| BACKGROUNDAtopic dermatitis(AD)is a chronic and recurrent inflammatory dermatosis which characterized by severe pruritus and eczematoid lesions.It is prone to children and belongs to the category of "Samanpung" and "Taeryumchang " in traditional Chinese medicine(TCM).In most countries of the world,AD has a high morbidity,especially on the rise in China in recent years.According to modern medicine in western,the pathogenesis of AD is related to heredity,environment,immunity and other factors.It is closely related to the damage of skin barrier function,congenital or acquired immune response disorder.According to theory of TCM,etiology and pathogeny of AD attribute to intolerant endowment,exogenous invasion,dietary unbalanced,under the influence of these factors,the fire of heart and deficiency of spleen Qi lead to onset.The expert consensus on the TCM diagnosis and treatment plan of atopic dermatitis in 2013 indicates protocols of treatment with syndrome differentiation.For pediatric patients,the most common situation is fire of heart and deficiency of spleen Qi interweave mutually.Therefore,in the treatment,attention is often paid to regulating the heart and spleen,clearing the fire of heart and tonifying the spleen Qi.To the patients who have heat accumulation,clearing and rushing down the endogenous pyrogen,as well as moistening the spleen.To the patients who have deficiency of spleen Qi,strengthening the spleen to avoid dampness accumulation.Piyan-? decoction was made based on theory of invigorating spleen and dampness,according to the clinical experience.It has good curative effect in reducing skin lesions and exudation,as well as alleviating clinical symptoms.In this study,firstly,we established the AD model of mice to study the characteristics of intestinal flora in the pathogenesis of AD,and the pharmacodynamic evaluation was made on the intervention of Piyan II decoction in the pathogenesis of AD.Secondly,we established the AD model of mice related to intestinal flora disorder,to study the role of intestinal flora disorder in the pathogenesis of AD,and the mechanism of Piyan II decoction in the intervention of intestinal flora disorder related AD.The purpose of our research is to lay a modern biological foundation for the application of the theory of invigorating spleen and infiltrating dampness in the treatment of AD,and expand the theory of"spleen as the foundation of postnatal development and main transportation" and its application.OBJECTIVE&SIGNIFICANCE1.Using the method of immune injury to established a mouse model of AD.From study the changes of intestinal flora in the pathogenesis of AD,to reveal the important role of intestinal flora disorder in the pathogenesis of AD.2.Using the method of immune injury combined with intragastric administration of antibiotics,to established a model of AD in mice with intestinal flora disorder.From study the inflammatory and pathological characteristics of AD under the condition of intestinal flora disorder,to further reveal the important role of intestinal flora disorder in the pathogenesis of AD.3.From evaluate the efficacy of Piyan ? decoction in the treatment of AD related or unrelated to intestinal flora disorder,to reveal the possible mechanism of Piyan ?decoction in the treatment of AD.Furthermore,to lay the foundation of modern medicine for the treatment of AD by Invigorating the spleen and infiltrating the dampness.METHODSExperiment 1:in 1st week,2%2,4-dinitrochlorobenzene(DNCB)was applied to the dorsal skin of BALB/c mice for two times of sensitization,the dosage was 200?l per time,with an interval of 3 days;in 2rd to 5th week,0.2%DNCB was applied to the same part for provocation,the dosage was 200?l per time,twice a week,with an interval of 3 days.In the 3rd to 5th week,the Piyan ? decoction was applied by intragastric administration with the concentration of 2.5g/ml and 5.0g/ml respectively.The dosage was 0.2ml per time,once a day.After animal model establishment of AD and drug administration,the severity of skin lesions was evaluated,as well as the level of Toll like receptors 2(TLR2),loricrin(LOR)and thymic stromal lymphopoietin(TSLP)were detected by Western blot.The serum levels of IFN-?,TNF-?,IL-1?,IL-4 and IL-5 were detected by flow cytometry,and the levels of IgE in serum were detected by ELISA.The total bacterial concentration and colony composition of colonic contents in mice were analyzed by DNA electrophoresis and fluorescence.Experiment 2:in the 1st week,the BALB/c mice were administrated with ceftriaxone sodium 200mg/ml by intragastric administration,the dosage was 0.2ml per time,twice a day,every time interval was 6 hours.In the 2rd week,2%DNCB was applied to the dorsal skin for sensitization twice,the dosage was 200?l per time,and the interval was 3 days.In the 3rd to 5th week,0.1%and 0.2%DNCB were used to sensitize the same part for provocation,the dosage was 200?l per time,2 times a week,and the interval was 3 days.At the 4th to 5th week,the Piyan ? decoction was applied by intragastric administration with the concentration of 2.5g/ml and 5.0g/ml respectively.The dosage was 0.2ml/time and once a day.the Piyan ? decoction is same as experiment 1.After animal model establishment of AD and drug administration,the skin lesion,histopathology,protein content in the skin,serum level of IgE and cytokines,as well as intestinal microflora were evaluated by the same method as the experiment 1.RESULTS1.After the external application of DNCB,the dorsal skin of mice could be seen to furfuration and scab;the epidermis thickening,hyperkeratosis,dermal inflammatory cell infiltration and other AD-like pathologically changes could be observed;the serum IgE,TNF ?,IL-1 ?,IL-4,IL-5 were significantly increased,and IFN-y was decreased(P<0.01).The mice model of AD was established.2.The Shannon index of intestinal microflora was significantly reduced(P<0.01)and the diversity of intestinal microflora was significantly decreased in mice model of AD.3.The AD mice model was established by the method of immune injury.Different dosages of the Piyan II decoction can improve the histopathology of the skin lesions and reduce the levels of serum IgE and cytokines.High dosage of the decoction can significantly reduce the levels of serum IgE,TNF-?,IL-1?,IL-4 and IL-5,in the same time increase the level of IFN-y(P<0.01).4.The mice model of AD with intestinal flora disturbance was established by intragastric administration of antibiotics combined with allergic substance.Different dosages of the Piyan ? decoction can improve the histopathology of skin lesions.The serum level of TNF-?,IL-1?,IL-4 and IL-5 were significantly decreased in high dosage(P<0.01).5.In terms of improving the Chao,ACE and Shannon indexes of AD mice model with intestinal flora disturbance,the drug effect of the Piyan ? decoction was significant(P<0.01).The dosage higher,in correspondence with more abundant and various of the intestinal microflora.CONCLUSIONS1.The mice model of AD was established by the method of immune injury,the mice model of AD with intestinal flora disturbance also established by combined with antibiotic intragastrical.2.In AD mice models related to or not to human intervention of intestinal flora,the Piyan ? decoction can effectively improve the histopathology of skin lesions,serum levels of IgE and cytokine,which is transform the immune response from Th2 to Th1.3.In the mice model of AD with intestinal flora disturbance artificially,the high dosage of drug can effectively improve the abundant and various of intestinal flora. |
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