The Research On The Efficacy And Mechanism Of Xiaoyin Recipe And Ebastine Therapy In Imiquimod Induced Psoriasis Mouse Model

Psoriasis is a common immune-mediated chronic inflammatory disease of the skin. Ithas a long course, easy to relapse, largely influences patients‘physical and mental health.The pathogenesis of psoriasis is complex, involved in many environmental and geneticfactors,mainly mediated by the T lymphocyte，of which the CD4+T lymphocytes play akey role in immune function. Studies a few years ago consider that there is an abnormalpresence of Th1/Th2balance in the course of psoriasis. In recent studies, another new CD4+T lymphocyte subsets-Th17cells have been discovered. Th17cells can producecytokines such as IL-17and IL-22, that play a key role in the pathogenesis of psoriasis.Th17cytokines such as IL-17A、IL-17F、IL-22,and Th1cytokines such as INF-γ、TNF-α,can activated keratinocytes, leading an excessive proliferation and produce a seriesof cytokines, that furtherly induces T cells, neutrophils to migrate to the inflammatory skin,creating a vicious cycle. Thus, Th1and Th17cells play a crucial role in the pathogenesisof psoriasis. Inhibition of Th1, Th17cell proliferation and the secretion of cytokinesdownstream can relieve psoriasis.The mice models of psoriasis induced by imiquimod have been widely used instudies recent years. Topical application of imiquimod (IMQ), can rapidly inducepsoriasis-like skin inflammation in mice,with similar clinical feactures, pathology andmolecular features to human disease pathogenesis of psoriasis.This model can effectivelysimulate human psoriasis, and can be used for elucidating the pathogenesis mechanism ofpsoriasis, assessing the efficacy of new therapies.Xiaoyin Recipe is a classical Chinese herbal medicine of ChangHai Hospital to treatpsoriasis.It can effectively control the disease and improve patients‘life quality,with a lowprice and less side effects. Ebastine is a non-sedating H1receptor antagonist.Recent studiesindicate that ebastine can inhibit T cell proliferation and the production of cytokines suchas IL-4, IL-5, IL-6, and TNF-α.The goal of our study is to construct a murine model of psoriasis by imiquimodinducement and then research the efficiency of Xiaoyin Recipe and ebastine, contrast thechange of Th1，Th17downstream cytokines,to explore the possible mechanism ofXiaoyin Recipe and the potential value of ebastine in treatment of psoriasis. Part One, Establishment and Evaluation of Psoriasis Animal Model Induced bythe Imiquimod[Objective] To establish imiquimod induced psoriasis mice model and evaluate theeffectivity of simulating human psoriasis, and to provide a reliable basis for further study.[Methods] We choose BABL/c mice as model animal. Take a2cm×1.5cm area hairremoval on its‘back. The blank control group were applied distilled water0.5ml once aday, while the model group applied5%imiquimod cream60mg once a day for6consecutive days.Observe changes in skin and take pictures every day. Aftermodeling,kill them by cervical dislocation. Then, take the lesion skin to do parafin sectionand HE stain,observe the thickness of epidermis and the infiltration of inflammatory cells.[Results] By visual observing, the skins of the blank group had neither erythema norscales，while the skins of the mice models had obvious erythema and adhesion of thesilver white scales.By microscopic observation of HE stain, the model group showedsignificant epidermal thickening and inflammatory cell infiltration, compared with blankcontrol group.[Conclusion] The imiquimod induced psoriasis mice model can effectively simulatehuman psoriasis.Part Two, Evaluation of the Efficacy of Xiaoyin Recipe and Ebastine Therapy byImiquimod Induced Psoriasis Mouse Model[Objective] To study efficacy of Xiaoyin Recipe and ebastine therapy in the treatmentof imiquimod induced psoriasis mouse model, and to further validate this mouse model ofpsoriasis.