Effects Of Exposure To SEB In Pregnant Rats On Notch Signaling Pathway In Thymus And Spleen Of The Offspring

BackgroundAccording to the"Notice on printing and distributing the national birth defect comprehensive prevention and control plan"issued newly by the National Health Commission,the annual incidence of birth defects among newborns is about 5.6%in China.Birth defects can be caused by genetic or environmental factors,or maybe by their interaction.A large part of the reasons of birth defects are caused by microbial infection during pregnancy.Staphylococcus aureus(S.aureus)as one of the most common pathogens causes not only bacterial infectious diseases,but also adverse pregnancy outcomes and abnormal fetal development in pregnant women.Staphylococcal enterotoxin B(SEB)is one of the important pathogenic substances produced by S.aureus.Notch signaling pathway is a high evolutionally-conserved signaling pathway that regulates a variety of biological processes including the immune system,which has become a focus in the fields of immunology,developmental biology,cellular biology and so on.In the immune system,Notch signaling pathway is one of the major signaling pathways to determine the development of immune cells.Notch signaling pathway regulates host immune function by regulating the development,differentiation and function of T cells,and plays an important role in the occurrence of various diseases and the maintenance of body homeostasis.The previous study by my supervisor’s research group found that exposure of pregnant rat to SEB can significantly affect the number and function of central and peripheral T cells in the offspring.However,it is unknown at home and abroad whether the effects of cellular immunity by prenatal SEB exposure have related with the changes of Notch signaling pathway.In present study,SEB was injected into pregnant rat during gestation,and the effects of SEB exposure on Notch signaling pathway in thymus and spleen of the offspring were studied by the methods of immunofluorescence,real-time PCR,western blot,and immunomagnetic bead cell separation.We hope this study provide an experimental basis and theoretical basis for better prenatal and postnatal care as well as fetal-origin diseases.ObjectiveTo study the effects of SEB injection to pregnant rat during gestation on Notch signaling pathways in thymus and spleen of the offspring.Methods1.Establishment of animal model and groupsAdult male and female SD rats(about 100 females and 40 males)mated in a 1:1ratio at about 18:30 every day.The female rats were considered to have been pregnant when plus was found in the next day,and this day was recorded as the first day of pregnancy(GD1).In GD16,pregnant rats were randomly divided into two groups.The rats in experimental SEB group was injected with 0.3 ml 50μg/ml SEB through the tail vein,and the control group with PBS.Then these pregnant rats were fed normally to let them natural delivery(a pregnant rat born 8 to 12 litters).One part of neonates was chosen for the following experimental studies and another was allowed to grow into adulthood(three months)for the corresponding studies.2.Preparation of cell suspension and magnetic bead sorting of spleen CD4~+T cells and CD8~+T cells2.1 Preparation of cell suspensionAfter rats of the neonatal and adult offspring in PBS and SEB groups were sacrificed under anesthesia and their abdomen was dissected under aseptic operation,the thymus and the spleen were removed and soaked in sterile PBS.The splenic or thymic tissues were gently ground via a 200-mesh filter and cell suspensions were collected.Then the same volume of lymphocyte separation liquid was added to cell suspensions.Lastly,the lymphocyte suspensions of both thymus and spleen were obtained by density gradient centrifugation.2.2 Sorting of spleen CD4~+T cells and CD8~+T cellsAfter being counted via abalone counting plates,10~7spleen lymphocytes transferred to centrifuge tubes were incubated and labelled with 20μl CD4 microbeads or CD8 microbeads and 80μl running buffer for 15 minutes in the dark.Then the labelled cells with microbeads were washed once with buffer and were re-suspended on the running buffer,the positive sorting was performed on sorter machine to obtain the CD4~+T cells or the CD8~+T cells.3.Effects of SEB injection to pregnant rat during gestation on Notch signaling pathway in the neonatal offspring3.1 Effect of SEB injection to pregnant rat during gestation on m RNA levels of Notch ligands in both thymus and spleen of the neonatal offspringPregnant rat during gestation was injected with SEB and was delivered naturally.On the 14th day of neonatal offspring in life,both thymus and spleen tissues were sterilely acquired from neonatal offspring in PBS and SEB groups,respectively.Their total RNA was extracted and was transcribed reversely into c DNA.The m RNA levels of Notch ligand DLL1,DLL3,DLL4,Jagged1 and Jagged2 were quantitatively detected by real-time PCR.3.2 Effect of SEB injection to pregnant rat during gestation on protein levels of Notch ligands in both thymus and spleen of the neonatal offspringBoth thymus and spleen tissues were sterilely acquired from neonatal offspring in PBS and SEB groups on the 14th day of neonatal offspring in life,respectively.Their total protein was extracted and the expression levels of Notch ligand DLL1,DLL3,DLL4,Jagged1 and Jagged2 were quantitatively determined by western blot.3.3 Effects of SEB injection to pregnant rat during gestation on the m RNA and protein levels of Notch receptor in thymic and splenic T cells of the neonataloffspringOn the 14th day of neonatal offspring in life,the aseptically obtained thymus and spleen tissues in PBS and SEB groups were made into single-cell suspension which were isolated to acquire their lymphocyte suspension with lymphocyte isolation solution.Single positive CD4~+T cells were sorted from splenic lymphocyte suspension by magnetic bead sorter.The total RNA and protein extracted from thymic lymphocytes and splenic CD4~+T cells were used to check quantitatively the m RNA and protein expression levels of Notch receptors