The Experimental Study Of The Therapeutic Effect Of Resiquimod On Mouse Atopic Dermatitis Model

Atopic dermatitis(AD)is a common inflammatory skin disease.Its main clinical manifestations are recurrent eczema-like lesions and severe pruritus.Most patients have allergic constitution or family history of allergic diseases.The incidence rate of AD is increasing year by year.About 20% of children and 10% of adults in Europe and the United States suffer from this disease.At present,the exact pathogenesis of AD is not definite,and genetic,immune,infection and environmental factors play great roles.The interaction of multiple factors leading to abnormal skin barrier and immune dysfunction,which are considered to be the key of the pathogenesis of AD.The immunological pathogenesis of AD is mainly related to the immune imbalance of Th1,Th2 and Treg cells,which eventually leads to the changes of immune response related to Th1 and Th2 and IgE mediated hypersensitivity.The increased expression of Th2 cytokines in AD lesions,such as IL-4,IL-5 and IL-13,can inhibit the repair of skin barrier and the secretion of antimicrobial peptides,promote the production of IgE,chemotactic eosinophils and lead to severe skin pruritus.In order to further explore the pathogenesis of AD and study the therapeutic effect of drugs,it is necessary to select appropriate AD animal models according to different research directions.At present,AD animal models can be roughly divided into the following three categories: 1.Inbred line model;2.Genetic engineering model with certain gene knockout or knockin;3.AD-like model induced by local application of exogenous drugs.Inbred mice can develop spontaneous eczema-like dermatitis under specific pathogen free conditions and enhance their immune response to percutaneous antigens,such as NC / Nga mice.This model reflects the natural process of human AD through enhancing the sensitivity of skin to hapten or allergen.Genetic engineering AD models include IL-4 transgenic mice,IL-31 transgenic mice,thymic stromal lymphopoietin(TLSP)transgenic mice,Rel B gene knockout mice,cathepsin gene knockout mice,etc.these models are of great significance to clarify the biological role of targeted molecules.The sensitizing substance induced AD model is the most commonly used model in the field of atopic dermatitis.The model may need to be sensitized repeatedly,but it is simple and affordable,and is suitable for all types of mice.Commonly used sensitizing substances include ovalbumin(OVA),dust mite,dinitrochlorobenzene(DNCB)and calcipotriol(MC903).MC903 can induce AD-like models in different strains of mice,and the serological manifestations are also similar to those of AD patients.The model has been widely used in laboratories at home and abroad in recent years.Therefore,MC903 model was used in this experiment.Resiquimod(R848),a TLR7 / 8 agonist,is an imidazoline immune response regulator,which can significantly enhance Th1 response and promote IFN-γ and IL-12 to play the role of anti-tumor and adjuvant.At the same time,it can inhibit Th2 response,reduce the production of IL-4 and IL-5,and decrease the IgE level of healthy people,allergic rhinitis and atopic dermatitis.It can be used to treat allergic diseases.The pathogenesis of AD is not clear,but the imbalance of Th1 / Th2 immune response and the increase of IgE all play an important role.In this experiment,we applied calcipotriol solution to the ears of mice,observed the inflammatory changes of mice ear lesions,and evaluated the stability of AD model.Then resiquimod was intraperitoneally injected into AD mice model.The improvement of mice ear lesions was observed and its therapeutic effect was evaluated.Part One The mouse model of atopic dermatitis was established with MC903Objective: To establish mouse model of atopic dermatitis and evaluate its stability.Methods: Twenty BALB/C mice were randomly divided into control group and model group.The mice ears in the model group were given 2nmol of calcipotriol solution once a day for 14 days.Mice in the control group were given 16μl of alcohol solution once a day for 14 days.On the 15 th day,the erythema,swelling and scale of ear lesions in the two groups were observed;The thickness of mice ears was measured with vernier caliper;HE staining and toluidine blue staining were used to observe the pathological morphology and inflammatory cell infiltration of skin lesions;The levels of serum IgE,IL-4 and IL-13 were detected by ELISA.Results: Compared with the control group,the ear lesions in the model group were swollen and red,with scales scattered on them,showing obvious dermatitis-like manifestations;The thickness of mice ears was significantly thickened;HE staining showed hyperkeratosis,parakeratosis,acanthosis,intercellular edema,telangiectasia and congestion with mixed inflammatory cell infiltration in the whole dermis.Toluidine blue staining showed scattered mast cells;The levels of serum IgE,IL-4 and IL-13 increased.Part Two Therapeutic effect of resiquimod on mouse AD model and its mechanismObjective: To observe the therapeutic effect of intraperitoneal injection of resiquimod on mouse model of atopic dermatitis and explore its therapeutic mechanism.Methods: Thirty BALB/C mice were randomly divided into blank group,model group and treatment group.The atopic dermatitis model was induced by calcipotriol solution in the model group and the treatment group.At the same time,the model group was intraperitoneally injected with 200μl PBS solution every day,and the treatment group was intraperitoneally injected with 50 nmol of resiquimod(dissolved in 200μl PBS solution)every day for 14 days.The blank group was not given any treatment.On the 15 th day,the erythema,swelling and scale of ear lesions in each group were observed;The thickness of mice ears was measured;HE staining and toluidine blue staining were used to observe the infiltration of inflammatory cells in skin lesions;The distribution of mast cells and TSLP in the skin were observed by immunohistochemistry;The levels of serum IgE,IL-4 and IL-13 were detected by ELISA;The mRNA expressions of TSLP and IFN-γ in skin lesions were detected by RT-PCR.Results: Compared with the model group,the degree of erythema,scale and swelling of ear skin lesions in the treatment group were significantly improved;HE staining showed that acanthosis,intercellular edema and inflammatory cell infiltration in dermis were significantly improved.Toluidine blue staining and immunohistochemical staining of CD117 showed that the number of mast cells decreased;TSLP staining showed that a large amount of TSLP was expressed in the whole epidermis of the model group and the treatment group;The levels of serum IgE,IL-4 and IL-13 decreased;There was no significant difference in the relative expression of TSLP mRNA between the model group and the treatment group,but the expression of IFN-γ mRNA in the treatment group increased with statistic significance.Conclusions:1.MC903 can increase the levels of serum IgE,IL-4 and IL-13,infiltrate inflammatory cells in mice ears,increase the number of mast cells,and make mice ears appear erythema,scales and increase the thickness.MC903 can establish a stable mouse AD model;2.Intraperitoneal injection of resiquimod can alleviate the clinical manifestations and inflammatory infiltration of AD mice;3.The levels of serum IgE,IL-4 and IL-13 in AD mice were significantly decreased by intraperitoneal injection of resiquimod;4.Intraperitoneal injection of resiquimod increased the expression of IFN-γ mRNA in skin lesions of AD mice,but had no effect on the relative expression of TSLP mRNA.