Regulation And Its Mechanism Of SIRT1 In The Pathogenesis Of Bronchial Asthma

Research BackgroundBronchial asthma(asthma)is the most common chronic airway inflammatory disease.The incidence rate of asthma increased by 50% every 10 years,and it is one of diseases with the most fast growing incidence rate around world.At present,there are about 235 million people with asthma all the world.China has about 20 million asthma patients and it's also a countrywith the highest mortality rate of asthma in the world.Therefore,the molecular mechanism of the occurrence and development of asthma is still a hot topic in basic and clinical research.Airway epithelium constitutes the first important protective barrier on the airway surface.Damage and shedding of airway epithelial cells are important pathologic features of asthma.Airway epithelial damage and dysfunctional led by a variety of factorsare initial factors of asthma;airway immunity,inflammation and remodeling are closely related to airway epithelial cell regulation.Clinical study found that more than 33.3% of asthma is mainly neutrophil inflammation.Th17 has the function of recruiting neutrophils,and is closely related to neutropenia-based chronic airway inflammation.Th17 cell is a new class of CD4~+T cell subset that specifically produces cytokine IL-17 and it can induce Th17 cell production and differentiationby activating specific transcription factor orphan receptor ?t(ROR?t).The expression levels of IL-17 A and IL-17 F in asthmatic patients were positively correlated with the severity of asthma.However,the key factors and molecular mechanisms that regulate the differentiation of CD4~+T cells into Th17 cells in asthma are unknown.Asthma is a chronic inflammatory disease associated with airway hyperresponsiveness,and the expression of these inflammatory proteins is closely related to the level of protein acetylation.Sirtuin 1(SIRT1)belongs to class ? deacetylase Sirtuin gene family members,it is an important intracellular deacetylase,involved in the body's various physiological and pathological processesincluding cell cycle,transcriptional regulation,DNA damage repair and aging.In a variety of tissues such as lung tissue,blood vessels,fat,fibroblasts,cerebrovascular disease,SIRT1 shows significant anti-inflammatory effect.SIRT1 also plays an important role in controlling the expression of various inflammatory genes in various types of chronic inflammation.However,does SIRT1 play a role in asthma airway inflammation? In recent years,it has been found that SIRT1 also plays an important role in immune regulation.SIRT1 regulates macrophage function,participates in T cell proliferation and differentiation,and plays a role in maintaining T cell tolerance.SIRT1 may also be mediated by CD38 pathway to regulate B cell function.A large number of literature reported that the occurrence of a variety of diseases are related to SIRT1 activity level.SIRT1 dysfunction can increase the incidence of inflammation and autoimmune diseases.At present,the immune regulation of SIRT1 in the pathogenesis of asthma and the specific mechanism are rarely reported.Based on the purpose,this study was to observe the expression of serum SIRT1 and IL-17 in asthmatic patients and to analyze the correlation between the clinical features and severity of asthma,especially of neutrophilicasthma.Further study about SIRT1 was done in vivo and vitro,andto explore the role and mechanism of SIRT1 in asthmatic airway epithelial cell injury and role in the differentiation of CD4~+T cells into Th17 cells.This topic is described in the following two parts.The first part: The relationship between expression of SIRT1 and asthma and and its correlation to the clinical characteristics of patients.Objective: To evaluate the clinical significance of SIRT1 in asthmatic patients by detecting the levels of SIRT1 and IL-17 in patients with asthma and evaluating the severity of asthma.Methods:The levels of serum SIRT1,IL-17,serum total Ig E,induced sputumgranulocytecount,lung function,and so on were measured in 93 patients(asthma group)and 104 healthy person(control group),and the correlation between serum SIRT1,IL-17 and serum total Ig E,induced sputum granulocyte count,lung function were evaluated.Patients with asthma were also assessed for the severity of the disease and analyzed.Results: The levels of SIRT1 in asthmatic group and control group were 2.88±0.41 ng/m L and 