Study On The Effect Of Electroacupuncture On Pruritus Behavior,serum Immunology And GRPR/TRPs Signal Pathway In AD Mice Induced By DNFB

【Purpose】Atopic dermatitis（AD）is a recurrent chronic,inflammatory skin disease that often occurs in infants and young children with a long course of disease and can be extended to adulthood.In addition to skin inflammation caused by atopic dermatitis,chronic pruritus caused by atopic dermatitis has a serious impact on the growth and development,quality of life and mental health of patients.Glucocorticoid is still the first choice for the treatment of atopic dermatitis,but the possible adverse reactions and side effects of glucocorticoid make patients have a lot of concerns.Traditional antihistamine therapy is not effective for chronic pruritus caused by atopic dermatitis.Acupuncture,as an external treatment under the guidance of the basic theory of traditional Chinese medicine,has been paid more and more attention by doctors and patients because of its relative safety.Modern medicine combines pulse electricity with traditional acupuncture,which has a good curative effect and is easy to popularize.The purpose of this experiment is to explore the effectiveness and mechanism of electroacupuncture"Quchi"and"Hegu"on AD.【Methods】C57BL/6J mice were selected to be AD model induced by back stimulation of 2-dinitrofluorobenzene（DNFB）.24 C57BL/6J mice were randomly divided into model group,electroacupuncture group,fake electroacupuncture group and normal control group.Model group,electroacupuncture group and fake electroacupuncture group were smeared with0.15%DNFB in the back shaving area to induce AD model,and The normal control group was treated with acetone without DNFB in the same area.The electroacupuncture group and the fake electroacupuncture group were treated with acupuncture at Hegu and Quchi points between the second DNFB smear and the 9th DNFB smear.The electroacupuncture group was connected with the electroacupuncture instrument,and the fake electroacupuncture group was only treated with acupuncture once every other day.The model group and the normal control group were fed normally without intervention,and the same grasping was done at the same time in the treatment of electroacupuncture group and the fake electroacupuncture group.Samples were taken after the last behavioral record.（1）In the course of the experiment,the skin lesions of mice were scored,and the scratching times of mice were recorded to observe the disease progress and behavioral effects of mice in each group.（2）HE staining was used to observe the skin histomorphological changes of mice in each group.（3）The changes of serum total IgE,IL-5 and ECP level were compared by ELISA.（4）The protein expression levels of TRPV1,TRPA1 in mouse dorsal root ganglion were detected by Western Blotting.（5）The mRNA levels of GRPR/TRPs in spinal cord and TRPs in skin tissue were detected by PCR.【Results】（1）At the end of the expriment,it can be seen that the back skin erythema of the electroacupuncture group and the fake electroacupuncture group subsided,the exudation decreased,and a small amount of crusts were occasionally seen,and the skin lesions were significantly better than those of the model group.The skin lesions of mice were scored,and the results showed that the scores of skin lesions in the electroacupuncture group and the fake electroacupuncture group were significantly different from those in the model group（P<0.01）.（2）Compared with the model group,the scratching times in the electroacupuncture group and the fake electroacupuncture group were significantly lower than those in the model group（P<0.01）.There was no significant difference in scratching times between electroacupuncture group and fake electroacupuncture group（P>0.05）.There was no significant difference in scratching times between the electroacupuncture group and the normal control group（P>0.05）.There was significant difference in scratching times between the fake electroacupuncture group and the normal control group（P<0.01）.（3）HE staining showed that obvious hyperplasia of epidermis,hypertrophy of spinous layer,spongy edema,keratosis or incomplete keratosis in stratum corneum,infiltration of a large number of inflammatory cells（mainly neutrophils and lymphocytes）in epidermis and dermis,accompanied by vasodilation and hyperemia were observed in the model group,which conformed to the histopathological changes of AD,indicating that the model was successful.Different degrees of proliferation and inflammatory cell infiltration could be seen in the epidermis in the electroacupuncture group and the fake electroacupuncture group,and the cell edema was obviously alleviated in the two grops.In the normal control group,the structure of epidermis and dermis was normal,and no hyperplasia,thickening,edema,hyperemia and lymphocyte infiltration were found.（4）Serum IgE level:Compared with the model group,the level of serum IgE in electroacupuncture group and fake electroacupuncture group decreased significantly（P<0.01）.Compared with the fake electroacupuncture group,the level of serum IgE in the electroacupuncture group was significantly higher than that in the fake electroacupuncture group（P<0.01）.Serum IL-5level:Compared with the model group,there was significant difference in the level of serum IL-5 between the electroacupuncture group and the sham electroacupuncture group（P<0.05）.Compared with the sham electroacupuncture group,there was no significant difference in serum IL-5 level between the electroacupuncture group and the sham electroacupuncture group（P>0.05）.Serum ECP level:Compared with the model group,there was significant difference in the level of serum ECP between the electroacupuncture group and the sham electroacupuncture group（P<0.05）.Compared with the sham electroacupuncture group,the level of serum ECP in the electroacupuncture group was significantly higher than that in the sham electroacupuncture group（P<0.05）.（5）The expression of TRPA1 in DRG of mice:there was significant difference between the electroacupuncture group and the fake electroacupuncture group compared with the model group（P<0.05）,and the two treatment groups were effective.The expression of TRPV1 in DRG of mice:there was significant difference between the electroacupuncture group and the fake electroacupuncture group compared with the model group（P<0.05）,and the two treatment groups were effective.（6）The mRNA level of GRPR in the spinal cord of mice:there was significant difference between the electroacupuncture group and the fake electroacupuncture group compared with the model group（P<0.01）,and the two treatment groups were effective.The mRNA level of TRPV1 in the spinal cord of mice:there was significant difference between the electroacupuncture group and the fake electroacupuncture group compared with the model group（P<0.01）,and the two treatment groups were effective.The mRNA level of TRPA1 in the spinal cord of mice:there was significant difference between the electroacupuncture group and the fake electroacupuncture group compared with the model group（P<0.05）,and the two treatment groups were effective.（7）The mRNA level of TRPV1 in the skin tissue of mice:there was significant difference between the electroacupuncture group and the fake electroacupuncture group compared with the model group（P<0.01）,and the two treatment groups were effective.The mRNA level of TRPA1 in the skin tissue of mice:there was significant difference between the model group and the normal control group（P<0.05）,and the model was successful,but there was no significant difference between the electroacupuncture group and the fake electroacupuncture group（P>0.05）.【Conclusion】1.Electroacupuncture at Quchi and Hegu points can significantly improve the skin lesion state of AD mice,reduce the scratching times of mice,reduce the levels of serum IgE and inflammatory factors,the protein expression of TRPs and the mRNA level of GRPR/TRPs in spinal cord and TRPs in skin tissue.2.Electroacupuncture of Quchi and Hegu may activate C fibers,reduce the levels of serum IgE and inflammatory factors,reduce the protein expression of TRPs and the mRNA levels of GRPR/TRPs in spinal cord and TRPs in skin tissue,change the distribution of Ca²⁺inside and outside the nerve cell membrane,inhibit the excitability of nerve cells,relieve itching and improve the skin lesion state of AD.