TLR7/8,which providing an idea for the treatmen of psoriasis and other autoimmune diseases. |
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