1.03±0.14 ng/m L respectively.The level of serum SIRT1 in asthmatic group was significantly higher than that in control group(P<0.01).The correlation analysis showed that serum SIRT1 was positively correlated with induced sputum neutrophil count,but was negatively correlated with pulmonary function FEV1 / FVC(%)(r =-0.290,P <0.01).Serum SIRT1 levels in asthma patients may be correlated to the severity of the disease,but not at the statistical level(r = 0.195,P = 0.056).The levels of serum IL-17 in asthmatic group and control group were 78.12 ± 19.23 pg / m L and 33.07 ± 11.74 pg / m L,respectively.The level of serum IL-17 in asthma group was significantly higher than that in control group(P <0.0l).The correlation analysis showed that serum IL-17 was negatively correlated to pulmonary function FEV1(% predicted)(r =-0.568,P<0.01),and positively correlated with the severity of disease(F = 24.62,P <0.01).And the level of serum SIRT1 was positively correlatedto that of IL-17 in asthma patients.In 22 patients who finish the induced sputum,13 patients(59.10%)were mainly neutrophils phenotype,and patients in the group SIRT1 has no relation to serum Ig E,Eosnophils count,but it was positively correlatedto IL-17(r=0.284,P=0.009).Conclusions: SIRT1 is significantly elevated in sera of asthmatic patients,and serum SIRT1 is associated with pulmonary function in asthmatic patients,patients who finished induced sputumwere mainly neutrophils phenotype(59.10%),and which was positively correlated with serum SIRT1;but in neutrophilic asthma patients SIRT1 was positively correlatedto IL-17,and was not correlatedto bloodeosnophils and serum Ig E,as suggest that SIRT1 may possible play an important role in the pathogenesis of asthma especially neutrophilicasthma.The second part :Regulation and its mechanism of SIRT1 in the pathogenesis of bronchial asthmaObjective:To investigate the role of SIRT1 in asthmatic airway epithelial cell injury and the differentiation of CD4~+T cells into Th17 cells by establishing cell model of airway epithelium injury and neutrophil asthmatic mouse model.Methods:Neutrophil asthmatic mice model was constructed.Theproportion of neutrophils in the bronchoalveolar lavage fluid were measured and the neutrophil infiltration was detected by HE staining.Immunohistochemistry,RT-PCR and immunoblotting were used to detect the expression level of SIRT1 in the peripheral Blood and airway epithelium.The expression of SIRT1 in HBE was knockout by si RNA,and the HBE were stimulatedby OVA and LPS.Real-time RT-PCR was used to detect neutrophil chemokines and inflammatory mediators.Under the condition of Th1,Th2 and Th17 polarized state,the expression of Th1,Th2 and Th17 was respectivelydetected by flow cytometry in Spleen-derived CD4~+T lymphocytesseparated from mice in which sirt1 is overexpressed byadenovirus infection.And the mice were further treated with SIRT1 recombinantprotein,to detect the differentiation of Th17 cells.Results: The neutrophil asthma model was successfully constructed.The number of alveolar lavage fluid(BALF)cells in asthma mice was significantly increased and the proportion of neutrophils was significantly increased.The histopathological changes of neutrophils were further revealed the increase of neutrephil infiltration.The expression of SIRT1 in peripheral blood was significantly higher than that in control mice(P<0.05).QRT-PCR and immunoblotting showed that the expression of SIRT1 in airway epithelium of asthma mice was significantly decreased(P<0.01,P<0.05).By si RNA and knockout of SIRT1 expression,and then to give OVA and LPS to stimulate HBE cell,the levels of neutrophil chemokines CXCL1,CXCL2 and IL-8 were significantly increased(P <0.01).The proportion of IL-17 A cells in SIRT1 overexpression was significantly higher than that in the control group under Th17 polarized state,while under Th1 and Th2 polarized state they were no significant difference between SIRT1 overexpression and control group(P> 0.05).SIRT1 recombinant protein was used to induce CD4~+T lymphocytes from spleen in mice.SIRT1 was used to promote the differentiation of CD4~+T lymphocytes into Th17 cells.Conclusions: SIRT1 may be involved in the pathogenesis of asthma from the following two aspects:SIRT1 plays a protective role in the pathogenesis of airway epithelium injury;SIRT1 may play anactive role in the process of differentiation of CD4~+T cells into Th17 direction.