| Objective1.To analyze retrospectively the hospitalized cases of moderate to severe atopic dermatitis admitted in recent years,summarize systematicly their clinical features,and to evaluate the actual clinical intervention effect and advantages of Chinese herbal compound Huailianyin decoction on moderate to severe atopic dermatitis,so as to provide practical experience and reference for the clinical diagnosis and treatment of atopic dermatitis.2.Through the intervention of Chinese herbal compound Huailianyin decoction in atopic dermatitis mouse model,we discussed the possible mechanism of its influence on the immune regulation of AD from the aspects of mouse scratching symptoms,skin lesion score,auricle swelling degree,skin tissue pathology,levels of serum and dermal T helper cells(Ths)related inflammatory cytokines,expression levels of regulatory T lymphocytes(Tregs),T helper 17(Th17)cells and T helper 9(Th9)cells in spleen and so on.Methods1.Retrospective clinical study:We collected a total of 117 adverse reactions and recurrence rate of the two groups were observed and analyzed,the average length of stay of the two groups were compared,simultaneously,the investigator,s global assessment(IGA)score and the scoring atopic dermatitis index(SCORAD)of the researchers were comparisoned before and after admission.2.Animal experiment:(1)We randomly divide female BALB/c mice into four groups(n=8),namely blank control group,model group,low dose Huailianyin group and high dose Huailianyin group.(2)In addition to the blank control group,the model group and the high and low dose Huailianyin groups need to make models,2,4-dinitrochlorobenzene(DNCB)was repeatedly stimulated to establish a mouse model of atopic dermatitis.Before the establishment of the model,the back skin of the mouse was prepared(about 2.0cm×1.5cm in area).On the first day of the establishment of the model,1% DNCB solution was applied to the skin on the back of the mouse and the skin on the back of the right ear to help the initial sensitization.The amount of DNCB in the back of the mouse was 150 ul,and that on the back of the right ear was 10 ul,after that,0.2% DNCB solution(100ul,10ul)was applied on the skin preparation area on the back of the mouse and the skin on the back of the right ear on the two consecutive days,four days apart,afterwards,7th,9th,11 th and 13 th days respectively,so the sensitized skin was repeatedly stimulated(secondary sensitization)to maintain hospitalized case information of atopic dermatitis patients in our hospital according to the inclusion and exclusion criteria from January2014 to January 2018.Devise the collection table of case information and establish database for statistical analysis.Overall clinical data of patients were tabulated and analyzed multidimensionally including age,sex,time of first onset,course of disease,clinical stage,season of onset,inducing factors,clinical characteristics,personal and family history of allergy and laboratory examination.At the same time,case control was set up according to the intervention measures of Chinese herbal compound Huailianyin decoction,among them,73 patients were treated with Huailianyin decoction and conventional western medicine,which was set as the combination group of traditional Chinese and Western medicine,44 patients were only treated with conventional western medicine,which was set as the Western medicine control group.The treatment efficiency,the mice were killed by rapid cervical dislocation.In the middle part of bilateral auricles,the ear pieces were obtained by 6mm mouse puncher,the quality of the ear pieces was detected by analytical balance,and the swelling degree of the auricles was calculated;the skin lesions of the back and right auricles were taken for routine skin pathological examination.Toluidine blue staining and carbon chromotrope 2R staining were used to test the infiltration of mastocyte and eosinophils;(6)The serum levels of helper T cell-related inflammatory factors(IL-4,IFN-γ,IL-10,IL-17,IL-9 and Ig E)were detected by enzyme-linked immunosorbent assay(ELISA),and the total RNA of skin lesions on the back of mice was extracted by Trizol method,and quantitative Real-Time PCR was employed to test the levels of IL-4,IFN-γ,IL-10,IL-17 and IL-9m RNA;(7)The spleen singlecell suspension was prepared.And then,isolating spleen lymphocytes,detecting expression levels of Th9,Th17 and Tregs by flow cytometry(FCM).Results1.Retrospective clinical study:(1)All the 117 hospitalized patients with atopic dermatitis included in the study were adolescents and adults with moderate and severe disease.The average age was(23.08±6.28)years,and the onset age wasearly,77.78% of patiens were within 1 year old.(2)More patients than in summer and winter were admitted to hospital with aggravation,and most of them had no obvious cause.70.94% of patients had AD-like inflammatory response.The left ear was coated with the same amount of1:4 acetone and olive oil matrix solution as the control.The blank control group was treated with depilation,only 1:4 mixture of acetone and olive oil was applied,meanwhile,the application time and dose were the same as the other groups.(3)The drug was administered on the first day of modeling for 14 consecutive days.The way of administration was gavage.The high and low dose Huailianyin groups were given twice the clinical dose(5.2g/kg·d)and the clinical equivalent dose(2.6g/kg·d)respectively.The blank control group and the model group were given the same volume of distilled water.