Study On The Mechanism Of Acupoint Sticking Therapy Of "Treating Winter Disease In Summer" In The Treatment Of Asthma Based On Multi-omics And Skin Barrier

Background"Treating winter disease in summer"(TWDS)is a unique therapy for lune disease prevention and treatment by Traditional Chinese Medicine.It reflects the theory of"Tian Ren He Yi" and "Chun Xia Yang Yang,Qiu Dong Yang Yin" in Traditional Chinese Medicine.The acupoint sticking therapy of "Treating winter disease in summer" has been applied for the prevention and treatment for many diseases,especially for the lung diseases,and its effect is always remarkble.Bronchial asthma is a chronic inflammatory disease,which is one of the most common chronic diseases all over the world,and its growing morbidity and mortality have received widespread attention.Due to the well clinical effect and low side effects,the acupoint sticking therapy of "Treating Winter Disease in Summer" has been widely used in the clinic to treat asthma in China.However,its mechanism of treatment has not been clarified yet.The metabolomics and microbiome may display the dynamic changes in vivo for the integral level of organism,avoiding the drawbacks which use the simplex or partial indicators to study their pathophysiological changes.Moreover,this study may provide a novel point to elaborate the mechanism of the treatment of "Treating Winter Disease in Summer" in asthma based on multi-dimension study.ObjectivesThe purpose of this study is to investigate the mechanism of treating asthma by using acupoint sticking therapy of TWDS.Firstly,to study the effects of this prescription on intestinal flora abundance and structure of asthmatic guinea pigs by 16Sr DNA sequensing,and screen the biomarkers of flora associated with asthma.Secondly,to explore the effect of this prescription on the metabolism of endogenous substances in the treatment of asthmatic guinea pigs,and to screen endogenous biomarkers which related to the onset and treatment of asthma.Thirdly,to study the effect of this prescription on the expression of inflammation-related proteins in asthma guinea pigs,and to clarify the anti-inflammatory mechanism of this prescription.Forthly,to investigate the effect of the Semen Sinapis Albae volatile oil on transdermal absorption for multiple ingredients in the prescription,and clarify its effect of promoting drugs transdermal absorption.Fifthly,by exploring the effects of the Semen Sinapis Albae volatile oil on the skin barrier and to study the mechanism of promoting percutaneous permeation,so that clerify the significance of "foaming"generated by white mustard seed in the TWDS therapy.MethodsFirstly,the guinea pigs were randomly divided into control group,OVA-sensitized asthma model group,positive control(DSP)group and acupoint of TWDS administration group.The guinea pig asthma model was established by OVA sensitization.Lung tissues of guinea pigs were evaluated by H&E and Masson staining.Secondly,DNA was extracted from the feces of guinea pigs,and the 16S rDNA V4 variable region gene was amplified by PCR and analysis by Illumina Hiseq sequencing.And then the bioinformatics analysis was performed to investigate the effect of the prescription on the intestinal flora diversity and bacterial community structure in asthmatic guinea pigs.Thirdly,the UPLC-ESI-QTOF-MS was used to detect the endogenous substances of serum in guinea pig,analytical methods such as the principal component analysis(PCA)were used for statistical analysis,to compare the original metabolomics maps and data of each group and seek for biomarkers.Fourthly,the levels of Th2 cytokines,IgE and NF-?B signaling pathway associated proteins in guinea pig were determined by molecular biology techniques.Fifthly,in vitro transdermal test and HPLC were used to determine the transdermal permeation of model drugs,to evaluate the effect of promoting transdermal penetration for others main marked ingredients by the Semen Sinapis Albae volatile oil.Finally,to study the effect of the Semen Sinapis Albae volatile oil on the structure of corneum,and the effect of the Semen Sinapis Albae volatile oil on the physiological and biochemical functions of active epidermal cells was explored with HaCaT cell line,the in vitro model of skin barrier.ResultsAcupoint sticking therapy of TWDS ameliorated asthma of guinea-pigs which sensitized by OVA.This therapy reduced airway and lung inflammation and collagen deposition in guinea pigs.The gut microbiota sequencing results showed that the diversity of intestinal flora in asthmatic guinea-pigs was increased after prescribing acupoint application compared with OVA group,but no significance.The abundance of Prevotellaceae,Prevotella,Fimicutes,Succinogenes,Arthrobacter Micrococcaceae in normal group were higher than other group,and Flexispira in OVA group was higher than other groups.Moreover,the abundance of Campylobacter,Rhizosphaerae,Clostridiaceae,Actinobacteria,Clostridium,Bifidobacterium,Parabacteroide,Bifidobacteriales,Bifidobacteriaceae,Stenotrophomonas,Lactobacillaceae,Lactoacillus,Distasonis,Coriobacteriales,Coriobacteriaceae,Blautia,Coriobacteriia,Xanthomonadaceae,Xanthomonadales in acupoint sticking therapy of TWDS group is highest among 4 groups.Additionally,the levels of phospholipids,sphingolipids,purines,amino acids,and epinephrine in the guinea pig of asthma were all abnormal,after treated with this prescription,the levels of substances above were recovered to normal,and the therapy inhibited sphingolipid metabolism by down-regulating ORMDL3.Moreover,the expression of IL-4,IL-5,IL-13 and IgE in guinea pigs were down-regulated after treated of TWDS,and the phosphorylation of p65 and I?B were inhibited.In our study,we also found that the Semen Sinapis Albae volatile oil significantly promoted the permeation absorption of the ingredients in TWDS prescription,the higher concentration,the better penetration promoting effect.Also,our results show that the Semen Sinapis Albae volatile oil altered the structure of corneum of rat skin and increased the concentration of Ca2+ and reduce the activity of Ca2+-ATP enzyme and reduce the membrane potential in HaCaT cell line.ConclusionThe therapy of TWDS effectively reduce the inflammation of lung,improve asthma symptoms and regulate the metabolism of endogenous of asthma in guinea pigs,and also 26 asthma-relative biomarkers were found.Our study showed that TWDS regulated the composition of intestinal flora in asthmatic guinea pigs to affect the body immunity,inhibited the Th2 reaction to make the balance of Thl/Th2.We also found that it reduced the inflammation by inhibiting the phosphorylation of NF-kB signaling pathway to achieve the effect of treatment of asthma.Moreover,the Semen Sinapis Albae volatile oil promote the transdermal absorption of the ingredients in TWDS prescription,as the main medicine of prescriptions,it play a dual role in the treatment and promotion of drug permeation,so as to achieve synergistic effect.And its mechanism is by changing the barrier properties of the epidermis and weakening the barrier function of the skin epidermis.This provide new ideas and methods for the study of the mechanism of transdermal enhancers.