Study On The Role And Mechanism Of MTOR-Mediated Autophagy Abnormality In Aggravating Lung Inflammatory Response Of Asthmatic Mice Exposed To Bisphenol A

Objective:Asthma is a chronic heterogeneous disease,which is often characterized by chronic airway inflammation,which further causes airway stenosis,affects lung ventilation,and leads to patients with breathing difficulties.The causes of asthma are complex.In addition to genetic susceptibility,environmental factors are an important reason for the increase in the incidence of asthma in recent decades.In recent years,epidemiological studies have reported that exposure to environmental endocrine disruptors-Bisphenol A(BPA)may be related to the increased risk of childhood asthma.For example,increased maternal urinary BPA concentration is associated with an increased risk of asthma symptoms and respiratory infections in children.Urinary BPA levels were associated with an increased risk of asthma in children aged 3,5 and 7 years.However,there are few reports on the effect of BPA exposure on lung function,and the specific mechanism of BPA exposure affecting the occurrence and development of asthma is still unclear.BPA is one of the environmental endocrine disruptors with estrogen activity,which is the main raw material in the production of baby bottles,food and beverage containers and other consumer goods.Under certain conditions,BPA can be seeping out of products and enter the human body through food and water.When BPA enters the body,it is mainly formed by the action of hepatic uridine diphosphate glucuronidase and then excreted through the kidneys in the urine.Therefore,the level of bound and/or free forms of BPA in urine may reflect the body's exposure level.The National Health and Nutrition Examination Survey from 2013 to 2016 found that 97.5%of children between the ages of6 and 19 had detected BPA in their urine.In 2012,84.9%of children aged 8-15 in Shanghai were detected with BPA in urine,with a geometric mean of 0.45 ng/m L.Therefore,the study on the adverse effects of BPA exposure on the body has attracted extensive attention.However,in recent years,studies on BPA mainly focus on reproductive function injury,nerve injury and metabolic disorders,and there are relatively few reports on the effect of BPA on immune function and the association of related allergic diseases.The pathogenesis of asthma is complex and still unclear,but the main pathological features are often accompanied by persistent chronic airway inflammation and abnormal immune function.Nuclear factor?B(NF-?B)signaling pathway is the main regulator of inflammation.Immune dysfunction was mainly manifested as T helper cell 1/2(Th1/Th2)and regulatory T cells(Treg)/Th17 cell imbalance.In recent years,studies have shown that epigenetic mechanisms play an important role in the pathogenesis of childhood asthma caused by environmental pollutants.Therefore,in recent years,the role and mechanism of environmental factors in the high incidence of childhood asthma has become a hot topic.NF-?B protein is an important transcription factor that regulates inflammatory response in vivo,and is also involved in innate and adaptive immune apoptosis,proliferative stress response and cancer progression.It has been suggested that the hepatotoxicity induced by BPA in male rats is induced by the activation of NF-?B through the increase of pro-inflammatory factors.It has not been reported whether BPA exposure can aggravate lung inflammation in asthmatic mice by activating NF-?B signaling pathway.The occurrence of asthma is often accompanied by increased serum Immunoglobulin E(IgE)levels.The decrease of Treg in children with allergic disease is linearly related to the increase of serum IgE level.The study showed that exposure to BPA in both prenatal and adult mice reduced the number of Treg cells.It suggested that BPA exposure may increase the risk of allergic diseases,which may be related to the decrease of Treg level and the increase of Th2 level.However,the effect of BPA on T cell differentiation and proliferation may vary with the dose pathway time and physiological status of BPA exposure.Therefore,the immunotoxic effect and mechanism of BPA still need to be further clarified.In recent years,it has been found that autophagy is correlated with inflammatory response.It has been