The Regulatory Mechanism Of PI3K/Akt/mTOR Signaling Pathway In Treg/Th17 Immune Imbalance Of Male Offspring Induced By Bisphenol A Exposure In Early Life

Objective:Bisphenol A（BPA）is common EDCs in the environment.It is the main raw material for the production of variety of consumer goods such as baby bottles,food and beverage containers.Under certain conditions,BPA can be exuded from products and enter the human body through food and water.Up to now,there have been many reports on reproductive toxicity,metabolic disorders and neurodevelopmental abnormalities caused by BPA exposure.Studies have shown that low-dose BPA exposure in early development can increase the risk of future reproductive dysfunction,obesity,diabetes and tumors in children.Recent epidemiological studies have found that childhood BPA exposure is positively correlated with IgE levels,increasing the risk of allergic diseases in children.Urinary BPA levels in pregnant women are associated with childhood asthma and allergic disease.Most animal studies support the idea that BPA exposure alters the activity of immune cells,and that the effects of exposure to BPA early in development are long-lasting.Therefore,the immunotoxicity of BPA is concerned.The toxic effect of BPA on mechanism may be related to Estrogen receptors,Estrogen receptors,ERs),aromatic hydrocarbon receptors（Aryl-hydrocarbon receptor,AhR）and peroxidase（pod）growth activated receptor（Peroxisome proliferator-activated receptor,PPAR）and other related signaling pathways.However,studies have shown that the adverse health effects and mechanisms of BPA can vary according to the dose,time,physiological status,age and gender of BPA.Currently,the immunotoxicity mechanism of BPA is still unclear.Therefore,it is of great practical significance to comprehensively explore the immunotoxicity mechanism of BPA in order to find effective control measures.In recent years,the incidence of allergic diseases among children and adolescents in China has been increasing year by year and showing a trend of younger age.One of the major immunological features of allergic diseases is the loss or suppression of Regulatory T cells（Treg）,which leads to the reduction of immune tolerance to allergens,thereby promoting the differentiation of initial CD4+T cells into Helper T cell（Th）2,leading to the rise of IgE and anaphylaxis.Since Mammalian target of rapamycin（mTOR）can directly regulate the proliferation and differentiation of Treg and thus affect its function,this study will take the mTOR signaling pathway as the research core to study the effect of BPA exposure in early life on the differentiation and functional regulation of Treg through the mtor-mediated pathway.MTOR is an important regulator of membrane receptor signal transduction into cells and plays an important role in cell growth,proliferation,metabolism and apoptosis.There are many effector molecules downstream of the mTOR signal transduction molecule phosphodylinositol-3-kinase（PI3K）,and Protein kinase B（Akt）,as an important effector factor downstream,is responsible for the transmission of biological information initiated by PI3K and can act on the downstream mTOR signal molecules.When the body in various growth factors,nutrients and energy signal changes,threonine and serine residues of Akt phosphorylation will happen into the cytoplasm and nucleus,and through the tuberous sclerosis complex activation of mTOR,pushing its downstream Ribosomal protein S6 kinase 1（Ribosomal protein S6kinase 1,S6K1）and eukaryotic translation initiation factor 4 E binding protein 1（EIF4E-binding protein 1,4 E-BP1）phosphorylation,Furthermore,it regulates mRNA translation,protein synthesis and energy metabolism,and plays a key role in cell proliferation,differentiation and growth.Studies have found that mTOR plays a key role in regulating Th immune response,and its mechanism is related to affecting the expression of Foxp3 gene,a tregs-specific marker molecule.Th17 cells are a subgroup of Th cells that produce IL-17 and are involved in the occurrence and development of many inflammatory reactions and autoimmune diseases.Studies have reported that IL-17,as a pro-inflammatory cytokine,induces increased secretion of other inflammatory