The Effective Mechanism Of Notch And Acupuncture About Imbalanced Transcription Factor Foxp3/RORγt In Airway Hyperrecation Rats

Bronchial Hyperresponsiveness refers to the airway producing excessive airway stenosis or bronchoconstriction, which causes only mild response reaction in normal or not. Airway hyperresponsiveness as the main feature of bronchial asthma is a common chronic disease in the current worldwide hazard to human health. The incidence rate is about 1%-18% of the general population (international asthma guidelines GINA2014). According to statistics, more than 300 million patients have asthma in the world. Although the work of prevention and treatment of asthma spreads broadly in recent years, the morbidity and mortality is still increasing.And airway hyperresponsiveness also causes chronic bronchitis, emphysema, allergic rhinitis, and upper and lower respiratory tract infections.Such patients prone to bronchospasm, edema and other crisis situations in the perioperative period, has become a serious public health problem affecting people’s physical and mental health. Studies have found that patients with reactive airway disease preoperative and postoperative incidence of bronchospasm mainly associated with endotracheal intubation. The incidence of airway hyperresponsiveness is 8.9% in intubation general anesthesia. It is much higher than that in without intubation general anesthesia. With the development of society, general anesthesia become more and more popular, therefore particularly important to prevent the occurrence of perioperative airway hyperresponsiveness, but the current mechanism of airway hyperresponsiveness is not very clear.Traditional Chinese medicine as a treatment for airway hyperresponsiveness, has been admitted by experts in respiratory department (Guidelines for the prevention and treatment of bronchial asthma, china,2013).Acupuncture is an important means of treatment with airway hyperresponsiveness in Chinese medicine. Past dynasties doctors and Modern clinical studies have confirmed that it can regulate immune function and treat airway hyperresponsiveness.But the mechanisms is unknow? Therefore, this study intends to establish ovalbumin (OV-albumin, OVA) sensitized airway hyperresponsiveness in BALB/c mouse model, observe the effect of traditional acupuncture therapy on airway hyperresponsiveness in OVA sensitized BALB/c mice, and to explore acupuncture by regulating Notch signaling mediated mechanisms of airway hyperresponsiveness Foxp3/RoRyt imbalance in mice.Chapter I The effect of acupuncture on OVA induced BALB/c mice with airway hyperresponsivenessObjectiveTo observe the effect of traditional acupuncture on OVA sensitized BALB/c mice with hyperresponsiveness.Methods1. Animal modelThirty BALB/c mice were randomly divided into three groups equally, including normal control group (group NC, n=10), airway hyperresponsiveness model group (group BHR, n=10) and acupuncture model group (group ACA, n=10).In this experiment the mouse in the group of airway hyperresponsiveness model was sensitized by i.p of ovalbumin(OVA)and Al(OH)3 on days 1,7,14 and challenged by intranasal inhalation OVA on days 21-23. The mice were bunded for 20 minutes each time from 17 to 21 days every other day.The normal control group were sensitized and challenged with saline instead.Acupuncture model group were acupunctured Zusanli (3mm) and Dazhui (3mm) 20 minutes each time from 17 to 21 days every other day.2. Observation parameters2.1 The general situation2.2 Record Baseline Perth value with Whole Body Plethysmograph;2.3 Observed the leukocyte classification and counting with microscope in bronchoalveolar lavage fluid;2.4 Observed the pathological changs of lung tissue with optical microscope.ResultsThe mice in normal control group were shiny coat, weight gain, respiratory frequency and amplitude of normal; The mice in airway hyperresponsiveness model group (group BHR) coat dull, weight loss, poor mental, respiratory rate increase. And the mice in acupuncture model group (group ACA) alleviated the symptoms compared with group BHR;Compared with the NC group, BHR Group airway hyperresponsiveness increased significantly in different concentrations of Mch excitation, when Mch concentrations greater than 3.12 mg/ml, airway hyperresponsiveness significantly increased, and the difference was significant (P<0.01); ACA group compared with BHR group, reduce airway hyperresponsiveness and methacholine concentration of 6.25mg/m,12.5 mg/ml,50 mg/ ml, the differences were statistically significant (P<0.01);The proportion of Eosnophils in BALF is greater in BHR group than in NC group, ACA group less than BHR group (P<0.01); compared with the NC group, BHR Group, ACA group the proportion of neutrophils was significantly higher than the normal group, and the difference was significant (P<0.01); compare BHR group and NC group, ACA group macrophages reduce the percentage, ACA group lower than BHR Group small margin, the