suggested that cigarette smoke can activate NF-?B by inducing autophagy enhancement of lung macrophages and thus induce airway inflammation.However,the correlation between autophagy and inflammation is different in different diseases.Autophagy activity is mainly regulated by the mammalian target of rapamycin(mTOR),the most representative of which are phosphatidylinositol-3-kinase(PI3K)-protein kinase B(Akt)and AMP-activated protein kinase(AMPK).BPA exposure can lead to decreased memory function in rats,which is related to the activation of autophagy and the enhancement of inflammation through the up-regulation of AMPK and down-regulation of mTOR.At present,there are few studies on the effects of BPA exposure on lung tissue inflammation and autophagy,so whether the increased risk of asthma caused by BPA exposure is related to the abnormal autophagy caused by BPA and thus the intensification of pulmonary inflammatory response is still unclear.Therefore,it is of great significance to study the correlation between autophagy and inflammation and the role of autophagy in aggravating the abnormal lung function in asthmatic mice exposed to BPA to further clarify the cause and mechanism of the high incidence of asthma.In addition to the regulation of autophagy,mTOR is also a negative regulator of the expression of Foxp3,a Tregs-specific marker molecule.In CD4~+T cells,inhibition of PI3K or mTORC1 increases Treg cell differentiation.Children with asthma are often accompanied by increased serum IgE and decreased Treg cells,suggesting that asthma is accompanied by increased pulmonary inflammatory response and systemic immune dysfunction.Thus,while BPA exposure affects lung inflammation in asthmatic mice,it may affect systemic immune function of the whole body.At present,there is no report on the comprehensive and systematic study of the effects of BPA exposure on the lung function and immune function of asthma animal models.In recent years,the mechanism of DNA methylation changes in the development of immunological diseases has attracted much attention.Research by Nadeau et al.found that air pollution can induce hypermethylation of Foxp3 gene locus,leading to differentiation and impaired function of Treg cells,thus increasing the risk of childhood asthma and the severity of asthma.However,the study on the epigenetic mechanism of allergic diseases is still in the initial stage,and there are few reports on the mechanism of gene methylation in the immunotoxicity caused by BPA exposure.Therefore,it is of great significance to explore the regulatory mechanism of DNA methylation in the abnormal Th immune response caused by BPA exposure to reveal the causes and molecular mechanisms of the high incidence of allergic diseases in children in recent years.In summary,we proposed the following hypotheses:1.Exposure to BPA in developing mice can aggravate OVA-induced pulmonary inflammatory response,and the mechanism is related to the abnormal autophagy caused by BPA through mTOR-mediated signaling pathway,thus aggravating inflammatory response.2.BPA can affect the differentiation and proliferation of spleen Treg cells through mTOR signaling pathway or epigenetic mechanisms,and reduce the level of Treg,leading to imbalance of Treg/Th17and Th1/Th2,causing systemic immune dysfunction,and inducing the occurrence and development of asthma.Methods:1.Overall animal experiment:Forty 4-week-old healthy female C57BL/6 mice were fed for 7 days and randomly divided into 5 groups:Control group(containing 1%ethanol solution),OVA group,0.1?g/m L BPA+OVA(L-BPA+OVA)group,0.2?g/m L BPA+OVA(M-BPA+OVA)group and 0.4?g/m L BPA+OVA(H-BPA+OVA)group.Eight mice in each group were exposed to BPA by drinking water for two months.Food intake and water intake were recorded on a weekly basis.OVA group of the first month in the drinking water is consistent with the control group.L-BPA+OVA group,M-BPA+OVA group and H-BPA+OVA group were exposed to corresponding BPA concentrations in the first month of the experiment.OVA sensitization and atomization tests were carried out one month after exposure.Airway hyperresponsiveness(AHR)experiment was conducted on the day before the mice were executed.Bronchoalveolar lavage fluid(BALF)test was performed on the left lung when the mice were collected.The right lobe was fixed,and the other lobes were