| Background and Objective:Atopic dermatitis(AD)is a prevalent chronic skin disorder affecting up to 20%of children and 5%of atopic dermatitisults globally.The clinical signs of atopic dermatitis are mainly eczema-like skin lesions such as skin redness,burning,exudation,and even erosion.The occurrence of atopic dermatitis is caused by the combined action of numerous complicated elements,genetic susceptibility,epidermal dysfunction,aberrant skin microbiota,and local neurologic and immune system diseases are all relevant elements in the onset and progression of atopic dermatitis.Histamine-independent itch is itch that does not respond to antihistamines,studies have shown its dominance in chronic itch caused by atopic dermatitis.Currently,the most common treatments for atopic dermatitis include topical moisturizers,corticosteroids,light therapy,and immunotherapy,among others,however,these treatments have little effect on moderate to severe atopic dermatitis and the accompanying chronic itching symptoms,and long-term use is commonly affected by serious toxic side effects.Therefore,it is of great value to find anti-inflammatory and anti-pruritic drugs with definite efficacy,few adverse reactions,and low cost for the treatment of atopic dermatitis and histamineindependent pruritus.Cortex Dictamni is the dried root bark of a rutaceae perennial Dictamnus dasycarpus.Cortex Dictamni has been used for thousands of years in traditional Chinese medicine to treat internal diseases such as asthma,rheumatism,jaundice,and skin diseases such as eczema,urticaria,and scabies.The main components of Cortex Dictamni are alkaloids,limonin,coumarin,flavonoids,steroids,and polysaccharides.Dictamnine(Dic)is one of the main active components separated from the Cortex Dictamni,as well as one of the index components used to assess the quality of the Cortex Dictamni.Dictamnine has been shown in recent studies to have anti-bacterial,anti-allergic,anti-cancer,and anti-inflammatory properties.However,the effects of Cortex Dictamni and Dictamnine on atopic dermatitis and histamine-independent itch have not been studied,and the effects of Dictamnine on the MrgprA3-mediated histamine-independent itch have not been reported.As a result,the purpose of this thesis is to certify the therapeutic effects of Cortex Dictamni and its main active ingredient-Dictamnine on atopic dermatitis and its accompanying chronic itch,as well as to investigate the relevant molecular mechanisms from animal,molecular,and cellular perspectives.This dissertation also aims to provide a scientific and reliable laboratory foundation for the use of Cortex Dictamni and Dictamnine for the treatment of clinical skin diseases.Research contents and methods:This thesis consists of four chapters:In the first chapter,data mining technology was used to analyze the medication rules of tratopic dermatitisitional Chinese medicine in the treatment of eczema in the past 20 years,and the frequently used herbs in the internal and external prescriptions were screened out,and then the properties,taste,and meridian of these frequently used herbs were analyzed.Then,we screened the high-frequency-used herbs to obtain the core herbs.Finally,we analyzed the medication rules of herbal treatment for severe itching,the common accompanying symptom of eczema.In the second chapter,acute pruritus behavioral experiments were conducted to investigate the effect of ethanol extract of Cortex Dictamni(ECD)on acute pruritus.1-fluoro2,4-dinitrobenzene(DNFB)was used to establish a mouse nape model of atopic dermatitis and chronic pruritus behavioral experiments were conducted to investigate the effect of ECD on chronic pruritus.The effect of ECD on the symptoms of atopic dermatitis was investigated by establish a mouse ear model of atopic dermatitis and observing the clinical manifestations of mouse ears and measuring their thickness.Secondly,we analyzed the thickness of the epidermis and dermis and the infiltration of eosinophils and mast cells in the ear of atopic dermatitis mice by analyzing the results of Hematoxylin-Eosin(H&E)staining and Toluidine blue(TB)staining of ear tissue sections,and the effect of the ECD on the number of CD4+T cells and CD8+T cells in the ear tissue of atopic dermatitis mice was detected by flow cytometry.Then,we examined the effects of ECD on inflammatory cytokine levels in the skin lesion area and serum of atopic dermatitis mice by ELISA technique.Finally,the effect of ECD on the Janus kinase(JAK)-Signal transducer and activator of transcription(STAT)signaling pathway in the ear tissue of atopic dermatitis mice was investigated by western blotting.In the third part,the effects of Dictamnine,the major active ingredient of Cortex Dictamni,on chloroquine-activated MrgprA3-mediated histamine-independent pruritus in mice were investigated using acute itch experiments.Primary neurons from pirtGCaMP3+/-transgenic mice were used for calcium ion imaging experiments to investigate the effect of Dictamnine on the excitability of MrgprA3+and Transient receptor potential ankyrin 1(TRPA1)+neurons,and to clarify the direct inhibitory effect of Dictamnine on the excitability of MrgprA3+and TRPA1+neurons.Molecular docking technology was used to examine the binding affinities of dictamnine with MrgprA3 and TRPA1.In the fourth part,we established a mouse model of atopic dermatitis on the