difference was significant (P<0.05);BALB/c mice lung tissue with HE staining showed that the bronchial epithelial cells in group of NC arranged in neat rows, no inflammation, no significant bronchial secretions clean; BHR group visible focal bronchial epithelial cells fall off, wrinkles increased mucosal edema, and pulmonary interstitial lumen endobronchial secretions visible, EOS focal infiltration; ACA group bronchial epithelial cells arranged in neat, mild mucosal congestion, see a little shedding of epithelial cells within the lumen.ConclusionThe traditional acupuncture has a good effect on OVA sensitized BALB/c mice with hyperresponsiveness.ChapterⅡ The impact of acupuncture with T cell differentiation and Foxp3/RORyt Balanceon OVA induced BALB/c miceObjectiveTo observe the effect of acupuncture on the differentiation of T lymphocytes in BALB/c mice with airway hyperresponsiveness;To observe the effect of acupuncture on regulation of Foxp3/RoRyt balance in BALB/c mice with airway hyperresponsiveness.Methods1. Animal modelThirty BALB/c mice were randomly divided into three groups equally, including normal control group (group NC, n=10), airway hyperresponsiveness model group (group BHR, n=10) and acupuncture model group (group ACA, n=10).In this experiment the mouse in the group of airway hyperresponsiveness model was sensitized by i.p of ovalbumin(OVA)and Al(OH)3 on days 1,7,14 and challenged by intranasal inhalation OVA on days 21-23. The mice were bunded for 20 minutes each time from 17 to 21 days every other day.The normal.control group were sensitized and challenged with saline instead.Acupuncture model group were acupunctured Zusanli (3mm) and Dazhui (3mm) 20 minutes each time from 17 to 21 days every other day.2. Observation parameters2.1 Detection the CD4+CD25+regulatory T cells and Th17 cells percentages in BALB/c mouse spleen with airway hyperresponsiveness by flow cytometry.2.2 Detection of the transcription factor of CD4+CD25+regulatory T cells and Th17 cells with immunohistochemistry.2.3 Detection of the Foxp3mRNA, RORyt mRNA expression with RT-PCR.ResultsCompared with the NC group, BHR Group and ACA group BALB/c mouse spleen CD4+CD25+percentage of regulatory T cells decreased (P<0.01); Compared with the ACA group, the percentage of regulatory T cells in BHR Group CD4+CD25+decreased obviously, and there was statistically significant (P<0.01). Compared with the NC group, the percentage of Thl7 cells in Group BHR and Group ACA were significantly higher (P <0.01); Compared with group BHR, the percentage of Th17 cells decline in group ACA(P <0.01).The immunohistochemical detection of lung tissue RORyt, Foxp3 protein expression results:The level of RORyt protein in lung tissue of BALB/c mouse was significantly higher in BHR group and ACA group than in NC group (P<0.01), the levels of protein expression was lower in ACA group, but still higher than NC group (P<0.01);The level of Foxp3 protein in lung tissue of BALB/c mouse was significantly lower in BHR group and ACA group than in NC group (P<0.01), levels of protein expression in ACA group was lower compared with BHR Group(P<0.01).RT-PCR detection of lung tissue RORyt, Foxp3mRNA expression results:BHR group and ACA group were significantly higher than in NC group (P<0.01), ACA group RORytmRNA content lower than the BHR group level, but still higher than the NC group (P<0.01);The content of Foxp3mRNA in lung tissue of BALB/c mice was significantly lower in BHR group and ACA group than in NC group (P<0.01), ACA group Foxp3mmRNA content decreased than in BHR group, but there was no statistical significance.Conclusion1. Treg/Th17, Foxp3/RoRyt imbalance in OVA-sensitized airway hyperresponsiveness with BALB/c mice, and are negatively correlated with airway hyperresponsiveness;2. Acupuncture therapy can affect T lymphocyte differentiation on OVA sensitized airway hyperresponsiveness in BALB/c mouse;3. Acupuncture can adjust the imbalance of OVA sensitized airway hyperresponsiveness in BALB/c mice.Chapter III The effect of mediate the balance of Foxp3/RoRγt with Notch in airway hyperresponsiveness and the intervention of acupuncturObjectiveTo explore the Notch signaling on airway hyperresponsiveness in BALB/c mice;To explore the Notch signaling on airway hyperresponsiveness in BALB/c mice through Foxp3/RoRγt imbalances role and the effect of acupuncture.Methods1. Animal modelThirty BALB/c mice were randomly divided int three groups equally, including normal control group (group NC, n=10), airway hyperresponsiveness model group (group BHR, n=10) and acupuncture model group (group AC A, n=10).In this experiment the mouse in the group of airway hyperresponsiveness model was sensitized by i.p of ovalbumin(OVA)and Al(OH)3 on days 1,7,14 and challenged by intranasal inhalation OVA on days 21-23. The mice were bunded for 20 minutes each time from 17 to 21 days every other day.The normal control group were sensitized and challenged with saline instead.Acupuncture model