[Methods]54BABLc mice were randomly divided into9groups (I~IX group),6rats in each group. Back hair removal. Group I was the normal control group, II~IXgroup were model groups which were applied5%imiquimod cream60mg once a day for6consecutive days. After modeling,these8model groups were still applied imiquimodevery two days in order to maintain the stability of psoriasis model. Regimen: Group II isthe model control group, do not give any medicine; Group III~IX were respectively givennormal saline (0.5ml/d), Xiaoyin Recipe (2.88g/kg.d), ebastine (3.03mg/kg.d), compoundglycyrrhizin (22.75mg/kg.d), tripterygium glycosides (9.1mg/kg.d), dexamethasone(1.02mg/kg.d), cyclosporine A(27.30mg/kg.d) liquid daily gastric lavage therapy for2weeks. After treatment, the mice were killed, take the lesion on the back, divide into threeparts,two of them immediately placed in80℃refrigerator for spare, the other one wasplaced in10%formalin fixed liquid. Photographed lesions erythema and scaling changes during the course of treatment. Make paraffin sections and do HE stain, record theinfiltration of inflammatory cells and the sectioned epidermis area values, and the datawere statistically analyzed.[Results] By visual observing, after two weeks treatment, lesions in Group IV，GroupVI and GroupVIII were improved, of which Group VIII were significantlyimproved, compared with Group II and Group III; erythema and scale in Group II, GroupIII, Group V, Group VII, and Group IX had no obvious improvement.Multiple comparisons of infiltrating cells number show that blank control group werelower than the other groups (P <0.05); the model control group and the saline group werehigher than the other groups (P <0.05); Xiaoyin Recipe Group was less than that ofebastine group and compound glycyrrhizin group (P <0.05), and it had no significantdifference with cyclosporine A group, tripterygium glycosides group and dexamethasonegroup; ebastine group was higher than that of Xiaoyin Recipe Group (P <0.05), and had nosignificant difference with compound glycyrrhizin group, cyclosporine A group,tripterygium glycosides group and dexamethasone group.Multiple comparisons of sectioned epidermis area values show that blank controlgroup were lower than those in the other groups (P <0.05); the model control group andthe saline group were higher than that of the other groups (P <0.05); Xiaoyin RecipeGroup Group was less than that of Compound Glycyrrhizin group, cyclosporine A group,ebastine group (P <0.05), and had no significant difference with dexamethasone group,tripterygium glycosides group; ebastine group was statistically different with all the othergroups,but only lower than that of the model control group and the saline group (P <0.05).[Conclusion] Imiquimod induced psoriasis mouse model can effectively simulatehuman psoriasis, and can be used for the study on pharmacodynamics. Xiaoyin Recipe andebastine in treatment of psoriasis in mouse models lead different degrees of efficacy, caneffectively reduce the number of inflammatory cell infiltration in a skin lesions of mousemodel, inhibit the excessive proliferation of epidermal cells.Part Three，Study on the Mechanism of Xiaoyin Recipe and Ebastine Therapyin Imiquimod Induced Psoriasis Mouse Model[Objective] To compare the difference of TNF-α, IFN-γ, IL17, IL22in lesions ofpsoriasis mouse model before and after Xiaoyin Recipe and ebastine treatment on gene andprotein levels， so as to explore their mechanism.[Methods] Take lesion samples that were placed in-80℃refrigerator mentioned in Part Two to extract RNA and total protein respectively。Use real-time quantitative PCR,Western blot to detect the TNF-α, IFN-γ, IL17, IL22mRNAand protein expression.[Results] The TNF-α, IFN-γ, IL17, IL22mRNA and protein expression in the blankcontrol group were significantly lower than those in the model group and saline group;After Xiaoyin Recipe treatment,Four kinds of cytokines mRNA expression in lesionalskin of mice models were significantly decreased, compared with model control group, thedecrease of IL-17mRNA expression was the most significant. After treatment withebastine, TNF-α, IFN-γ, IL-17mRNA expression was significantly decreased, but nosignificant change in IL-22mRNA. The protein expression of TNF-α, IFN-γ, IL17, IL22were in accord with mRNA expression.[Conclusion] Xiaoyin Recipe and ebastine can effectively reduce the expression ofmRNA and protein in psoriasis-like lesions of mouse model. The effect of Xiaoyin Recipeis more comprehensive,it can significantly decrease both Th1cytokines TNF-, IFN-γ andTh17cytokines IL17, IL22. Ebastine also can inhibit Th1and Th17cell pathway，it cansignificantly decrease TNF-α、IFN-γlevel in psoriasis-like lesions.