(4)On the first day,the eighth day and the15 th day in the course of the experiment,the scratching times and skin damage scores of mice in each group were observed and recorded;(5)The mice were anesthetized in depth within 24 hours(day 15)after the experiment finished.The blood was taken from the eyeball extraction method,and then significant(P<0.01).Compared with the western medicine control group,the IGA score,SCORAD score and average length of stay in the combined Chinese and Western medicine group were also significantly decreased(P<0.05).2.Animal experiment:(1)At the end of modeling,the scratching times,skin damage score and auricular swelling of the model group were significantly higher than those of the blank control group(P<0.01).Compared with the model group,the scratching times,skin damage score and auricle swelling degree of mice decreased significantly after the intervention of low or high dose Huailianyin decoction(P<0.01).The skin histopathology of the low and high dose Huailianyin group showed that the thickness of spinous layer was less than that of the model group,the infiltration of eosinophils and the count of mast cells were also significantly decreased(P<0.01);(2)Compared with the blank control group,thelevels of serum Ig E,IL-4,IL-17 and IL-9 in the model group were significantly increased(P<0.01),while the serum IFN-γand IL-10 had no significant changes(P>0.05).The contents of Ig E,IL-4,IL-17 and IL-9 in the low and high-dose Huailianyin group were significantly lower than those in the model group(P<0.01),and the levels of IL-4 and IL-9 were close to those in the blank control group,while the levels of IFN-γ and IL-10 were not significantly different from those in the model group(P>0.05);(3)The results of real time quantitative polymerase chain reaction(Real-Time PCR)of skin lesions on the back of mouse showed that the m RNA expression levels of IL-4,IL-9,IL-17 and IFN-γin the model group were significantly higher than those in the blank control group(P<0.01),while a personal or family history of allergy;Most of the patients had increased serum total Ig E level(67.52%)and EOS count(69.23%),and the combination of house dust mite and dust mite with allergen inhalation group had the highest frequency of serum specific Ig Epositive.(3)Some clinical features have clinical diagnostic value,among 117 patients with moderate to severe AD,dry skin of the whole body(109 cases),flexural dermatitis/eczema(100cases),pruritus during sweating(80 cases),erythema of the face(69cases),and white scratch of the skin(65 cases)were the most common ones.(4)The total effective rate,incidence of adverse reactions and recurrence readmitted rate in the integrated Traditional Chinese and Western medicine group were 86.30%,10.96% and 28.77%,respectively,which were better than those in thewestern medicine control group(70.45%,31.82% and54.55%),and the differences between the two groups were statistically(P<0.01),while the proportion of CD4+IL-17+cells(Th17)was increased markedly(P<0.01),and the proportion of CD4+IL-9+cells(Th9)was no significant change(P>0.05).Compared with the model group,the proportion of Tregs in the spleen of low and high dose huailianyin group was significantly increased(P<0.01),while the proportion of Th17 cells was significantly decreased(P<0.01),and the proportion of Th9 cells was slightly decreased compared with the model group,with no statistical difference(P>0.05).Conclusions1.(1)The hospitalized cases of AD are usually adolescents and adults with moderate to severe symptoms.They have a long history with early onset,and most of them have atopic constitution and some clinical characteristics with diagnostic value;(2)Application of Chinese herbal compound Huailianyin decoction in the treatment of moderate and severe atopic dermatitis can improve the condition,shorten the hospitalization time,slow down the recurrence,and has good safety,which is worthy of wide clinical application and promotion.2.(1)The atopic dermatitis model was successfully prepared by exogenous stimulation of DNCB in BALB/c mice.It can be seen from clinical manifestations,immunological serology and skin histopathology that Chinese herbal compound Huailianyin decoction can effectively improve the skin lesion of AD like mouse model,relieve scratching symptoms,and also reduce the infiltration of inflammatory skin cells;(2)One of the therapeutic mechanisms of Huailianyin decoction is to down-regulate the the expression level of IL-10 m RNA was significantly decreased(P<0.05);Compared with the model group,the m RNA expression levels of IL-4,IL-9,IL-17 and IFN-γin the low and high dose Huailianyin group were significantly decreased(P<0.01);Compared with the model group,the expression level of IL-10 m RNA in the high-dose Huailianyin group was significantly increased(P<0.05),while the expression of IL-10 m RNA in the low-dose Huailianyin group was increased with no significant difference between the two groups(P>0.05),the results showed that there was a dose effect relationship between Huailianyin group and IL-10 m RNA expression;(4)Compared with the blank control group,the proportion of CD4+CD25+Foxp3+cells(Tregs)in the spleen of the model group was decreased significantly content of Ig E,IL-4,IL-17 and IL-9 and the expression level of IL-4,IFN-γ,IL-17 and IL-9m RNA in skin lesions,enhance the expression level of IL-10 m RNA in skin lesions,correct the above-mentioned cytokine disorder and restore immune homeostasis in vivo;(3)The proportion of Tregs in spleen of mice in the low and high dose Huailianyin group was significantly increased,while the proportion of Th17 cells was significantly decreased.This indicates that Huailianyin decoction may play a multi-target comprehensive therapeutic effect by enhancing the negative immune regulation mechanism in vivo and regulating the imbalance of Th17/Treg. |
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