stored in a refrigerator at-80?for follow-up indicators.At the same time,the weights of liver and spleen were weighed and recorded,and the corresponding organ coefficients were calculated.The amount of CD4~+CD25~+Foxp3(Treg)in spleen tissue was determined by flow cytometry.The airway resistance of mice was measured by noninvasive animal airway detection system.The total number of white blood cells in BALF of mice and the white blood cells were counted by blood cell counting apparatus.IL-4,IL-5,IL-6 and TNF-?in BALF and specific OVA-IgE levels in serum were measured by ELISA kit.The histopathological changes of lung were observed by HE staining and PAS staining.The expression of LC3 protein in mice lung was detected by immunohistochemistry.The expression levels of AMPK and PI3K/Akt signaling pathways,autophagy-related proteins and genes,NF-?B signaling pathway and immune-related proteins,and methyltransferase gene in mice were detected by Western blot and RT-PCR.The methylation level of Foxp3 gene in mice spleen was detected by bisulfite sequencing.2.In vitro cell experiment:Mouse leukemia monocytes/macrophages RAW264.7cells were cultured in DEME medium(containing 10 m M HEPES 10%heat inactivated fetal bovine serum 100?g/m L streptomycin and 100 U/m L penicillin).The MTS method was used to determine the dose of BPA(0.25,0.5 and 1?M)and CQ(7.5?M)for cell experiments.In all experiments,cells were treated with different concentrations(0.25,0.5and 1?M)of BPA,CQ(7.5?M)and BPA(0.25?M)±CQ(7.5?M)in the medium for 24h,respectively.At the end of the experiment,the effects of BPA exposure on mitochondrial endoplasmic reticulum and the formation of autophagosomes were observed by transmission electron microscopy.The supernatant was collected to detect the levels of IL-6 and TNF-?.Cells were collected to detect protein and gene expression levels in autophagy and inflammatory signaling pathways.Results:1.Overall animal experiments:(1)Physiological conditions of mice:compared with the control group,the body weight of mice in the OVA group was not significantly different,the amount of food intake and water intake were significantly reduced,and the coefficients of lung and spleen were significantly increased;Compared with the OVA group,there was no significant difference in body weight and organ coefficient of each dose of BPA group,while food intake increased and water intake decreased.(2)Successful establishment of a mouse model of asthma induced by OVA:Clinical phenotypes of asthma appeared in mice after sensitization.Compared with control group,the specific airway resistance(s Raw)value of the OVA group was significantly increased,the serum IgE level was significantly increased,and the total number of leukocytes,eosinophils,neutrophils,monocyte and lymphocyte and IL-4,IL-5,IL-6 and TNF-?levels in BALF were significantly increased.The number of eosinophils and lymphocytes in the airway connective tissue was significantly increased.(3)BPA exposure can aggravate lung ventilation resistance and inflammatory response in asthmatic mice:Compared with the OVA group,the s Raw value,serum IgE level,total number of white blood cells,eosinophils,neutrophils,monocytes and lymphocytes,as well as the levels of IL-4,IL-5,IL-6 and TNF-?in BPA+OVA group were significantly increased.(4)The effects of BPA exposure on the expression of inflammation-related proteins in lung and spleen of asthmatic mice:Compared with the control group,the expression of TLR4 and NF-?B protein in lung and spleen of mice in OVA group was significantly increased,and the expression of I?B?was significantly decreased.Compared with the OVA group,the expression of TLR4 and NF-?B protein was significantly increased in the BPA+OVA group,while the expression of I?B?was significantly decreased.(5)The effects of BPA exposure on the expression of autophagy-related proteins and genes regulated by AMPK and PI3K/Akt signaling pathways:Compared with the control group,the protein expression levels of TSC1,TSC2,AMPK,p-ULK1,Beclin1 and LC3 and the gene expression levels of Prkaa2,Ulk1,Becn1 and Map1lc3b in lung tissues of mice in the OVA group were significantly increased.The expression levels of p-PI3K,p-Akt(Ser473 and Thr308),p-mTOR and p62 proteins and Pik3r1,Akt1,Mtor and p62/Sqstm1 genes were significantly decreased.Compared with the OVA group,the protein expression of p-ULK1 decreased in the H-BPA+OVA group,the protein expression of TSC1,TSC2,AMPK,Beclin1 and LC3and the gene expression of Prkaa2,Ulk1,Becn1 and Map1lc3b were significantly up-regulated in the L-BPA(or M-BPA)+OVA group.The expression levels of p-PI3K,p-Akt(Ser473 and Thr308),p-mTOR and p62 proteins and Pik3r1,Akt1,Mtor and p62/Sqstm1genes were significantly decreased.