| ObjectiveThe purpose of this study is to establish an animal model of asthma and to explore the effect of acupuncture on autophagy mediated NMU-ILC2 immune regulation.Methods(1)The autophagy inhibitor 3-methyladenine(3MA group)and agonist rapamycin(RAPA group)were used to intervene BEAS-2B cells for 36 hours,respectively.There is no special intervention in the control group(CON group).The NMU-ILC2 s immune proteins and autophagy related proteins were detected by Western blot.RT-PCR was used to detect the expression of NMUR1 and RORα gene.The inflammatory cytokines IL-4 and IL-13 in cell culture medium were detected by ELISA.(2)Thirty female C57 mice were randomly divided into 5 groups: negative control group(CON),asthma model group(AHR),acupuncture treatment group(ACU),autophagy inhibitor treatment group(3MA)and positive control group(DM).HDM and OVA was intraperitoneal injected for asthma model establishment.The ACU group was treated with Feishu,Dazhui and Zusanli acupuncture.The 3MA group and DM group were treated with3-methyladenine and dexamethasone intraperitoneal injection,respectively.The airway resistance test was performed within 24 hours of the last sensitization.HE staining was used for the observation of pathological of the mice lung tissue.NMU-ILC2 s immune proteins and autophagy related proteins were detected by Western blot,and NMU,NMUR1,RORα genes were detected by RT-PCR.Peripheral blood and alveolar lavage fluid were collected for inflammatory factors detection.Results(1)The expressions of proteins NMU,NMUR1 and RORα in RAPA group were significantly increased in comparison with the CON group,(P<0.05),and down-regulated in 3MA group(P<0.05).The expression of NMUR1 and RORα genes was consistent with the protein expression.The secretion of IL-4 and IL-13 was increased in RAPA group(P<0.05),and decreased in 3MA group(P<0.05).(2)The percentage of lung resistance increase in AHR group was significantly higher than that in the CON group(P<0.05).The percentage of lung resistance changes in the ACU group,the 3MA group and the DM group all decreased compared with group AHR(P<0.05).(3)The observation of HE staining showed that the bronchial wall structure of mice in AHR group was incomplete,and the bronchial wall was thickened.A lot of exudate could be seen in the narrowed lumen with inflammatory cells infiltrated around.The bronchial structure of ACU group tended to be complete,and the bronchial wall was thinner than that of AHR group.The exudate in the lumen was reduced as well as inflammatory cells.In the3 MA group,the bronchial structure was complete,and the epithelial cells were arranged in a complete way.There was no exudate in the lumen,and a few inflammatory cells were infiltrated around it.In DM group,the bronchial wall became thinner than that of AHR group,and the exudate in the lumen decreased as well as inflammatory cells.(4)The expressions of proteins Atg5,P62 and LC3 in ACU and 3MA groups were significantly lower than those in AHR group(P<0.05),and the change of proteins expression in DM group was not statistically significant.NMU,Nmur1,RORα protein expression was down regulated in ACU group and 3MA group than that of AHR group(P<0.05),and there was no significant difference of the expression in DM group(P<0.05).The expression changes in NMU,NMUR1,RORα genes was consistent with the protein.(5)Compared with CON group,the expressions of IL-4 and IL-13 in peripheral blood and alveolar lavage fluid of mice in AHR group were significantly increased(P<0.05).The expressions of IL-4 and IL-13 were down-regulated in the ACU group(P<0.05).conclusionAcupuncture treatment can inhibit the expression of NMU-ILC2 s immune related proteins and genes as well as type 2 cytokines by downregulating autophagy,and then relieve airway hyperresponsiveness and airway inflammation in asthmatic mice. |
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