Nourishing And Eliminate Wind Drink Of Allergic Contact Dermatitis And Immunologic Mechanisms

Objective:This thesis evaluates the pharmacological effects of experienced prescription of the wind Yangxue drink on acute and chronic ACD from the perspective of pathological changes and histomorphology by adopting mice of acute and chronic allergic contact dermatitis(ACD) model,probes immunological mechanism on the level of cells and molecules by examines the relevant cells and its mRNA,so that to provide objective basis for the application of this experience experienced prescription's better prevention and treatment of acute and chronic ACD clinically.Materials and Methods1.Experimental materials(1) Experimental animals:Healthy male Kunming mice.(2) Experimental medicine:The method of oral drug delivery is used.The experienced prescription of Yangxue Xiaofeng Drink is adopted in Chinese medical treatment and the control group of Western medicine used loratadine tablets,dissolved with distilled water made turbid liquid;blank group,model group gavage with distilled water contour.2.Research Methods(1)Model approach:To use the methods of DNCB skin to prepare acute and chronic ACD animal models of mice.(2)To make preliminary evaluation of effect of ACD- inhibition by observing the Yangxue Xiaofeng Drink on acute and chronic ACD ear swelling disease in mice,the percentage of swelling inhibition,and spleen index and thymus index.(3)To analyze the effects of Yangxue Xiaofeng Drink on acute acute lesions histomorphology through the light microscope and transmission electron microscopy observation of Yangxue Xiaofeng Drink lesions of acute ACD in mice and the pathological changes in tissue ultrastructure.(4)To determine the levels of IFN-γand IL-4 in the serum of ACD mouse model by using ELISA method,using RT-PCR used to detect lesions local IFN-γmRNA,IL-4mRNA expression and serum cytokine protein expression and mRNA expression in lesions of the correlation analysis,from the Th1 / Th2 cytokine balance in the perspective of the network to explore further in-depth Yangxue Xiaofeng Drink ACD drink immunological inhibition mechanism.(5)To detect content of TNF-α,NF-κBp50,Fas / Fas-L of chronic lesions of cytokines in ACD mouse model by using immunohistochemical methods,and explore the possible role of drinking wind mechanisms and links by studying the pathogenesis of chronic skin lesions of ACD positions cytokine expression in the above-mentioned changes in consumer Yangxue.(6)To study the percentage of early apoptosis by Annexin V-FITC apoptosis detection method. and using flow cytometry in chronic ACD mouse spleen lymphocytes,and to explore whether drinking Yangxue Xiaofeng Drink reduced intensity of immune response,thereby inhibiting the occurrence of ACD from cell level by inducing lymphoid ways of apoptosis.Results1.Yangxue Xiaofeng Drink can reduce acute and chronic ear disease model mice ACD weight difference and swelling degree.Acute and chronic ACD model of the ears of mice were significantly higher than the weight difference between the blank group(P＜0.01),Yangxue Wind-drink low-,medium-, high-dose group and the group of loratadine ear weight difference with the model group were significantly reduced compared to(P＜0.01 or P＜0.05).With the range of this experiment dose used,Yangxue Xiaofeng Drink on the role of acute ACD model no significang amount- effect dependence on the role of chronic ACD model was a certain amount - effect dependence.2.Yangxue Xiaofeng Drink can be reduced acute and chronic mouse model of ACD spleen index and thymus index.Acute and chronic ACD model group mice spleen index,thymus index were significantly higher(P＜0.01):Yangxue Xiaofeng Drinking group and loratadine can be significantly reduced acute and chronic mouse model of ACD and the spleen index Thymus index(P＜0.01);Yangxue Xiaofeng Drink on the impact of both showed a certain amount - effect dependence.3.Yangxue Xiaofeng Drinking mice reduce acute ACD partial lesion histopathology and ultrastructure of the extent of injuryThe mice back skin biopsy of ACD model group showed:loss of partial necrosis of epidermal cells,the cells edema;dermal papilla edema,dermal infiltration of inflammatory cells in large numbers;gap widened collagen fibers;vasodilator,endothelial cell swelling. After the intervention of Yangxue Xiaofeng Drink,the basic structure of full-thickness skin integrity,cells and intercellular edema significantly reduced and inflammatory cell infiltration in the dermis decreased significantly.All levels of damage are less than those of the ACD model group.The number of dermal inflammatory cell infiltration in Loratadine group is more than that of Yangxue