(6)The effect of BPA exposure on the regulation of T cell differentiation by mTOR-mediated signaling pathway in spleen of asthmatic mice:Compared with the control group,the proportion of Treg cells in spleen of mice in OVA group was significantly decreased,the protein levels of Fox P3,T-bet and p-AMPK were significantly down-regulated,and the protein levels of ROR?T,GATA3,p-PI3K,p-Akt(Ser473 and Thr308),p-mTOR and p70S6K were significantly up-regulated.Compared with the OVA group,the proportion of Treg cells was significantly decreased in the BPA+OVA group,the protein levels of GATA3,ROR?T,p-PI3K,p-Akt(Ser473 and Thr308),p-mTOR and p70S6K were significantly increased,and the protein levels of T-bet,Fox P3 and p-AMPK were significantly down-regulated.(7)The effect of BPA exposure on the methylation level of Treg gene in spleen tissue of asthmatic mice:Compared with the control group,the gene expression levels of methylation transferases Dnmt1,Dnmt3a and Dnmt3b in spleen of mice in OVA group were significantly increased,and Foxp3 methylation level was up-regulated.Compared with the OVA group,the gene expression levels of Dnmt1,Dnmt3a and Dnmt3b in BPA+OVA groups were significantly increased.However,only the L-BPA+OVA and M-BPA+OVA groups significantly increased the total methylation level of Treg gene.In addition,methylation rates of Cp G at 143 and 159 sites in Foxp3 were significantly increased in the M-BPA+OVA group,and H-BPA+OVA group had no significant effect on the methylation level of Treg gene,the reason for which needs further study.2.In vitro cell experiments:(1)Effects of BPA exposure on inflammatory and autophagy-related proteins or genes and corresponding signaling pathways in RAW264.7cells:Compared with the control group,the levels of TNF-?and IL-6 in RAW264.7 cells increased with the increase of BPA exposure dose.The protein expression of TLR4,NF-?B,LC3,Beclin1 and p-ULK1 was significantly up-regulated,while the protein expression of I?B?,p62 and p-mTOR was down-regulated.Map1lc3b,Becn1,Ulk1,Tlr4 and Nfkb1gene expression levels were significantly up-regulated,while p62/Sqstm1,Nfkbia and Mtor gene expression levels were gradually down-regulated.In the 0.25?M BPA treatment group,the ultrastructure of cells was significantly changed,and irregular organelles were damaged and the nuclear membrane was incomplete,and bilayer vesicles were observed.(2)Effects of BPA on the expression levels of inflammation and autophagy related proteins and genes in RAW264.7 cells after using autophagy inhibitor Chloroquine(CQ):Compared with BPA group,TNF-?and IL-6 levels were significantly down-regulated in BPA+CQ group.The number of autophagosomes increased.The protein expression levels of TLR4,NF-?B,Beclin1 and p-ULK1 were significantly decreased,while the expression levels of p-mTOR,I?B?,p62 and LC3 were significantly up-regulated.The expression levels of Mtor,Map1lc3b,p62/Sqstm1 and Nfkbia were up-regulated,and the expression levels of Becn1,Ulk1,Tlr4 and Nfkb1 were gradually down-regulated.Conclusion:1.BPA exposure can aggravate lung inflammation in asthmatic mice,and the mechanism is related to the autophagy activation by BPA down-regulation of PI3K/Akt signaling pathway and up-regulation of AMPK protein expression.2.BPA exposure increased the expression levels of inflammatory cytokines,inflammatory proteins and autophagy related proteins in RAW264.7 cells.After autophagy was inhibited,the inflammatory response was reduced.We further verified that the mechanism of BPA exposure aggravating the pneumonia response in asthmatic mice was related to the activation of autophagy.3.BPA exposure can increase serum IgE and decrease the proportion of Treg cells in spleen of asthmatic mice.The mechanism may be related to the activation of mTOR mediated signaling pathway and epigenetic abnormalities.Therefore,BPA can affect the occurrence and development of allergic diseases through multiple pathways.