| Airway Hyperrecation(AHR)refers to the excessive response of the trachea and bronchus to specific antigen stimulation and non-specific stimulation,which is one of the important characteristics of bronchial asthma patients different from normal people.Bronchial asthma,which is characterized by high airway response,is one of the most common chronic diseases that endanger human health.More than 300 million people are suffering from asthma worldwide.Asthma often has a family tendency,and is affected by genetic factors.With the change of lifestyle,its morbidity and mortality are on the rise.As a traditional therapy of Chinese medicine,the clinical efficacy of acupuncture has been widely recognized by Chinese and Western respiratory experts.However,there are few studies on the specific mechanism of acupuncture treatment of AHR,which restricts acupuncture treatment of AHR is recognized and applied in a wider range.It is currently widely believed that inflammation is one of the important mechanisms leading to AHR.When the airway is stimulated by allergens or other external factors,lymphocyte ratio and autophagy imbalance,elevated levels of inflammatory cytokines,inflammatory cell infiltration,airway epithelial cell damage and other factors lead to AHR.Endoplasmic reticulum stress(ERS)and autophagy are important mechanisms of cell damage resistance.They can participate in the proliferation/differentiation and inflammatory response of T lymphocytes through a variety of pathways,including AHR.The autophagy gene ATG5 plays an important role in AHR.Our team,in previous studies,confirmed that acupuncture can improve the symptoms of AHR in mice model by regulating the balance of CD4 T lymphocyte subsets.Therefore,this study intends to establish an ovalbumin(OVA)induced AHR model in C57 mouse and conditional ATG5 knockout mice,and observe the effects of acupuncture at Dazhui,Feishu and ZuSanli on OVA-induced AHR.Based on these,the mechanism of acupuncture-mediated endoplasmic reticulum stress-autophagy in CD4 T lymphocyte homeostasis will be studied,in order to provide theoretical and laboratory evidence for the clinical application of acupuncture in AHR.Chapter I Literature review1.Advances in asthma research2.Application of acupuncture in the treatment of asthma3.The relationship between endoplasmic reticulum stress and asthma4.The relationship between autophagy and asthma5.The relationship between autophagy gene ATG5 and asthmaChapter II Research studySection 1.Effects of acupuncture on AHR and inflammation of the trachea in OVA-induced miceObjectiveTo observe the effects of acupuncture at Dazhui,Feishu and ZuSanli on AHR and inflammation of trachea in OVA-induced C57 miceMethods1.AHR model construction and grouping intervention18 C57 mice were randomly divided into 3 groups:Sham group,AHR group(airway high response model group),and AAHR group(acupuncture treatment group),with 6 mice in each group.Sham group:0.2 ml of sterile saline was injected into the abdominal cavity on the 0th,7th and 14th day of the experiment.The mice were anaesthetized with Pelltobarbitalum Natricum on the 15th,once every other day,until the end of the 27th day.On days 28,29,and 30,the mice were aerosolized daily with sterile saline for 20 min/day for 3 consecutive days.AHR group:0.2 ml of physiological saline containing 10?g OVA and 1 mg Al(0H)3 was injected into the abdominal cavity at 3 points on the 0th,7th and 14th day of the experiment.The mice were anaesthetized with Pelltobarbitalum Natricum on the 15th,once every other day,until the end of the 27th day.The sensitized mice were aerosolized daily with 1%OVA saline(70 mg OVA in 7 ml sterile saline)for 20 min/day for 3 consecuti-ve days.AAHR group:The airway high-response model was constructed in the same AHR group.On the 15th day,Dazhui,Feishu(double)and Zusanli(double)were selected for acupuncture,and acupuncture was performed once every other day for 7 times to 27th.The sensitized mice were atomized on the 28th,29th,and 30th,and the method was the same as the AHR group.2.Measurement1)Non-Invasive Airway Mechanics sites(NAM)records the sRaw%value after methacholine stimulation.2)HE staining observed the morphological changes of airway and lung tissue in the pathological sections of lung tissue of each group.3)Wright' s staining observed