| BACKGROUNDAtopic dermatitis(AD)is a common chronic inflammatory skin disease.The clinical manifestations are severe itching,exudative tendency,and eczema-like skin lesions.Atopic dermatitis,asthma,food allergies,etc.often occur with a high probability which performed.At present,the prevalence rate of AD in children has exceeded 20%,and the prevalence rate in adults is 3%～5%.With the development of urbanization,the incidence rate is increasing year by year,bringing a huge burden to patients and families.AD treatment drugs concentrate on glucocorticoids,antihistamines,calcineurin inhibitors and biological agents,or are expensive,or long-term use can cause side effects such as skin atrophy,pigmentation,relapse of drug withdrawal,and drug dependence.AD belongs to the category of "milk ringworm" and "blood wind sores" in Chinese medicine.The physicians of the past believe that the disease is due to insufficient congenital endowment,fetal poison and heat,and the body is too strong;or the acquired spleen and soil are insufficient,the health and luck is abnormal,the internal heat of dampness,external wind,Humidity,heat evil,internal and external combination of evil,fight on the skin and get sick.Mr.Zhao Bingnan,the dean of TCM Dermatology Surgery,believes that internal damp-heat and damp-heat and external evils converge,which is the essence of the disease.The "damp" evil runs throughout,and the dampness is heavy and turbid,which is difficult to remove.Cause the delay to be difficult to heal.Professor Zhao Bingnan,based on more than 30 years of clinical experience,transformed the "Longdan Xiegan Decoction" into Qingre Chushi decoction(QRCSD),which treats the acute stage of AD,the damp heat is prosperous,the skin accumulates,and the heat is heavier than the damp heat.It can effectively improve the appearance of skin lesions in patients with AD attack,reduce exudation,erosion and itching,and is safe and effective.Traditional Chinese medicine compound prescriptions have the characteristics of multiple components,multiple targets and multiple pathways in the treatment of diseases,and the exploration of the mechanism is often difficult.In order to realize the optimization of the compound prescription,the pharmacological analysis of drug molecules,genes and protein networks is used to reveal the effective components of the compound prescription of traditional Chinese medicine and its regulatory effect on the body network through modern bioinformatics technology The onset of AD is closely related to genetic background,environmental stimulation,skin barrier function,body immunity and bacterial infection.As an allergic inflammatory skin disease,the effect of mast cells is irreplaceable.After being stimulated,they are activated and degranulate,releasing a large amount of inflammation.Medium,chemotaxis inflammatory cells,cause capillary dilation,increase vascular permeability,cause tissue damage,appearance of erythema,edema,erosion,exudation and itching,etc.,which is not the same as the skin lesions caused by "damp heat hitting the skin" Conspiracy.The detection of mast cells in the skin lesions of AD patients is significantly increased,and the proportion of activated degranulation is increased.There is no literature report on the degranulation effect of QRCSD and its active ingredients on mast cell activation.This study first establishes and evaluates AD mouse models of different strains to provide a basis for subsequent model selection;then uses in vivo and in vitro experiments to explain the role of QRCSD in treating AD mouse models and regulating mast cell degranulation from multiple perspectives;then through the Internet Pharmacology explores the active ingredients of QRCSD and the "component-disease-target" network for the treatment of AD,pointing out the direction for the exploration of the mechanism of the Chinese medicine compound;finally observe the effect of the active ingredients on the AD mouse model and the activation and degranulation of mast cells,with the hope that The follow-up prescription optimization research and clinical application provide theoretical basis and directionOBJECTIVES1.Use different strains of