Effect Of Yangxue Jiedu Recipe And Sagrassin On Abnormal Activation Of Psoriasis - Like Mice And IL - 17 + T Cells

Objective:To observes the effects of Yang xue jie du fang andits main pharmaceutical composition--astilbin on mice psoriasis-like lesions induced by Imiquimod (IMQ). To study the effects ofastilbin on Thl7 cells and gamma delta T cells cytokine expression and production in vitro.To explore the exact immunological mechanisms ofYang xue jie du fang and Astilbin in potential psoriasis treatment.Methods:Balb/c male mice were randomly assigned to groupsrControl group(topical using vaseline on the back and NS oral administration once a day for 8 days), Model group(topical 4% Imiquimod (IMQ) 42mg and NS oral administration once a day for 8 days), Yang xue jie du group(water decoction of Yang xue jie du fang converted into mice doseoral administration, with topical IMQ as Model group), Liang xue jie du group(water decoction of Liang xue jie du fang converted into mice doseoral administration, with topical IMQ as Model group), Huo xue jie du group(water decoction of Yang xue jie du fang converted into mice doseoral administration, with topical IMQ as Model group), Astilbin High group (10mg/kg astilbin solution oral administration, with topical IMQ as Model group), Astilbin Low group (5mg/kg astilbin solution oral administration, with topical IMQ as Model group),Methotrexate group (lmg/kg methotrexate solution oral administration, with topical IMQ as Model group), Tripterygium glycosides group (GTW, 10mg/kgGTW solution oral administration, with topical IMQ as Model group). C57BL/6J mice, Stat3 overexpression mice were randomly divided into normal control group (wild type Wild Type-Ctrl, WT-Ctrl), wild type model group (WT-IMQ), wild type of Yang xue jie du fang in high dose group (WT-Yang Xue Jie Du decoction-High, WT-YH), wild type Yang xue jie du fang low dose group (WT-Yang Xue Jie Du decoction-Low, WT-YL), wild typemethotrexate group (WT-MTX), Stat3overexpression control group (Stat3-Ctrl), Stat3overexpression model group (Stat3-IMQ), Stat3 overexpression Yang xue jie du fang high dose group (Stat3-YH), Stat3 overexpression Yang xue jie du fang low dose group (Stat3-YL), Stat3 overexpression methotrexate group (Stat3-MTX). At day 8, skin lesions were cut for pathological examination. The lesions were evaluated according to the Psoriasis area and severity index (PASI). The histology and epidermal thicknesses were observed by light microscope. Proliferating cell nuclear antigen (PCNA) expression was investigated by immunohistochemical staining. Meanwhile, CD3 positive expression were counted by immunohistochemical staining for inflammatory infiltration of T lymphocytes.CBA method to measure the concentration of serum inflammatory cytokines content; IL-17 cytokines and Stat3 signaling pathway related protein expression in lesions detected by RT-PCR and Western blot.CD3+gamma delta T cells in the lymph nodes, spleen and bone marrow were detecte by flow cytometry. At the same time, to observe inhibitory effect of astilbin on the selecting gamma delta T cells in vitro by flow cytometry.Results:1. Compared with Model group, the cutaneous symptoms of the Yang xue jie du group and Liang xue jie du group were alleviated, with PASI scores decreased, epidermal parakeratosis and epidermal over-proliferation relived, the numbers of dermal T lymphocytes reduced significantly.2. Compared with wild type mice, Stat3 overexpression transgenic mice appearance had no significant difference, skin epidermal layer slightly thicker, marked thickening of the dermis,IL-17 A,Il-22, ROR gamma T mRNA expression respectively about two times 16 times and 3 times as high as that the wild type miceskin. Comparison of two IMQ group can be found that Stat3-IMQ mice lesions erythema deeperthan WT-IMQ mice, scaly patches and easy to fall off, thickening of the skin infiltration is more significant, thickness and proliferation of epidermal cell number, dermal CD3+ cells significantly more than WT-IMQ mice, Stat3-IMQ mice spleen enlarged 2 times as WT-IMQ spleen, in the lesions of IL-17A,Il-22, ROR gamma T, expressed mRNA respectively compared with mice WT-IMQ up to 24 times,8 times,2.5 and 2 times. Imiquimod induced by two kinds of mice both in appearance of psoriasis like lesions or epidermal proliferation, T lymphocytic infiltration of the dermis, the expression of inflammatory factors in skin lesions and peripheral blood and secretion content