and counted the percentage change of white blood cells in BALF.Results1.The non-invasive airway mechanics sites showed that compared with Sham group,the percentage of sRaw changes in mice in AHR group increased when the concentration of Mch was 3.125 mg/ml to 50 mg/ml,and the difference increased statistically significant-After acupuncture treatment,the percentage of sRaw changes in AAHR group decreased than that in AHR group at each concentration of Mch stimulation,and the difference was statistically significant.2.HE staining results showed that normal C57 mice lung tissue bronchial mucosa epithelial cells arranged regularly,no inflammatory cell infiltration,no obvious secretions in the bronchi;AHR group bronchial mucosa showed congestive edema,epithelial cells visible Focal detachment,wrinkles increased,obvious secretions in the bronchial lumen and interstitial lung;AAHR group collateral mucosal epithelial cells tend to be neatly arranged,mucosal congestive edema is weakened,bronchial lumen and interstitial secretion significantly reduced.3.Wright' s staining in BALF showed that the total number of white blood cells in BALF significantly increased in AHR group,and significantly reduced in AAHR group.At the same time,the percentage of eosinophils and neutrophils in the AHR group increased significantly compared with that in the sham group,and the macrophages significantly decreased compared with the Sham group(both P<0.05).After acupuncture intervention,the percentage of eosinophils and neutrophils decreased significantly and the percentage of macrophages increased significantly.ConclusionIn this study,airway hyperresponsiveness models in C57 mice was successfully prepared.Acupuncture at Dazhui,Feishu and ZuSanli have significant improvement in the clinical symptoms of AHR and inflammation of the trachea in OVA-induced C57 mice,which is an effective treatment.Section 2.Effects of acupuncture on T lymphocyte subsets in Airway Hyperrecat ion miceObjectiveTo observe the effects of acupuncture at Dazhui,Feishu and ZuSanli on Thl/Th2 and Treg/Th17 balance in airway hyperresponsiveness miceMethods1.AHR model construction and grouping intervention2.Measurement1)Flow cytometry detected regulatory T cells,Th17 cells,Thl cells and Th2 cells in spleen,and the changes of T lymphocyte subsets were observed.2)The levels of TGF-?,IL-17,IFN-y and IL-4 in serum and alveolar lavage fluid(BALF)were detected by ELISA.Results1.The results of flow cytometry showed that compared with the sham group,the proportion of regulatory T cells cells and Thl cells in AHR group was significantly decreased,and the proportion of Th17 cells and Th2 cells was significantly increased.After acupuncture intervention,compared with the model group(AHR group),the proportion of regulatory T cells cells and Thl cells in the acupuncture group was significantly increased,and the ratio of Th17 cells and Th2 cells was significantly decreased(P<0.05).2.In the serum and BALF,the levels of TGF-?,IL-17,and IL-4 in the AHR group were higher than those in the sham group,while the IFN-y content in the AHR group was lower than that in the Sham group(P<0.05).Compared with the AHR group,the TGF-?,IL-17,and IL-4 in the acupuncture AAHR group were significantly decreased,and the IFN-? content was significantly increased.ConclusionThe AHR model showed the imbalance of Thl/Th2 and Treg/Th17 cell ratios,accompanied by the corresponding disturbance of cytokine secretion.Acupuncture at Dazhui,Feishu and Zusanli points could regulate the proportion of T lymphocyte and the secretion of cytokine,thus alleviating airway inflammation.Section 3.Effects of acupuncture on endoplasmic reticulum stress and autophagy in Ai rway Hyper recat i on miceObjectiveTo observe the effects of acupuncture at Dazhui,Feishu and ZuSanli on endoplasmic reticulum stress and autophagy in airway hyperresponsiveness miceMethods1.AHR model construction and grouping intervention2.Measurement1)RT-PCR and Western blot detected the expression changes of ATG5,LC3II,PERK1,IRE-1,ATF6 and Grp78.2).The ultrastructure of lung tissue was observed by electron microscopy.Results1.The mRNA and protein expression levels of ATG5,LC3II,PERK1,IRE-1,ATF6,Grp78 in AHR group were significantly elevated,compared with the Sham group(both P<0.05).After acupuncture intervention,the