The Effect Of Electroacupuncture On Airway Inflammation Based IL33-ILC2 Axis In Mouse Asthma Model

Asthma is one of the major public health challenges in our country and one of the diseases that is growing at the fastest rate.Described as a heterogeneouir disease,asthma is a chronic airway inflammatory disorder that is caused by a variety of different cells(eosinophils,neutrophils,mast cells,T cells,and airway epithelial cells).According to a study published by Lancet in 2019,there are 45.7 million adults over the age of 20 with asthma in our country.Asthma is a complex disease with a variety of pathogenesis,pathophysiology,and clinical manifestations.Recent studies have shown that group II innate lymphoid cells(ILC2)are closely related to the onset of asthma.ILC2 lacks specific antigen receptors,so when stimulated by allergens,it does not need to bind specifically to antigens.It is rapidly activated by alarmin cytokines(IL33,IL25,TSLP)secreted by airway epithelium,thus releasing inflammatory factors such as IL5 and IL13.Asthma is mediated through interactions with eosinophils,mast cells,neutrophils,dendritic cells,B cells,macrophages,and epithelial cells.Interleukin 33(IL33),one of the alarmin cytokines secreted by airway epithelium,plays a variety of roles in tissue homeostasis,prevention of parasitic infection,induction of allergic inflammation.It has been confirmed that the expression of IL33 in the airway of bronchial asthma increased with the severity of the disease,and that the level of IL33 in the airway correlated with the level of type 2 cytokines and was positively correlated with the number of eosinophils in patients with asthma.Although several genes have been associated with asthma in different large-scale genetic studies,the gene encoding IL33 and its receptor ST2(IL1RL1)are two of the very few genes that can be repeatedly identified as a major susceptibility site in all the Genome-wide association studies(GWAS)of asthma,supporting the critical role of the IL33-ILC2 axis in human asthma.As a treasure of traditional Chinese medicine,acupuncture has long been used clinically for asthma.It has been demonstrated in several studies that acupuncture could reduce airway hyperresponsiveness,and airway inflammation and improve patients’ quality of life.In our previous study,we found that acupuncture at Dazhui,Feishu,and Zusanli points could regulate endoplasmic reticulum stress and induce airway hyperresponsive T lymphocyte subsets homeostasis by regulating the imbalance of Th1/Th2 and Th17/Treg subsets in asthmatic mice,thus reducing airway hyperresponsiveness and inflammation.During electroacupuncture,an electric current is passed through a needle,similar to traditional manual acupuncture,but it is more commonly used than traditional manual acupuncture.It has been reported that electroacupuncture is effective for asthma in some studies,however,fewer studies have been conducted on electroacupuncture.Consequently,we speculate that electroacupuncture may reduce airway inflammation by affecting IL33-ILC2.To confirm the above hypothesis,this study will establish a mouse model of asthma and explore the effect of electroacupuncture at Dazhui,Feishu,and Zusanli on asthmatic airway inflammation,the number,and function of ILC2 through expanding ILC2 and inhibiting IL33.ObjectiveTo observe the effect of electroacupuncture on airway inflammation of asthmatic mice,and to explore the mechanism of electroacupuncture intervention on airway inflammation of asthmatic mice around the IL33-ILC2 axis.Methods1.Ovalbumin(OVA)was used to establish the model of asthma mice,which were divided into 5 groups:blank control group,asthma group,manual acupuncture group,electroacupuncture group and dexamethasone group.The changes of body weight of mice in each group were detected,lung tissue inflammation was detected by HE staining,goblet cell proliferation of mice airway epithelial cells was detected by PAS staining,and the changes of IL5 and IL13 concentrations in alveolar lavage fluid and serum IgE concentrations in mice were detected by ELISA.To observe whether electroacupuncture can reduce airway inflammation of asthmatic mice in Dazhui,Feishu and Zusanli.2.Three different allergens were used to establish the model of asthma mice:OVA,house dust mite,IL33.Each model method was divided into three groups:blank control group,asthma group and EA group.The proportion of eosinophils in mouse alveolar lavage fluid,the level of ILC2 in mouse lung tissue and the function of IL5 and IL13 secreted by ILC2 in mouse lung tissue were analyzed by flow cytometry.mRNA expression levels of IL5,IL13 and GATA3 in mouse lung tissue were detected by qPCR,and the changes of ILC2 in mouse lung tissue were observed by immunofluorescence co-localization.To investigate the effect of electricity on ILC2 in asthmatic mice.3.IL2/Jes6-1 was used to expand ILC2 in asthmatic mice induced by HDM and electroacupuncture.They were divided into 5 groups:blank control group(Control group),asthma group(HDM group),electroacupuncture group(EA+HDM group),asthma+IL2/Jes6-1 group(HDM+IL2 group),electroacupuncture+IL2/Jes6-1 group(EA+HDM+IL2 group).Flow cytometry was used to detect the level of ILC2 in mouse lung