Study On The Effect Of MTOR And AhR In Impact Of Bisphenol A Exposure During Pregnancy And Lactation On Treg/Th17 Balance And Autophagy In Offspring Female Mice

Objective: Bisphenol A(BPA)exposure in the perinatal period can pass through the placenta and breastfeeding into the offspring,and the exposure to BPA in early life is more harmful in the body,which needs more attention.In recent years,it has been found that Regulatory T cell(Treg)/ Helper T cell 17(Th17)imbalance plays an important role in the occurrence of allergic diseases and perinatal BPA exposure can affect the levels of Treg and Th17 cells in the spleen of offspring,but the relevant mechanism remains unclear.Studies have found that both Mammalian target of rapamycin(mTOR)and Aryl hydrocarbon receptor(Ah R)can regulate the differentiation of Treg and Th17 cells.Moreover,the activation of mTOR and Ah R proteins can inhibit the autophagy process in cells and affect the homeostasis of cells.Some scholars believe that BPA exposure can lead to abnormal autophagy,but relevant reports are mostly seen in tumor studies,and there are few reports on the role of autophagy in the effect of BPA exposure on immune function.To study the effects of pregnancy and lactation different doses of BPA exposure in female offspring on Treg/Th17 balance,the possible effects of spleen and thymus tissues mTOR,Ah R and autophagy level in Treg/Th17 imbalances induced by BPA exposure.Methods: Twenty-four pregnant C57 mices were put into control group(CON,n=8),lowdose BPA group(LBPA,0.2 μg/m L BPA,n=8)and high-dose BPA group(HBPA,2.0μg/m L BPA,n=8)at random.They were exposure by drinking water from Gestational Day6 to postpartum Day 21(End of lactation).After lactation,the progeny females(randomly selected one offspring from each litter and form different progeny groups)continued to drink normal water for one month.Feed and water consumptions were measured at 8 am every day.Weight change was measured weekly based on the first day of delivery.At 21-29 days after lactation,0.2 m L 0.1mg/m L Ovalbumin was intraperitoneally injected,30 days after lactation,the progeny female mice were anesthetized with ether and sacrificed by spinal dislocation.The spleens and thymus were taken out,weighed and recorded.The levels of Treg and Th17 cells in some fresh spleens were detected by flow cytometry,and the rest of the spleens and thymus were stored in-80℃ deep freeze refrigerator.The forkhead box P3(Foxp3),retinoic acid-associated orphan receptors γt(RORγt),mTOR,pmTOR,Ah R,p62 and microtubule-associated protein 1 light chain 3(LC3)protein expression levels were detected by Western Blot.Results: 1.Each offspring female mice weight has no obvious difference(P > 0.05),in addition to the 4 weeks of age,food and water intake of female offspring mice in HBPA group were significantly lower than the other two groups(P < 0.05),no obvious difference was found in feed and water consumptions among each group(P > 0.05).There were no statistical differences in weight and coefficient of spleen and thymus of offspring female mice among each group(P > 0.05).2.With the increase of BPA exposure dose,the number of Th17 cells in spleen of offspring female mice significantly increased,while the number of Treg cells decreased significantly(P < 0.05).The expression of ROR γt protein in spleen rose,and the expression of Foxp3 protein dropped along with the rise of BPA exposure dose,and the differences between the three groups were statistically significant(P < 0.05).3.With the rise of BPA exposure dose,the expression of mTOR and p-mTOR protein in spleen of offspring female mice were increased,and the differences between each group had statistical significant(P < 0.05);the expression of Ah R protein in HBPA group was significantly higher than the other groups(P < 0.05);the expression level of LC3 protein decreased with the increase of BPA exposure dose and there was significant difference between the HBPA group and the CON group(P < 0.05);the expression of p62 protein in the HBPA group was significantly higher than that in the other groups(P < 0.05),and there was no significant difference between the LBPA group and the CON group(P > 0.05).4.In female mice thymus,the expression of RORγt protein in LBPA group is significantly higher than the other group(P < 0.05),the expression of RORγt protein between HBPA group and the CON group has no significant difference(P > 0.05);compared with the CON group,the expression of Foxp3 protein was significantly decreased in the dose groups(P< 0.05),but there was no significant difference in the expression of Foxp3 protein between the dose groups(P > 0.05).5.The expression of mTOR and p-mTOR protein in thymus of offspring female mice in LBPA group were significantly higher than those in CON group and HBPA group(P < 0.05),but there was no significant difference between the HBPA group and the CON group(P > 0.05);compared with the CON group,there was no significant change in the expression of p62 protein in the LBPA group(P > 0.05),but the expression of p62 protein in HBPA group was significantly increased(P < 0.05),and the difference between the two dose groups was also statistically significant(P < 0.05);the expression of LC3 protein was firstly decreased and then increased with the increase of BPA exposure dose,but there was no significant difference in LC3 protein expression among each group(P > 0.05).Conclusions: BPA exposure during pregnancy and lactation can induce Treg/Th17 imbalance and autophagy inhibition in spleen tissue of offspring female mice,which may be related to that BPA actives mTOR and Ah R signals in spleen tissue and then downregulates the expression of Foxp3 and LC3 protein,and up-regulates the expression of RORγt and p62 protein.Low BPA can also activate mTOR signal in thymus tissue,resulting in decreased expression of Foxp3 protein and increased expression of RORγt and p62 protein.