expression levels of the above genes and proteins showed different degrees of down-regulation,and the differences were statistically significant(P<0.05).2.The ultrastructure of lung tissue in AHR group showed more typical autophagosomes,which showed a closed,spherical double-layer membrane structure,and the number of autophagosomes in AAHR group was significantly reduced.ConclusionThe expression of genes and proteins related to ER stress and autophagy increased in lung tissue of AHR model.Acupuncture can improve airway hyperresponsiveness and airway inflammation by regulating the expression of ER stress-autophagy related genes and proteins.Section 4.The roles of autophagy gene ATG5 in acupuncture-regulated ER stress-autophagy induced T lymphocyte homeostasis in AHR miceObjectiveTo observe the roles of ATG5 in acupuncture regulating ER stress-autophagy-T lymphocyte subsets balance in AHR mice.Methods1.Preparation of CD4+cells conditional ATG5 knockout miceThe ATG5 gene was subjected to flox modification by homologous recombination of fertilized eggs to prepare ATG5flox/floxCD4+Cre+mice and its control ATG5flox/floxCD4+Cre-mice.2.AHR model construction and grouping intervention18 ATG5flox/floxCD4+Cre+mice were randomly divided into 3 groups:Sham(+)group,AHR(+)group(AHR model group)and AAHR(+)group(acupuncture treatment group),with 6 mice in each group.18 ATG5flox/floxCD4+Cre-mice were randomly divided into 3 groups:Sham(-)group,AHR(-)group and AAHR(-)group,with 6 mice in each group.AHR model construction and grouping intervention were the same as section 1.3.Measurement1)Identification of genotypes of ATG5 knockout mice.2)Invasive Resistance/Compliance(RC)System record the RI%change value after methacholine stimulation.3)HE staining observed the morphological changes of airway and lung tissue in the pathological sections of lung tissue of each group.4)Wright' s staining observed and counted the percentage change of white blood cells in BALF.5)Flow cytometry detected regulatory T cells,Th17 cells,Thl cells and Th2 cells in spleen,and the changes of T lymphocyte subsets were observed.6)The levels of TGF-?,IL-17,IFN-? and IL-4 in serum and alveolar lavage fluid(BALF)were detected by ELISA.7)RT-PCR and Western blot detected the expression changes of ATG5,LC3II,PERK1,IRE-1,ATF6 and Grp78.8)The ultrastructure of lung tissue was observed by electron microscopy.Results1.82 F3 generation mice were obtained by laboratory breeding,among which flox homozygous and Cre-positive mice(ATG5flox/floxCD4+Cre+)19,flox homozygous and Cre-negative mice(ATG5flox/floxCD4+Cre-)20.18 mice were selected for subsequent experiments.2.Invasive pulmonary function test results:When ATG5flox/floxCD4+Cre-mice and ATG5flox/floxCD4+Cre+mice were stimulated with different concentrations of Mch,the RI increase percentage of AHR group was significantly higher than that of Sham group(P<0.05);after acupuncture treatment,the RI change percentage of Cre-mice and Cre+mice at each concentration of Mch was smaller than that of AHR group,the difference was significant(P<0.05);Meanwhile,in AHR group and AAHR group,the RI%change of ATG5flox/floxCD4+Cre+mice was significantly lower than that of ATG5flox/floxCD4+Cre-mice(P<0.05).The results showed that the airway hyperresponsiveness of model mice was enhanced,while the airway hyperresponsiveness was weakened by Acupuncture and ATG5flox/floxCD4+Cre+mice.The airway resistance of ATG5flox/floxCD4+Cre+mice after acupuncture was similar to that of normal control group.3.HE staining of the lung histomorphology:The lung histomorphology of the sham group in ATG5flox/floxCD4+Cre-mice was similar to that of sham group in ATG5flox/floxCD4+Cre+mice.The infiltration of inflammatory cells of the AAHR group in the ATG5flox/floxCD4+Cre+ mice was significantly weaker than that of AAHR group in ATG5flox/floxCD4+Cre-mice.4.Wright's staining of the BALF:For ATG5flox/floxCD4+Cre-mice and ATG5flox/floxCD4+Cre+ mice,the results of Wright staining showed that the total number of white blood cells,eosinophils and neutrophils in BALF of AHR group were significantly higher than those of Sham group,while macrophages were significantly lower than those of Sham group.The AAHR group improved.In AHR group,compared with ATG5flox/floxCD4+Cre-mice,ATG5flox/floxCD4+Cre+mice had significantly