are with the CTRL group has statistically significant difference. Stat3-Y group of mice than Stat3-IMQ mice skin lesions symptoms significantly alleviated, PASI scores decreased, keratinocyte proliferation decreased, dermal T lymphocyte infiltration weakened, the skin moisture and oil content was higher than the Stat3-IMQ group. At the same time, Stat3-Y mice spleen weight, lesions of IL-17A,Il-22, ror gamma T, expressed mRNA expression and outer peripheral blood inflammation related cell factors (IL-17, IL-6 and TGF alpha, IL-2, IL-4, IFN gamma) was significantly lower than that Stat3-IMQmice. Analysis of mouse skin lesions of Stat3 protein expression can be found:Stat3-Ctrl STAT3, p-STAT3, JAK3, P-Jak3 protein expression group was higher than that of WT-Ctrl; p-STAT3, P-Jak3 protein expression in Stat3-IMQgroup was higher than that of WT-IMQgroup; with the CTRL group comparison, elevated IMQ group of Stat3, p-Stat3, JAK3, P-Jak3 protein expression; Stat3-Y group and WT-Y group expression of p-STAT3 and P-Jak3 protein are IMQ group than in the corresponding low.3. Mice were administered 5 to 10 mg/kg astilbin. Inflammation of psoriasis-like lesions was assessed by histology, circulating levels of T cells were assessed by flow cytometry and cytokines by bead-based immunoassay. Jak/Stat3 in isolated T cells was assessed by western blotting and RORyt expression was assessed by RT-PCR. Administration of astilbin ameliorated IMQ-induced keratinocyte proliferation, infiltration of CD3+ cells to psoriatic lesions and ameliorated elevations in circulating CD4+and CD8+ T cells and inflammatory cytokines (IL-17A, TNF-a, IL-6, IFN-y and IL-2). In vitro, astilbin inhibited Th17 cell differentiation and IL-17 secretion of isolated T cells, and inhibited Jak/Stat3 signaling in Th17 cells, while up-regulating Stat3 inhibitor SCOSE3 expression in psoriatic lesions4. Purified naive CD4+T cells were cultured in vitro under Thl 7 polarizing condition, Th17 cell differentiation was markedly increased. Compared with Thl 7 polarizing condition group, Astilbin (10μg/ml, 1μg/ml,0.1μg/ml, 0.01μg/ml) had an influence on the differentiation of Th17 (CD4+IL-17+) cells (P<0.05). Higher expression of IL-17AmRNA, IL-17FmRNA, TNF-amRNA, RORytmRNA and decreased expression of IL-22mRNA、IFN-ymRNA, IL-4mRNA, IL-10mRNA were observed in Th17 polarizing condition group. In the presence ofAstilbin, the expression of IL-17AmRNA, IL-17FmRNA, TNF-amRNA was inhibited by Astilbin treatment, which correlated with decreased RORytmRNA.5. Compared with Model group, the cutaneous symptoms of the Astilbin group and Methotrexate group were alleviated, epidermal parakeratosis and epidermal over-proliferation relived, the numbers of dermal lymphocytes reduced significantly. IL-17 related gene expression in psoriasis lesions tends to be normal, and the percentage of CD3+ gamma delta T cells was down in the immune organs, at the same time, astilbin significantly suppress the gamma delta T cells induced by in vitro.Conclusions:1. Yang xue jie du fang and Liang xue jie du fang improved imiquimod-induced murine psoriasis-like lesions probably by inhibiting keratinocytes over-proliferation, parakeratosis and inflammatory infiltration mediated by Toll receptor activation.2. Imiquimod induced Stat3 overexpression transgenic mice model in the pathological change, cellular level and molecular level is more close to the change of human psoriasis, as a simulation of psoriasis is more appropriate animal model, for drug screening and to explore the mechanism and so on.Yangxuejiedu decoction can significantly ease the imiquimod induced mouse psoriasis like lesions, improve skin barrier function, model to regulate the immune disorder, reduce skin lesions and week serum IL-17 related inflammatory cytokines expression, regulate the activity of STAT3 protein phosphorylation expression. Yangxuejiedu decoction can inhibit the the excessive activation of Stat3 signaling pathway and epidermal cell proliferation, and regulat IL-17 related immune response.3. Astilbin could ameliorate imiquimod-induced skin lesions. The protection achieved byastilbin in imiquimod-induced psoriasiform mice is most probably due to its ability to downregulate immune response of IL-17A through inhibition of Stat3 phosphorylation. It is also having an influence on the differentiation and secretionof Th17 (CD4+IL-17+) cellsand gamma delta T cells.Astilbin as potential drug is remarkable for the treatment of psoriasis.