group were acupunctured Zusanli (3mm) and Dazhui (3mm) 20 minutes each time from 17 to 21 days every other day.2. Observation parameters2.1 Detection of lung tissue Notch1mRNA, Notch3mRNA, CSLmRNA content with RT-PCR;2.2 Detection of lung tissue Notchl, Notch3, CSL protein expression changes with Western blot;2.3 Investigate the effect of acupuncture on airway hyperresponsiveness in mice Notch signaling.ResultsCompared with the NC group, the expression of Notch1mRNA in BHR Group significantly increased (P<0.01), the expression of NotchlmRNA in ACA group increased (P<0.05); Compared with the NC group, the expression of Notchl3mRNA decreased in BHR Group and ACA group (P<0.01); CSLmRNA expression of the three groups was not statistically significant.Compared with the NC group, the expression of Notchl protein in BHR group and ACA group significantly increased (P<0.01), the expression of Notchl protein ACA group were lower than BHR, and the difference was statistically significant (P<0.01); Compared with the NC group, the expression of Notch3 protein in BHR group and ACA group significantly decreased (P<0.01), the expression of Notch3 protein in ACA group is higher than that in BHR group, the difference was statistically significant (P<0.01); CSL protein three groups was not statistically significant.ConclusionNotch signal mediated BALB/c mice airway hyperresponsiveness occurs;Notchl and airway hyperresponsiveness were negatively correlated with Foxp3/RoRγt;Notch3 and airway hyperresponsiveness were positively correlated with Foxp3/RoRγt ratio in BALB/c mice;Traditional acupuncture therapy can affect OVA sensitized airway hyperresponsiveness in BALB/c mouse through the expression of Notchl, Notch3.ChapterⅣ The mechanism of mediate the balance of Foxp3/RoRγt with Notch in airway hyperresponsiveness and the intervention of acupuncturObjectiveExplore acupuncture regulating Foxp3/RoRγt balance mechanism mediated by Notch signaling.Methods1. Animal modelThirty BALB/c mice were randomly divided int three groups equally, including normal control group (group NC, n=10), airway hyperresponsiveness model group (group BHR, n=10) and acupuncture model group (group ACA, n=10).In this experiment the mouse in the group of airway hyperresponsiveness model was sensitized by i.p of ovalbumin(OVA)and Al(OH)3 on days 1,7,14 and challenged by intranasal inhalation OVA on days 21-23. The mice were bunded for 20 minutes each time from 17 to 21 days every other day.The normal control group were sensitized and challenged with saline instead.Acupuncture model group were acupunctured Zusanli (3mm) and Dazhui (3mm) 20 minutes each time from 17 to 21 days every other day.2. Observation parametersChromatin Immunoprecipitation experiments to determine the amount of bound RORγt and Foxp3 with Notch.ResultsBHR Group with lung tissue RORγtmRNA Notch binding capacity was significantly higher than NC group in BALB/c mice, ACA group was lower than in BHR group, but still higher than the NC group(P<0.01); Foxp3mRNA Notch combined was lower in BHR group than in NC group (P<0.01), ACA group was higher than in BHR group (P<0.01).The ratio of Foxp3/RORγt combined with Notch and lung tissue Foxp3/RORγt protein and gene expression ratio were positively correlated.ConclusionAcupuncture regulate airway hyperresponsiveness with Foxp3/RoRyt balancing through Notch signaling may be through bound to each other directly.Chapter V The effect of acupuncture on OVA induced BALB/c mice with airway hyperresponsivenessObjective:To observe the hemodynamics in patients with general anesthesia induction period and the influence of the lung compliance with acupuncture.Methods:40 patients scheduled for surygery under general anesthesia requiring orotracheal intubation were randomly allocated to two groups. Acupuncture group (group A, n=20) offered to 2/15 HZ density wave electroacupuncture bilateral internal clearance with Quchi and Neiguan, stimulation intensity as the current 0.5-1 ma, time for 30 minutes 30 min before the preoperative; The control group (group C) not electroacupuncture simply by intravenous general anesthesia induction. Observe patient general condition, hemodynamic index and pulmonary ventilation related index.Results:There was no significant difference in the two groups of patients in demographic data. (P<0.05). Compared with base value preinduction, BP, HR and RPP were significantly decreased in both groups. (P<0.05) Tracheal intubation caused significant increases in BP, HR, and RPP as compared with postinduction values. (P<0.05) RPP value of the two groups after intubation were significant.rise in two groups, but the duration of the RPP increased in group A is the shorter of that in group C. (P<0.05) And the RPP after 10 min of induction was lower than those of group C. (P<0.05)Conclusion:Compared with control group, the application of acupuncture group can be obviously improved with the hemodynamic indexes and the related parameters of lung ventilation.