nape of the mice to investigate the effect of Dictamnine on chronic pruritus and skin lesions caused by atopic dermatitis,and we analyze the histopathological changes and immune cell infiltration(CD4+T cells and CD8+T cells)in the skin lesions by histopathological staining and immunohistochemistry.Next,the effect of Dictamnine on the distribution density of MrgprA3+fiber and MrgprA3+neuron in the skin and dorsal root ganglion(DRG)of MrgprA3EGFP-Cre;ROSA26tdTomato+/-transgenic mice was detected.qRT-PCR and Western blot were used to detect the expression of MrgprA3,TRPA1,P2X purinergic ion channel type 3 receptor(P2X3R)at mRNA levels and TRPA1,P2X3R at protein levels in the skin and DRG of atopic dermatitis mice.Results:In the first chapter,through the data mining and analysis of medical records related to the treatment of eczema in the Chinese journal literature database in the past 20 years,we found that Glycyrrhiza uralensis,Cortex Dictamni,Raw rehmannia glutinosa and Sophora flavescens were the most frequently used oral prescriptions,and Sophora flavescens,Phellodendron amurense,Belvedere fruit,Osthole seed and Cortex Dictamni were most frequently used in external prescriptions.Through further analysis,it was found that the property and taste of these high-frequency drugs were mainly ’bitter and cold’ and these drugs mainly belong to’liver’,’lung’,’spleen’ meridian.We also found that the traditional Chinese medicine Cortex Dictamni was used frequently in the treatment of eczema and pruritus and that its clinical efficacy was significant.These high-frequency drugs have certain reference value for antiinflammatory and anti-pruritic drugs in clinical skin related diseases.In the second chapter,we demonstrated that ECD pretherapy significantly inhibited histamine or chloroquine(CQ,an agonist of MrgprA3)induced acute pruritus.Then,the treatment of ECD also significantly inhibit the symptoms and chronic pruritus induced by atopic dermatitis.At the same time,the ECD can effectively alleviate the histological changes of skin barrier damage and epidermis thickening caused by repeated scratching in atopic dermatitis mice,and ECD can reduce the levels of inflammatory cytokines in atopic dermatitis mice ear tissue and serum,as well as the phosphorylation of the JAK1-STAT3/STAT6 pathway in ear tissue.These results suggest that ECD has a good inhibitory effect on acute and chronic pruritus and can inhibit the inflammatory symptoms of atopic dermatitis by regulating the JAK1-STAT3/STAT6 signaling pathway.In the third part,both dermal and intragastric administration of Dictamnine significantly inhibited chloroquine-induced acute pruritus in mice,suggesting the anti-histamineindependent effect of Dictamnine on pruritus.We demonstrated that the inhibitory effect of Dictamnine on the excitability of chloroquine-activated MrgprA3+neurons or allyl isothiocyanate(AITC,an agonist of TRPA1)activated TRPA1+neurons using calcium imaging experiments.The results of molecular docking experiments showed that MrgprA3 has a good binding affinity with Dictamnine.These results suggest that Dictamnine can inhibit histamine-independent pruritic signal transduction by directly regulating the MrgprA3-TRPA1 pathway.In the fourth part,we also found that chronic pruritus induced by DNFB was significantly inhibited by Dictamnine in atopic dermatitis model.In addition,Dictamnine could effectively alleviate skin barrier damage and thickening of the epidermis caused by repeated scratching in atopic dermatitis mice.The distribution of MrgprA3+fiber and MrgprA3+ neuron in the skin and DRG of atopic dermatitis mice was significantly inhibited by the treatment of Dictamnine.The mRNA expression levels of MrgprA3,TRPA1,and P2X3R and the protein expression levels of TRPA1 and P2X3R were also significantly reduced by Dictamnine.These results suggest that Dictamnine may have a good therapeutic effect on atopic dermatitis and its chronic pruritus by regulating MrgprA3 and its downstream ion channels.Conclusion:In conclusion,firstly,we analyzed the clinical medication rules of traditional Chinese medicine in the treatment of eczema and screened out the core drug-Cortex Dictamni,which may have a good anti-inflammatory and anti-pruritic effect.we have shown that ECD has both in vitro and in vivo therapeutic effects on atopic dermatitis symptoms and histamineindependent chronic pruritus.Secondly,we identified that Dictamnine,the main active substance of Cortex Dictamni,can inhibit histamine-independent itching signal conduction by regulating the MrgprA3-TRPA1 pathway,and has a good therapeutic effect on acute histamine-independent itching caused by chloroquine and chronic itching caused by atopic dermatitis.It also has a good therapeutic effect on the inflammatory symptoms of atopic dermatitis and shows a certain safety in vivo.This study is helpful to clarify the effect and mechanism of Cortex Dictamni and Dictamnine in the treatment of atopic dermatitis and chronic itch and to provide a theoretical basis for the clinical treatment of atopic dermatitis and pruritus.At the same time,this study also helps to deepen our understanding of histamineindependent itch to a certain extent and provides a reference for the research and development of effective drugs for the treatment of histamine-independent itch. |
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