| Atopic dermatitis(AD)is a chronic,recurrent,inflammatory skin disease,which recurrent,lingering refractory,and the incidence increased year by year,has been a serious harm to human health.At present,the pathogenesis of AD is not clear,there are some limitations in modern medical treatment.Traditional Chinese medicine(TCM)treatment of atopic dermatitis has its own advantages for a long history and rich clinical experience.The Longmu decoction which is created by professor Huang of Xiyuan Hospital have a better clinical efficacy.In this study,we established AD model mice to give oral administration of Longmu decoction to observe its curative effect,and to explore its possible therapeutic mechanism through the change of immunological index.Objective1.To explore the preparation of AD model mice and model evaluation.2.Efficacy of Longmu Decoction in Treating AD Model Mice3.To explore the immunoregulatory mechanism of Longmu Decoction in AD model mice.MethodsStduy Ⅰ Preparation and evaluation of AD model mice1.BALB/c mice were stimulated by DNCB exogenous stimuli in the study of AD-like model mice with reference to domestic and foreign literatures.Twelve 6-week-old male BLAB/c mice were randomly divided into model group and control group.2.Modeling method:At the frist week,the model group was treated with 1%DNCB solution,using a micro-pipette and brush with the uniform smear in the back of the mouse and the right ear,each 50ul;At the same time,the control group was treated with acetone olive oil mixture(1:4),coated with the model group.At the frist week,mice were brushed by once a day,continuous smear 2 days,interval 4 days,starting from the 7th day,the model group to 0.2%concentration of DNCB solution,while the control group with the former.After every other day,smear once.Modeling time for a total of 3 weeks,repeated smear a total of 10 times.3.The clinical symptoms(the number of scratching,the condition of skin lesion and the degree of ear swelling)were observed at different time points(1,7,13,21 days,after 1h of applying reagent).After the external application of the solution to stimulate on the 21th day,we took blood from the mice eyes for examination within 24 hours,and to take local lesions pathological examination.Comprehensive clinical symptoms,changes in skin pathology and changes in serum inflammatory factors,it’s a time to evaluate whether the model was successfully prepared.Study Ⅱ Efficacy of Longmu decoction in treating AD model mice1.We bought another 18 BALB/c mouses,and took out 6 mouses randomly that is regared as blank group,the left 12 mouses were molded as the previous method.When the mode was modeled successfully,we divided the 12 mouses into the TCM group and the model group.The TCM group was given a gavage using Longmu decoction,while the model group and the blank group were given a gavage using the 0.9%NS.After 3 weeks contrasted the 3 groups to verify the efficacy and detected the curative effect of Longmu decoction.2.The expression of serum IL-4,IgE and IFN-γ in three groups of mice(TCM group,model group and blank group)was detected by mouse serum ELISA kit.The effect of Longmu decoction on serum inflammatory factors was analyzed by LSD statistical method.3.Composite the clinical characteristics and the immunology,we use multi-indexes such as lesion score,the thickness of auricle,and the blood index ect.to discuss the characteristics.Study Ⅲ Immunological mechanism of Longmu decoction in AD model mice1.After the intervention of the drug,we take back skin fron the model group and the TCM group(12 mice),and SP immunohistochemical staining was used to analyze the expression of TLR4 and NF-κB p65 in the skin lesions.The correlation between TLR4 and NF-κB p65 was analyzed by Spearman correlation analysis.2.The protein expression of TLR4 and NF-κB p65 in the back lesions of the three groups of mice was counted.The data obtained were immunohistochemical staining positive signal cumulative optical density(IOD),IOD value as a quantitative standard for tissue expression.Combinating the expression of inflammatory factors(IL-4,IgE,IFN-γ)in sera of three groups of mice,the immunoregulatory mechanism of Longmu decoction in AD model mice was investigated by partial correlation analysis.ResultsStudy Ⅰ Preparation and evaluation of AD model mice1.compared with the control group,the pathological features of the back lesions in AD model group were hyperkeratosis,hyperkeratosis,acanthosis,inflammatory cells infiltration in the epidermis and dermis,telangiectasia.2.compared with the control group,the serum levels of IgE and IL-4 in the AD model group were significantly higher(P<0.01),while the expression of IgE and IL-4 did not change significantly(P>0.05).3.After the model was finished,compared with the control group,the clinical symptoms(scratching times,ear swelling degree and back skin lesions score)of model group mice increased significantly,the difference was statistically significant(P<0.05).The severity of clinical symptoms in model group mice increased significantly with the increase of modeling time(P<0.01).Study Ⅱ Efficacy of Longmu decoction in treating AD model mice1.After 3 weeks of intragastric administration intervention,the frequency of scratching,ear swelling and skin lesion severity scores of TCM group were lower in the model group,the difference was statistically significant(P<0.05),but still higher than the control group,the difference was statistically significant(P<0.05).2.After 3 weeks of intragastric administration intervention,compared with the control group,the expressing levels of IgE and IL-4 in the serum of the model group and the treated group were all higher,the difference was statistically significant(P<0.01).Compared with the model group,the expressing levels of IgE and IL-4 in the serum of the treated group decreased,and the difference was statistically significant(P<0.05).There was no significant difference in the expressing levels of IFN-γ in the serum between the three groups(P>0.05).Study Ⅲ To explore the immunological mechanism of Longmu Decoction in the treatment of AD mice1.With LSD statistical analysis:the expressing levels of TLR4,NF-κB and p65 in the three groups were different(P<0.05),the highest model group,Chinese medicine group followed,the control group minimum.2.The expressing levels of p65,TLR4 and NF-κB protein in lesions of AD mouse were analyzed by Spearman correlation.It was found that the expression of TLR4,NF-κB and p65 were positively correlated(r=0.618,P<0.05).3.We analyzed the correlation between serum inflammatory factors(IL-4,IgE,IFN-γ)and TLR4/NF-κBp65 pathway,results of statistical analysis:IL-4 and TLR4,NF-κB p65 was positively correlated,correlation coefficient is respectively 0.485,0.569(P<0.05);IgE and TLR4,NF-κBp65 was positively correlated,correlation coefficient is:0.651,0.721(P<0.05);IFN-γ and TLR4、NF-κBp65 was positively correlated,correlation coefficient is respectively 0.55,0.634(P<0.05).Conclusions1.AD mouse could be successfully prepared by DNCB induced BALB/c mouse.2.Longmu decoction is effective in the treatment of AD mice,can improve the clinical symptoms,serum index and dermatological pathology etc.3.Longmu decoction has probably affected the expressing level of TLR4 and blocked NF-κB p65 pathway released of inflammatory factors,so as to balance the Thl/Th2 cell subsets and play a role in immune regulation. |
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