Xiaofeng Drink group,and other changes is about the same with Yangxue Xiaofeng Drink group.In the study of ultrastructure of the skin,basal cell skin lesions and spine cells of ACD mouse model shrink and the electron density of epidermal keratinocyte slightly enhanced, inter-connection of epidermal basal cells disappear and within the cell,cytoplasm was vacuolated changes nuclear heterochromatin condensed into a mass distribution to the nuclear periphery.The lesions basal cell skin and spine cells of Yangxue Xiaofeng Drinking mice kept structural integrity,cells with mild atrophy,intercellular is liked with desmosomes and the nuclear chromatin basically uniform distribution.Mitochondria and rough endoplasmic reticulum are of structure integrity,little vacuolization,and cell injury is less than the model group.Loratadine group part of the nuclear chromatin was mass distribution,and other changes is about the same with Yangxue Xiaofeng Drink group.4.Yangxue Xiaofeng Drink can raised level of IL-4 and reduced levels of IFN-γin serum of ACD model,while increase expression level of of IL-4mRNA reduced the expression level of IFN-γmRNA in ear tissue.Levels of IFN-γsignificantly increased in serum of chronic ACD mouse model and the level of IL-4 decreased significantly(P＜0.01),there is high expression level of IFN-γmRNA in ear lesions organizations and expression level of IL-4mRNA reduced expression(P＜0.01).The ratio of serum IFN-γ/IL-4 ear tissues were significantly higher IFN-γmRNA/IL-4mRNA ratio(P＜0.01) in model mice group;Both Yangxue Xiaofeng Drink and loratadine ACD in mice significantly reduced serum levels of IFN-γas well as the ear tissue of IFN-γmRNA expression level(P＜0.01);Yangxue Xiaofeng Drink can significantly increase serum IL-4 levels(P＜0.05) as well as the ears tissue expression of IL-4mRNA level (P＜0.01),while loratadine increase serum levels of IL-4 on the ear lesion tissue expression of IL-4mRNA the level of no effect;Yangxue Xiaofeng Drink or loratadine group,while the impact of the ratio of IFN-γ/IL-4 is of no significant difference between the two groups.5.Yangxue Xiaofeng Drink ACD significantly reduced expression levels of TNF-α.and NF-κBp50 in chronic skin lesions of mice,and markedly increase that of Fas and FasL. TNF-αand NF-κBp50 expression were significantly increased(P＜0.01) in chronic ear organizations of ACD mouse model;Yangxue Xiaofeng Drink organizations can significantly lower TNF-α,NF-κBp50 expression(P＜0.01);TNF-αexpression was not obvious(P＞0.05) in tissue of Loratadine group,but can significantly reduced NF-κBp50 expression(P＜0.05); and the interference role of Yangxue Xiaofeng Drink is superior to that of loratadine group(P＜0.05 or P＜0.01).Fas and FasL expression were significantly increased(P＜0.01) in chronic ear organizations of ACD mouse model;Yangxue Xiaofeng Drink organizations can significantly increase Fas and FasL expression(P＜0.01);and loratadine group has no significant effects on the two(P＞0.05).TNF-αand NF-κBp50,Fas and Fas-L expression in ear skin of mice were positively correlated(correlation coefficients were:0.783,P＜0.01;0.747,P＜0.01).6.Yangxue Xiaofeng Drink chronic drinking can significantly promote the ACD mouse spleen T lymphocytes in early apoptosis.Percentage of early apoptosis of lymphocytes is significantly increased in the spleen of model of mouse(P＜0.01);Yangxue Xiaofeng Drink or loratadine may significantly increase percentage of early apoptosis of lymphocytes in the mouse spleen,Yangxue Xiaofeng Drinking group was stronger than the loratadine group(P＜0.01).Percentage of early apoptosis of lymphocytes in Mouse spleen and Fas.Fas-L expression in mouse ears organizations were positively correlated(correlation coefficients were:0.783.P＜0.01;0.785,P＜0.01).Conclusion:1.Yangxue Xiaofeng Drink has inhibition effect on acute and chronic ACD of mice and can reduce the swelling of the ear,inhibit the swelling response of immune organs in the course of changes and reduce thymus index and spleen index.2.Yangxue Xiaofeng Drink can effectively reduce the injury degree of ultrastructure and local tissue pathological lesions of acute ACD group in mice.3.Yangxue Xiaofeng can lower the cytokine of Th1 and promote the secreting of the cytokine of Th2,so that adjust network Th1/Th2 balance of mouse model of chronic ACD.4.Yangxue Xiaofeng Drink can significantly lower the TNF-α,NF-κBp50 expression levels in skin lesions of chronic ACD mice,and may inhibit inflammation at the transcriptional level and suppress immune response.5.Yangxue Xiaofeng Drink can significantly promote Fas / FasL expression level in the Fas / FasL expression level and promote apoptosis of lymphocytes in mouse spleen.