factors,such as tumor necrosis factor（TNF-α）and IL-6,and up-regulates Th2 immune response,and is involved in the occurrence and development of allergic asthma,rhinitis,specific dermatitis and other allergic diseases.The differentiation and activity of Th17 cells depend on the expression of the transcription factor RORγt.Therefore,the occurrence of allergic diseases in children not only involves Th1/Th2 imbalance,but also Treg/Th17 imbalance plays an important role in the early inflammatory development of allergic diseases.To sum up,this study comprehensively elaborated the regulatory mechanism of PI3K/Akt/mTOR signaling pathway in Treg/Th17 immune imbalance of offspring male mice induced by BPA exposure in early life through the method of combined in vivo animal experiments and in vitro cell experiments.Based on the lowest visible harmful exposure level of BPA in animals（50 g/kg bw/day）,an animal model of low and high BPA exposure in early life was established to study the effects of maternal BPA exposure on the development and function of offspring Trge and Th17 cells,and the role of mTOR signaling pathway in regulating Th immune response.Methods:1.Overall animal experiment:30 pregnant C57 mice were randomly divided into a control group（drinking distilled water solution containing 1%anhydrous ethanol）,a low-dose group（0.2μg/mL BPA solution）,and a high-dose group（2.0μg/mL BPA solution）.10 animals in each group were poisoned with free drinking water on the7th day of gestation.The weight,food intake and water intake of pregnant rats were recorded weekly.During pregnancy,maternal blood circulation of offspring male mice was stained with BPA.From birth to before weaning（21 days after birth）,offspring of male mice were exposed to BPA in their breast milk.Offspring of male mice after weaning,the second phase of infected（i.e.,continuous infected after weaning）,specific process is as follows:move each child male mice（the control group,low dose group and high dose group）is further divided into two groups,namely the control group is divided into drinking distilled water containing 1%ethanol solution group and drink 0.2μg/mL BPA solution group,low dose group is divided into drinking distilled water containing 1%ethanol solution group and drink 0.2μg/mL BPA solution group,high dose group was divided into drinking distilled water containing 1%ethanol solution group and drink 0.2μg/mL solution set of BPA,until the end of a month after weaning.The weight,food intake and water intake of male pups were recorded weekly.Immune induction was conducted before the end of the poisoning.Each mouse was injected with 0.2 mL ovoalbumin（OVA,0.1 mg/mL）by intraperitoneal injection for 1week.After 1 week,0.2 mL OVA was injected again.2.In vitro cell experiments:（1）Peripheral blood mononuclear cells extraction:the rats were anesthetized with ether,the thoracic cavity was opened,the abdominal aorta was dissected,and the abdominal aorta was sampled with medical blood vessels.The collected blood was diluted with PBS in a 1:1 ratio,and then the mixed blood was slowly added into the centrifuge tube containing the lymphocyte separation solution to maintain a clear interface between the blood and the separation solution.The centrifuge was performed at 2000 rpm/min for 20 min,and white cells in the middle layer,namely peripheral blood T lymphocytes,were observed after removal.（2）Stimulation of peripheral blood monocytes:the extracted monocytes were placed in a centrifuge tube and supplemented with D-hanks liquid or PBS to 10mL.250 g,centrifuge for 10 min.Discard the supernatant and add d-hanks liquid or PBS to 10 mL.250 g,centrifuge for 10min.Discard the supernatant and add 1 mL（10%FBS）1640 growth solution to the centrifuge tube to suspend the deposited cells.Take a small amount of cell suspension diluted,counting cells,adjust cell concentration to 1×10⁵/mL,vaccination in cells in a petri dish,add 10μg/mL PHA stimulants,at 37℃,5%CO₂ incubator in 24 h.