mice to establish AD models and compare them to provide a basis for subsequent model selection and application2.To evaluate the intervention effect of QRCSD on AD mouse model and mast cell degranulation model.3.Explore the active ingredients of QRCSD and potential targets for the treatment of AD,and provide directions for mechanism research4.Evaluate the intervention effect of QRCSD effective active ingredients on AD mouse models,combine with target prediction,verify the possible mechanism of action,clarify the effective active ingredients and action links of QRCSD.and provide theoretical support for QRCSD prescription optimization and clinical efficacy.5.Evaluate the intervention of QRCSD effective active ingredients on mast cell activation and degranulation,target signal pathways,clarify the possible molecular mechanisms of effective active ingredients,and enrich the pharmacodynamic research of active ingredients,which is a new strategy for medical transformation and clinical treatment The proposal provides new ideas.METHODSExperiment 1:Adopting Nc/Nga mice and BALB/c mice,applying DNCB on the back/ears to establish an AD model and compare and evaluate it.Twelve Nc/Nga mice were randomly divided into 2 groups,each with 6 mice,namely the normal control group and the model group The model group mice were used to apply DNCB on the back and ears to establish an AD model Week 1 1-3 days,apply 1%(0.5%)DNCB on the back(ears)every day to stimulate inflammation,and rest for 4 days;in the second and third weeks,apply 0.5%(0.25%)DNCB on the back(ears)3 times a week to maintain the skin inflammatory response,and draw materials on the 22nd day of the model.Twelve BALB/c mice were randomly divided into 2 groups,each with 6 mice,namely the normal control group and the model group.The mice in the model group were smeared with 1%DNCB on the back of each group on the first,fourth,and seventh day of the first week.100μl of solution,or apply 20μl of 0.5%DNCB solution to the right ear for sensitization,rest in the second week,and start from the third week,apply 100μl of 0.25%DNCB solution on the back the next day,or apply 20μl of 0.25%DNCB solution to the right ear to stimulate and maintain the inflammatory reaction of the back skin for 2 weeks,and samples were taken on the 29th day of modeling.In the normal control group,the same amount of substrate was smeared at the same place at the same time.Observation indicators:(1)Observe the general condition,scratching behavior and inflammation degree score of mice;(2)HE staining and toluidine blue staining to observe the pathological changes of mouse skin lesions;(3)ELISA method to detect serum IgE,IL-4 and IFN-γ levels;(4)Calculate the spleen index and use flow cytometry to detect the ratio of Thl/Th2 in the spleen Experiment 2:Use Nc/Nga mice to establish an AD model to observe the effects of QRCSD on AD;use IgE and C48/80 to stimulate RBL-2H3 and P815 to establish a degranulation model of mast cell activation to study the effect of QRCSD on mast cell activation.Forty-eight Nc/Nga mice were randomly divided into 6 groups,each with 8 mice,namely the normal control group.the model group,the prednisolone(positive drug)group,and the QRCSD high,medium,and low dose groups.Except for the normal control group,mice in other groups used DNCB on the back and ears to establish AD models.During the first week,1%(0.5%)DNCB was applied to the back(ears)continuously every day to stimulate inflammation and rest,4d:In the 2nd and 3rd weeks,smear 0.5%(0.25%)DNCB on the back(ears)3 times a week to maintain skin inflammation.From the 2nd week,the mice in the treatment group were given the corresponding drug gavage treatment,and the normal control group and the model group were given an equal volume of distilled water once a day,and the samples were taken on the 22nd day of the model building.Observation indicators:(1)Observe the general condition and skin lesions of mice and score;(2)HE staining and toluidine blue staining to observe the pathological changes of mouse skin lesions;(3)ELISA method to detect serum IgE levels;(4)Immunohistochemical method to detect the expression levels of tight junction transmembrane proteins Claudin-1(CLDN1)and Occludin(OCLN)in the skin lesions;(5)Multi-factor protein chip technology to detect the differential expression of 40 related inflammatory factors in the skin lesions;(6)qPCR method to verify the mRNA expression of differential expression factor transcription level;(7)using IgE and C48/80 methods to induce RBL-2H3 and P815 mast cell degranulation models;(8)using CCK-8 method to detect QRCSD Cytotoxicity,the safe concentration of QRCSD is screened according to cell viability.