tissue,the function of ILC2 secreting IL5 and IL13,and the proliferation of ILC2.Flow cytometry was used to analyze the proportion of eosinophilic granulocyte in mouse alveolar lavage fluid,and HE staining was used to detect the inflammation of mouse lung tissue.To observe whether the effect of electroacupuncture on reducing airway inflammation in asthmatic mice was reversed by IL2/Jes6-1,and to explore whether electroacupuncture could reduce airway inflammation in asthmatic mice by ILC2.4.Anti-IL33,an IL33 inhibitor,was added based on the previous step.They were divided into 6 groups:blank control group(Control group),asthma group(HDM group),electroacupuncture group(EA group),HDM+IL33 inhibitor group(anti-IL33 group),electroacupuncture+IL33 inhibitor group(EA+anti-IL33 group),Electroacupuncture+IL33 inhibitor+ILC2 expansion agent group(EA+anti-IL33+IL2 group).ELISA was used to detect the concentration of IL33 in mouse alveolar lavage fluid,flow cytometry was used to analyze the level of ILC2 in mouse lung tissue,the function of ILC2 secreting IL5 and IL13 and the proportion of eosinophils in mouse alveolar lavage fluid.The inflammation of mouse lung tissue was detected by HE staining.To observe whether the airway inflammation of asthmatic mice was reduced by electroacupuncture through the IL33-ILC2 axis.Results1.The body weight of asthmatic mice was significantly lower than that of control group.The inflammation of lung tissue,goblet cell hyperplasia of airway epithelial cells,the concentration of IL5,IL13 and serum IgE in alveolar lavage fluid of asthmatic mice were significantly higher than that of blank control group.The body weight of mice in the manual acupuncture group,electroacupuncture group and dexamethasone group were significantly higher than those in the asthma group.The inflammation of lung tissue,goblet cell hyperplasia of airway epithelial cells,IL5 and IL13 concentrations and serum IgE concentrations were significantly lower than those in the asthma group.2.The proportion of eosinophil in alveolar lavage fluid of asthmatic mice,the level of ILC2 in mouse lung tissue,the secretion of inflammatory cytokines IL5 and IL13 by ILC2 in mouse lung tissue,mRNA levels of IL5,IL13 and ILC2 key transcription factors GATA3 in mouse lung tissue were significantly higher induced in OVA group,HDM group and IL33 group than those in blank control group,and were effectively reduced after electroacupuncture intervention.The increase of ILC2 in lung tissue of asthmatic mice induced by HDM was observed by immunofluorescence co-localization,and the decrease of ILC2 in lung tissue after electroacupuncture intervention.3.The level of ILC2 in the lung tissue of mice,the function of secreting inflammatory cytokines IL5 and IL13 by ILC2 in the lung tissue of mice,the proliferation of ILC2 in the lung tissue of mice,the proportion of eosinophil in the alveolar lavage fluid of mice,and the inflammation of the lung tissue of mice were significantly higher in the HDM+IL2 group than those in the HDM group.The above indexes of the EA+HDM+IL2 group were significantly higher than those of the EA+HDM group,and there was no difference between the HDM+IL2 group and the EA+HDM+IL2 group.4.Compared with the HDM group,the IL33 of alveolar lavage fluid in EA and antiIL33 groups was decreased.Compared with EA group,IL33 of alveolar lavage fluid in antiIL33 group and EA+anti-IL33 group was decreased.Compared with EA+anti-IL33 group,there was no difference in alveolar lavage fluid IL33 between EA+anti-IL33 group and EA+anti-IL33+IL2 group.Compared with the HDM group,the level of ILC2 in the lung tissue of mice,the function of secreting inflammatory cytokines IL5 and IL13 by ILC2 in the lung tissue of mice,and the proportion of eosinophils in the alveolar lavage fluid of mice were decreased in the EA group and the anti-IL33 group.There was no difference between EA group and anti-IL33 group,EA group and EA+anti-IL33 group,anti-IL33 group and EA+anti-IL33group.Compared with the EA+anti-IL33 group,the EA+anti-IL33+IL2 group increased.Compared with HDM group,the lung tissue inflammation score of EA group and antiIL3 3 group was reduced.Compared with EA group,the lung tissue inflammation score of anti-IL33 group and EA+anti-IL33 group was decreased.Compared with the anti-IL33 group,there was no difference in lung tissue inflammation in the EA+anti-IL33 group.Compared with EA+anti-IL33 group,the lung tissue inflammation score of EA+anti-IL33+IL2 group was increased.Conclusions1.The OVA model of mouse asthma was established successfully,and it was proved that electroacupuncture can effectively reduce airway inflammation of asthmatic mice in Dazhui,Feishu and Zusanli.2.Three different allergens,OVA,HDM and IL33,were used to establish different asthma mouse model.The results showed that the reduction of airway inflammation by electroacupuncture was related to ILC2.3.The important role of ILC2 in reducing airway inflammation by electroacupuncture in asthmatic mice was confirmed,indicating that the effect of electroacupuncture in the treatment of asthmatic mice airway inflammation depends on ILC2.4.It was demonstrated that electroacupuncture reduced airway inflammation in asthmatic mice by IL33-ILC2.