（3）Poisoning of peripheral blood monocytes:prepare the BPA reserve solution1mmol/L in advance,dilute the BPA reserve solution to the working solution（the concentration is 20,40,80,160,320μmol/L）,remove the culture dish after 24 hours,inoculate the peripheral blood monocytes to the 96-well plate,add the working solution of BPA of different concentrations,and collect the cells for subsequent detection after 30min.Rap reserve solution 1 mmol/L was prepared in advance,and Rap reserve solution was diluted to working solution（concentration 0.2,0.4,0.8,1,2,4,8 nmol/L）.Peripheral blood mononuclear cells were inoculated into 96-well plates,and Rap working fluids with different concentrations were added.Cells were collected for subsequent detection after acting for 30 min.Results:1.During pregnancy（w1-w3）,maternal body weight,food intake and water intake significantly increased with time,and maternal water intake during lactation was significantly higher than that during pregnancy.Body weight,food intake and water intake of offspring male mice exposed to BPA early in life increased over time,but there was no difference between the groups.Serum IFN-γlevels decreased gradually and IL-17 and TNF-αlevels increased gradually in male offspring exposed to BPA in early life.Treg cells gradually decreased and Th17 cells gradually increased in the spleen tissues of male offspring exposed to BPA in the early stage of life.Compared with the control group,groups of 0.2 and 2.0μg/mL showed statistical differences.After weaning;there was no difference in the corresponding indicators between the uninfected group and the infected group.2.Western blot method to detect fault early life exposure to BPA on rat mTOR and mTOR signaling pathway upstream related protein expression levels,according to the results of not infected after ablactation and infected group,compared with control group,0.2,2.0μg/mL of BPA group male young mice spleen cells PI3K,p-Akt（Ser473）,p-Akt（T308）,mTOR and p-mTOR protein expression levels were significantly increased relatively,and the differences were statistically significant;The relative expression levels of TSC1 and TSC2 proteins in the spleen cells of male offspring were significantly decreased in the groups of 0.2 and 2.0μg/mL BPA before and after weaning,and the differences of TSC2 proteins were statistically significant.Foxp3 and t-bet proteins decreased with the increase of the exposure dose in the unexposed and exposed groups after weaning.On the contrary,RORγt and GATA3 proteins increased with the increase of the exposure dose,and the differences were statistically significant.BPA exposure significantly increased the expression levels of ER,TLR4,AhR and NF-κB protein in the spleens of male offspring mice.3.A low dose group of 80μM BPA and a high dose group of 160μM BPA were selected,and there was no difference between the two groups after exposure to 0.8 nM Rap for 30 min.Compared with the control group,the relative expression levels of mTOR,Foxp3,T-bet,RORγt and GATA3 proteins in the 80 and 160μM BPA groups were significantly increased.The relative expression levels of mTOR,Foxp3,T-bet,RORγt and GATA3 proteins in the 0.8 nM Rap group were not significantly changed.Compared with the groups of 80 and 160μM BPA,the relative expression levels of mTOR,RORγt,GATA3 and Foxp3,T-bet proteins in the 0.8 nM Rap intervention group were significantly decreased,and the relative expression levels of Foxp3 and T-bet proteins were significantly increased.Conclusion:1.Exposure to BPA during pregnancy and lactation can cause immune imbalance of Treg/Th17 and cause immune inflammatory responses in male mice of the offspring.There was no statistical difference in the effects of continuous exposure of low-dose BPA and non-exposure of BPA on Treg/Th17 balance after weaning,suggesting that pregnancy and lactation are critical periods for the development of Treg and Th17 cells.2.Early life exposure of BPA activates the PI3K/Akt/mTOR signaling pathway by ER,causing changes in Th1/Th2 and Treg/Th17 cell-specific transcription factors,suggesting that early life exposure of BPA can affect Th cell differentiation.The mechanism is related to the enhanced inflammatory response caused by the activation of AhR and TLR4/NF-B signaling pathway by BPA.3.In vitro cell culture has verified that BPA can affect Th cell differentiation through mTOR mediation,thus resulting in imbalance of Th1/Th2 and Treg/Th17,suggesting that BPA has immunodevelopmental toxic effects.