(9)ELISA method was used to detect the effect of QRCSD on the release levels of β-hexosaminidase(β-HEX),histamine(HIS),IL-4 and TNF-α in the supernatant of activated mast cells.Experiment 3:Obtain the target intersection of inflammatory skin diseases(keywords are psoriasis,atopic dermatitis,eczema,inflammation)through database mining(Drug Bank,OMIM,Gene Cards,etc.)and literature search,and construct Related target database.Use the TCMSP database to obtain the effective compounds and target genes of QRCSD,and screen the effective ingredients of QRCSD decoction according to the properties of the drug molecule ADME and the compatibility of the QRCSD formula.Through the intersection of QRCSD target gene and inflammatory skin disease target.QRCSD-disease target is obtained,PPI network is constructed,and protein interaction network is drawn.The DAVID database was used to visually analyze the core targets,and through GOBP analysis and KEGG enrichment,the QRCSD treatment inflammatory skin disease signal pathway was obtained,the possible mechanism of QRCSD treatment of AD was revealed,and the analysis results were analyzed.Experiment 4:Using BALB/c mice to establish an AD model to verify the effect and mechanism of the active ingredients of Qingre Chushi Decoction,Gentiopicroside,Baicalin and Luteolin in the treatment of AD.66 male BALB/c mice were randomly divided into 11 groups,6 in each group,which were the normal control group,model group,prednisolone(positive drug)group,and gentiopicroside high,medium,and low dose groups.Baicalin high,medium and low dose groups,luteolin high,medium and low dose groups.In addition to the normal control group,60 BALB/c mice were sensitized by applying 100 μl of 1%DNCB solution on the back of each group on the first,fourth,and seventh days of the first week,or applying 0.5%DNCB solution on the right ear of 20 μl.Rest for 2 weeks.Starting from the third week,apply 100μl of 0.25%DNCB solution on the back the next day,or apply 20μl of 0.25%DNCB solution on the right ear to stimulate and maintain the inflammatory response of the back skin for 2 weeks.From the 3rd week,the mice in the treatment group were given the corresponding drugs by gavage,and the normal control group and the model group were given an equal volume of distilled water once a day,and the samples were taken on the 22nd day of the model building.Observation indicators:(1)Observe the general condition and skin lesions of mice and score them;(2)Observe the pathological changes of mouse skin lesions by HE staining and toluidine blue staining;(3)Detect serum IgE levels by ELISA;(4)Flow cytometry to detect the Th1/Th2 ratio in the spleen;(5)qPCR method to detect the expression levels of inflammatory factors IL-4,IL-5,IL-6 and IL-17 in the skin lesions;(6)Western blot method to detect the skin lesions The expression level of MAPK signal pathway proteins(p-p38/p38,p-ERK/ERK,p-JNK/JNK)in tissues.Experiment 5:IgE and C48/80 were used to stimulate RBL-2H3 and P815 to establish a model of mast cell activation and degranulation,to study the effect of Gent on mast cell activation,and to explore possible mechanisms.(1)Two methods,IgE and C48/80,were used to induce the degranulation model of RBL-2H3 and P815 mast cells.(2)The CCK-8 method was used to detect the cytotoxicity of Gent,and the safe concentration of Gent was screened according to cell viability.(3)ELISA method was used to detect the effect of Gent on the release levels ofβ-hexosaminidase(β-HEX),histamine(HIS),IL-4 and TNF-α in the supernatant of activated mast cells.(4)qPCR method was used to detect the effect of Gent on the expression levels of IL-4 and TNF-α mRNA in activated mast cells.