lower total leukocyte count,eosinophil,neutrophil percentage and higher lymphocyte percentage,while in AAHR group,compared with ATG5flox/floxCD4+Cre-mice,ATG5flox/floxCD4+Cre+mice had significantly lower eosinophil and neutrophil percentage.The percentage of lymphocyte and lymphocyte increased significantly.ATG5flox/floxCD4+Cre+mice AAHR group was similar to Sham group.5.Flow cytometry results:In ATG5flox/floxCD4+Cre-mice and ATG5flox/floxCD4+Cr e-mice,compared with Sham group,the proportion of regulatory T cells and Thl cells in AHR group decreased significantly,and the proportion of Th 17 cells and Th2 cells increased significantly.After acupuncture interve ntion,compared with AHR group,the proportion of regulatory T cells and Thl cells in AAHR group increased significantly,while that of Th17 cells and Th2 cells decreased significantly(P<0.05).In AHR group,ATG5flox/floxCD4+Cre+mice had significantly higher proportion of regulatory T cells and T h1 cells and lower proportion of Th17 cells and Th2 cells than ATG5flox/floxCD 4+Cre-mice(all P<0.05);in AAHR group,compared with the ATG5flox/floxCD4+Cre-mice,the proportion of regulatory T cells and Thl cells in ATG5flox/floxCD4+C re+mice increased significantly,the proportion of Th17 cells and Th2 cell s decreased significantly(all P<0.05);and in ATG5flox floxCD4+Cre+mice,the pr oportion of spleen T lymphocytes in acupuncture treatment group was simil ar to the normal control group.6.Elisa results:In ATG5flox/floxCD4+Cre-and ATG5flox/floxCD4+Cre-mice,the serum and BALF levels of TGF-p,IL-17,and IL-4 in AHR group were higher than those in Sham group,while the levels of IFN-? in AHR group were lower than those in Sham group(P<0.05).Compared with AHR group,the levels of TGF-?,IL-17,IL-4 and IFN-? in AAHR group were significantly decreased and increased(P<0.05).Compared with ATG5flox/floxCD4+Cre-mice,in the AHR group ATG5flox/floxCD4+Cre+mice showed serum IL-4 content decreased,IFN-? content increased;BALF TGF-?,IL-17,and IL-4 significantly decreased,IFN-? content signific antly increased;BALF TGF-?,IL-17,and IL-4 significantly decreased,IF N-? content significantly increased;In the AAHR group ATG5flox/floxCD4+Cre m ice,compared with ATG5flox/floxCD4+Cre-mice,L-17 and IL-4 decreased signifi cantly,while IFN-? increased significantly.ATG5flox/floxCD4+Cre+AAHR mice h ad a lower degree of inflammation than Sham mice.7.RT-PCR and Western blot results:In ATG5flox/floxCD4+Cre-and ATG5flox/floxCD4+Cre+mice,the expression levels of autophagy genes ATG5,LC3II,endoplasmic reticulum stress-related genes PERK1,IRE-1,ATF6 and Grp78 in airway hyperresponsiveness model group were significantly higher than those in Sham group.After acupuncture intervention,the expression levels of the above genes and proteins showed different degrees of downregulation,with statistical significance(P<0.05).The expression levels of ATG5 and LC3 in ATG5flox/floxCD4+Cre+mice AHR group and the contents of autophagic protein ATG5 and endoplasmic reticulum stress proteins PERK1,IRE-1,ATF6 and Grp78 were lower than those inATG5flox/floxCD4+Cre-mice.In AAHR group,compared with ATG5flox/floxCD4+Cre-mice,ATG5flox/floxCD4+Cre+mice had lower levels of autophagy genes ATG5,LC3 and endoplasmic reticulum stress genes PERK1,autophagy proteins ATG5,LC3 and endoplasmic reticulum stress proteins PERK1,IRE-1,ATF6 and Grp78(P<0.05).ATG5flox/floxCD4+Cre+AAHR mice had lower levels of autophagy and endoplasmic reticulum stress,and were closer to sham mice..8.Electron microscopy results:In ATG5flox/floxCD4+Cre-mice,the ultrastru cture of lung tissue in AHR group showed more typical autophagosomes,and the number of autophagosomes in lung tissue of AAHR group was significan tly reduced.In the ATG5flox/floxCD4+Cre+mice,the ultrastructure of lung tis sue in the AHR group showed a very small number of autophagosomes,while the acupuncture group did not find a typical autophagosome structure.ConclusionThe autophagy gene ATG5 mediates endoplasmic reticulum stress-autophagy and regulates T lymphocyte subsets to improve symptom of AHR and inflammation of the trachea.Acupuncture at Dazhui,Feishu(double)and Zusanli can regulate the expression of ATG5 gene,and then promote T lymphocyte homeostasis by regulating ER stress-autophagy. |
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