Effect Of SEB Exposure To Pregnant Rats During Pregnancy On The Splenic Treg Of Their Offspring And Its Mechanism

In my country,the annual birth defect rate of newborns is about 5.6%.One main cause of this birth defect is owing to the microbial infection during pregnancy.SEB(Staphylococcal enterotoxin B)is the most common and important category in the superantigen family and belongs to a toxin.Preliminary research by the supervisor’s research group found that perinatal SEB administration could largely affect the composition of central CD4/CD8 T cells in the offspring.In addition,the SEB administration induced a certain increased amount of CD4+CD25+FoxP3+T cells(Treg cells)in the offspring’s thymus.It also had a significant impact on FoxP3 and cytokines in the center of the adult offspring.However,there is no research on the effect of SEB administration during pregnancy on splenic Treg cells in the offspring of pregnant rat.In this experiment,the pregnant rats on the 16th day of pregnancy were injected with 0.3ml 50μg/ml SEB through the tail vein.After delivery,the spleens were obtained from the newborn offspring and the adult offspring that neonates were allowed to be raised until three months old.Then,the number of Treg cells in the spleen was analyzed by flow cytometry,the expression level of FoxP3 mRNA was detected by real-time PCR while its protein expression level was detected by Western blotting,the inhibitory function of Treg was determined by immunosuppressive method,and the methylation status of FoxP3 was detected by DNA immunoprecipitation qPCR.These experiments aimed to clarify the influence of spleen treg cells and its mechanism when pregnant rats were given SEB during pregnancy.The research results will provide extremely valuable theoretical and experimental basis for exploring the influence of exposure to SEB during pregnancy on the immune regulation of offspring,and good prenatal and postnatal care.Objective:To investigate the effect of perinatal SEB exposure on pregnant rat on the splenic Treg cells of their offspring and its mechanismMethods:(1)Establish the model of SD rats during pregnancy and inject SEB to pregnant rats;(2)Divide the newborn or the adult offspring into an experimental group and a control group;(3)Aseptically take out the spleens from the neonatal offspring on the life of 1,3,and 5 days,or the adult offspring that part of neonates allow to be raised to adulthood,and prepare a single cell suspension by grinding,then separate the mononuclear cells with the lymphocyte separation solution,and then use the fluorescent antibody CD3-FITC,CD4-APC,CD25-PE were used for surface staining,and FoxP3-percy5.5 antibody was used for intracellular staining.Flow cytometry was used to detect the proportion of Treg cells in the spleen of the experimental group and the control group,and calculate their numbers;(4)Obtain the spleens of the newborn offspring and the adult offspring after birth,extract total RNA from the spleen tissue,and detect the level of FoxP3 mRNA in the spleen by qPCR;(5)Obtain the spleens of newborn or the adult offspring,and extract the total protein in the sample,then use western blotting to quantitatively detect the expression level of FoxP3 protein in the spleen;(6)Collect the spleens from the newborn or adult offspring in both the experimental group and the control group,then use immunosuppression test to evaluate the ability of Treg suppression.(7)Obtain the spleens of newborn or adult offspring in experimental group and control group aseptically,and detect the methylation level of FoxP3 DNA by MeDIP-qPCR.Results:(1)The effect of perinatal SEB exposure to pregnant rats on the absolute number of Treg cells in the spleen of newborn offspring is that this could significantly increase the absolute number of Treg cells compared with the control group;(2)Perinatal SEB exposure to pregnant rats significantly increased the expression levels of FoxP3 mRNA and protein in the spleen of the five-day offspring;(3)The pregnant rats in the experimental group were injected with SEB,and some of the newborn mice continued to be raised to adulthood.The results showed that the absolute number of Treg cells in the offspring spleen in SEB group was significantly higher than that in PBS group;(4)The effect of perinatal SEB exposure to pregnant rats notedly increased the mRNA and protein levels of FoxP3 in the spleen of the experimental offspring compared with the competition group;(5)Perinatal SEB exposure to pregnant rats decreased significantly the level of FoxP3 DNA methylation in the spleen of the newborn or the adult offspring compared with the PBS group;(6)Compared with the PBS group,perinatal SEB exposure to pregnant rats remarkably enhanced the inhibitory ability of Treg cells in the spleen of the newborn or adult offspringConclusion:(1)Exposure of pregnant mice to SEB during perinatal period can significantly increase the absolute number of Treg cells and the expression levels of FoxP3 mRNA and protein in the spleen of newborn offspring;(2)Exposure to SEB in pregnant mice during perinatal period can significantly increase The absolute number of Treg cells in the spleen of adult offspring,the expression level of FoxP3 mRNA and protein,that is,the effects of SEB exposure during the perinatal period on Treg cells and FoxP3 can be retained from the newborn offspring to the adult offspring to form an imprinting effect;(3)Exposure of perinatal pregnant mice to SEB significantly enhanced the immunosuppressive function of Treg cells in the spleen of offspring;(4)Exposure of pregnant mice to SEB during the perinatal period reduced the methylation level of FoxP3 DNA in the offspring’s spleen and exerted an effect on Treg.Regulation of cells.