(5)Western blot method was used to detect the effect of Gent on the expression of MAPK signal pathway proteins(p-p38/p38,p-ERK/ERK)in activated mast cells.RESULTSExperiment 1:After applying DNCB to Nc/Nga mice and BALB/c mice,eczema-like skin lesions,edema and exudation gradually appeared,and they worsened over time,with erythema and scab gradually appearing,with itching and scratching.The performances are consistent with the clinical manifestations of AD.Histopathology showed that the inflammatory cells in the model group were significantly infiltrated,the thickness of the epidermis and the number of mast cells increased,which were consistent with the pathological characteristics of AD skin lesions.The spleen index of mice in the model group increased significantly,and the Th1/Th2 ratio of the spleen was unbalanced,and the Th2 type was dominant.The ELISA results showed that the serum IgE,IL-4 and IFN-γ levels of the model group increased.Compared with the skin lesions of Nc/Nga mice and BALB/c mice,the skin lesions of Nc/Nga mice appeared earlier and the erythema was more obvious,but the difference was not statistically significant Experiment 2:Compared with the normal control group,the mice in the model group showed AD-like skin lesions such as erythema,erosion,and skin thickening,accompanied by an increase in the number of scratches(P<0.005).Compared with the model group,the QRCSD treatment group had alleviated skin lesion symptoms,decreased SCORAD score,decreased epidermal thickness,and decreased mast cell infiltration(P<0.05),and the QRCSD high-dose group had the most obvious effect.The results of immunohistochemistry showed that the expression level of tight junction protein(OCLN,CLDN1)in the model group decreased,and the level of tight junction protein increased in the QRCSD high-dose group(P<0.05).In the model group,the serum IgE level and the spleen index increased,and the Th1/Th2 ratio of the spleen was unbalanced(decreased).QRCSD high-dose treatment could reduce the IgE expression,lower the spleen index,and reverse the Th1/Th2 ratio imbalance(P<0.05)Compared with the normal control,multi-factor protein chip detection showed that the differentially expressed factors in the skin lesions of the model group included IL-4,IL-5,IL-6.IL-17 and Eotaxin.etc.,and the relative expression of IL-4 and IL-6 mRNA Increased(P<0.05),QRCSD high-dose group can inhibit the expression of inflammatory factors IL-4 and IL-6 at the secretion level and gene transcription level(P<0.05).The safe concentration range of QRCSD screened by CCK-8 method is 0-2000mg/ml.QRCSD can inhibit the release level of mast cell inflammatory mediators β-HEX,HIS)in a dose-dependent manner.Experiment 3:Obtain 75 QRCSD compounds with OB≥ 30%and DL≥ 0.18 through data mining,and 100 potential target proteins of traditional Chinese medicine,which are combined with inflammatory skin diseases(psoriasis,atopic dermatitis,eczema,inflammation).Obtained 19 AD-related targets,constructed a QRCSD-disease-target visualization network,and obtained the top 10 targets as follows:PTGS2,BCL2,MAPK8,TNF,AKT1,TP53,IL6,CASP3,ICAM1 and NFKBIA,indicating these targets The point plays a role in the pathological process of AD immune imbalance and inflammation.Based on the target bioinformatics analysis of the DAVID database,it is found that QRCSD is mainly involved in regulating the inflammatory response,regulating the immune response process and the inflammatory response to antigen stimulation.The enrichment analysis of GOBP and KEGG shows that the MAPK signaling pathway is one of the important signaling pathways involved in the treatment of AD by QRCSD.At the same time,combined with the QRCSD prescriptions,effective active ingredients including gentiopicrin,baicalin and luteolin are obtainedExperiment 4:Compared with the normal control group,the mice in the model group showed AD-like skin lesions such as erythema,erosion,and skin thickening,accompanied by an increase in the number of scratches(P<0.005).Compared with the model group,the skin lesion symptoms of mice in each group of gentiopicrin,baicalin and luteolin were all relieved,the SCORAD score was reduced,the thickness of the epidermis was reduced,and the infiltration of mast cells was reduced(P<0.05),all of which were higher.The dose group has the most obvious curative effect,and gentiopicroside has the best effect.Flow cytometry showed that the ratio of Th1/Th2 in the gentiopicroside group was increased(P<0.05).The qPCR result of the gentiopicroside group was able to inhibit the expression of inflammatory factors IL-4 and IL-6 mRNA(P<0.05).Western blot showed that the expression levels of p-ERK and p-p38 in the skin lesions of AD mice increased(P<0.05),and gentiopicrin could inhibit the expression levels of p-ERK and p-p38(P<0.05),but p-There was no difference in the expression of JNK(P>0.05)Experiment 5:IgE-induced RBL-2H3 cell degranulation model conditions:DNP-IgE monoclonal antibody(100μg/ml)overnight,DNP-BSA(500μg/ml),stimulation time 1h:C48/80 induced P815 cell degranulation model The conditions are:10μg/ml,and the stimulation time is 30min.After activation and degranulation of mast cells,compared with untreated blank control cells,the release levels of β-HEX,HIS,IL-4 and TNF-α in the supernatant increased,and the expression of IL-4 and TNF-α mRNA in the cells increased,the phosphorylation level of p38/ERK MAPK signaling pathway protein increased(P<0.05).The biosafety concentration of Gent for RBL-2H3 is 0-100μM,and the biosafety concentration for P815 is 0～200μM,and there is no cytotoxicity in this range.100μMGent can inhibit the degranulation of RBL-2H3 mast cells,reduce the release levels of β-HEX,HIS,IL-4 and TNF-α in the supernatant of degranulation cells(P<0.05),and reduce the activation of mast cell inflammatory factors IL-4 and IL-4 TNF-α mRNA expression(P<0.05),and p38/ERK MAPK pathway protein phosphorylation level(P<0.05).200μMGent can inhibit the degranulation of P815 mast cells,reduce the release levels of β-HEX,HIS,IL-4 and TNF-α in the supernatant of degranulation cells(P<0.05),and can reduce mast cell inflammatory factors IL-4 and TNF-α mRNA expression(P<0.05),inhibit the phosphorylation of ERK pathway proteins.CONCLUSION1.Both Nc/Nga mice and BALB/c mice can be used to construct AD models,and the skin lesions are in line with clinical characteristics;however,the skin lesions of Nc/Nga mice appear early,the erythema is more obvious,and there is a genetic background,which can be used for Research on the genetic factors of AD.However,BALB/c mice have low price and wide sources,and can be widely used.In the follow-up study,the model can be selected according to the needs2.QRCSD can significantly improve DNCB-induced AD eczema-like skin lesions in Nc/Nga mice,reduce edema,exudation and itching,reduce mast cell infiltration,reduce epidermal thickness,reduce serum IgE levels,repair skin barriers,reduce spleen index,and improve Th1/Th2 immune imbalance can inhibit the expression of inflammatory factors in skin lesions;QRCSD can improve mast cell degranulation,inhibit the release of inflammatory mediators,and clarify QRCSD’s inhibition of inflammation and immune regulation functions3.Gentiopicrin,baicalin and luteolin can significantly improve DNCB-induced AD eczema-like skin lesions in BALB/c mice,reduce edema,exudation and itching,reduce mast cell infiltration,reduce epidermal thickening,and use gentian Picroside works best.Gentiopicrin can reduce serum IgE levels,inhibit the expression of inflammatory factors in skin lesions,and clarify the therapeutic effect of gentiopicroside on AD.It may inhibit inflammation and regulate immunity by inhibiting the activation of p38 MAPK and ERK signaling pathways.4.Gentiopicrin has an inhibitory effect on the activation and degranulation of RBL-2H3 and P815 mast cells induced by IgE and C48/80 pathways,can reduce the release of inflammatory mediators,and can inhibit the MAPK family(p38 and ERK)Protein phosphorylation level,which may be one of its mechanisms for the treatment of AD,enriches the anti-inflammatory,anti-allergic and immunomodulatory effects of gentiopicroside,and is used to treat immune inflammatory and allergic skin diseases similar to the pathogenesis of AD Provide ideas. |
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