Notch1,Notch2,Notch3 and Notch4 by real-time PCR and western blot.3.4 Effects of SEB injection to pregnant rat during gestation on m RNA andprotein levels of Notch target gene in thymic and splenic T cells of the neonatal offspringThe total RNA and protein were extracted from thymic lymphocytes and splenic CD4~+T cells in PBS and SEB groups,and were used to determine quantitatively the m RNA and protein expression levels of Notch target genes Hes1 and Hes5 by real-time PCR and western blot.4.Effects of SEB injection to pregnant rat during gestation on Notch signaling pathway in the adult offspring4.1 Effect of SEB injection to pregnant rat during gestation on m RNA levels of Notch ligands in thymus and spleen of the adult offspringPregnant rat during gestation was injected with SEB and was delivered naturally.After neonates were reared to grow into adult offspring in PBS and SEB groups,their thymus and spleen tissues were sterilely harvested,respectively.Their total RNA was extracted and was transcribed reversely into c DNA.Then the m RNA levels of Notch ligand DLL1,DLL3,DLL4,Jagged1 and Jagged2 were quantitatively detected by real-time PCR.4.2 Effect of SEB injection to pregnant rat during gestation on protein levels of Notch ligand in thymus and spleen of the adult offspringThymus and spleen tissues were sterilely acquired from adult offspring in PBS and SEB groups,respectively.Their total protein was extracted and the expression levels of Notch ligand DLL1,DLL3,DLL4,Jagged1 and Jagged2 were quantitatively determined by western blot.4.3 Effects of SEB injection to pregnant rat during gestation on m RNA and protein levels of Notch receptors in thymic and splenic T cells of the adult offspringThe aseptically obtained thymus and spleen tissues of the adult offspring in PBS and SEB groups were made into single-cell suspension which were isolated to acquire their lymphocyte suspension with lymphocyte isolation solution.Splenic CD4~+T and CD8~+T cells were sorted from splenic lymphocyte suspension by magnetic bead sorter.The total RNA and protein extracted from thymic lymphocytes and splenic CD4~+T and CD8~+T cells were used to test quantitatively the m RNA and protein expression levels of Notch receptors Notch1,Notch2,Notch3 and Notch4 by real-time PCR and western blot.4.4 Effect of SEB injection to pregnant rat during gestation on effector proteins in Notch signaling pathway in the adult offspring spleenThe aseptically obtained spleen tissues of the adult offspring in PBS and SEB groups were fixed with paraformaldehyde,dehydrated by sucrose solution with different concentrations and embedded with OCT embedding agent.Tissue sections were performed by frozen microsectioner,stained with immunofluorescence staining(API-nucleus,FITC-CD8~+Tcell,RODIN-NICD)and detected for effector protein by living cell workstation.4.5 Effects of SEB injection to pregnant rat during gestation on m RNA andprotein levels of target genes and transcription factors in thymic and splenic T cells of the adult offspringThe total RNA and protein extracted from thymic lymphocytes and splenic CD4~+T and CD8~+T cells in PBS and SEB groups were used to determine quantitatively the m RNA and protein expression levels of Hes1,Hes5,Gata3 and T-Bet by real-time PCR and western blot.Results1.Effect of SEB injection to pregnant rat during gestation on Notch signaling pathway in the neonatal offspring(1)SEB injection to pregnant rat during gestation significantly decreased the m RNA and protein levels of Jagged2,but notably increased the m RNA level of DLL1in thymus tissues of the 14-day-old neonatal offspring.(2)Compared with PBS group,the m RNA and protein levels of Notch1,Notch2,Notch4 and Hes5 were clearly decreased in thymus lymphocytes of the 14-day-old neonatal offspring in SEB groups.(3)SEB injection to pregnant rat during gestation obviously reduced the m RNA and protein levels of DLL3and the m RNA levels of Jagged1 in splenic tissues of the14-day-old neonatal offspring,but notely increased the m RNA levels of DLL1 and DLL4.(4)The m RNA and protein levels of and Hes5 in splenic CD4~+T cells of the 14-day-old neonatal offspring in SEB group were markedly lower than those in PBS group,while the m RNA levels of Notch1,Notch2 and Notch3were also lower.2.Effects of SEB injection to pregnant rat during gestation on Notch signaling pathway of the adult offspring(1)SEB injection to pregnant rat during gestation significantly increased the m RNA and protein levels of DLL3 and DLL4 in thymus tissues of the adult offspring.(2)Compared with PBS group,the m RNA and protein levels of Notch2,Notch3,Notch4 and Hes1 were clearly increased in thymus lymphocytes of the adult offspring in SEB group.(3)SEB injection to pregnant rat during gestation markedly reduced the m RNA and protein levels of DLL4 in spleen tissues of the adult offspring,but significantly increased the m RNA level of Jagged1.(4)The m RNA and protein levels of Notch1,Notch3,Hes1 and T-bet were significantly lower in splenic CD4~+T cells of the adult offspring in SEB group than those in PBS group,while Notch2 m RNA level was also lower.(5)SEB injection to pregnant rat during gestation noticeably increased the m RNA and protein levels of Notch1,Notch3 and Hes1 in splenic CD8~+T cells of the adult offspring,as well as clearly increased the protein level of Notch2 and the m RNA level of Hes5.(6)SEB injection to pregnant rat during gestation caused the notable increase of effector protein NICD in spleen tissue of the adult offspring.Conclusion1.SEB injection to pregnant rat during gestation can inhibit Notch signaling pathway of thymus lymphocytes in the neonatal offspring.2.SEB injection to pregnant rat during gestation can activate Notch signaling pathway of thymus lymphocytes in the adult offspring.3.SEB injection to pregnant rat during gestation can inhibit Notch signaling pathway of splenic CD4~+T cells in the neonatal offspring,and retains it until adulthood to form imprinting effect.4.SEB injection to pregnant rat during gestation can activate Notch signaling pathway of CD8~+T cells in the spleen of the adult offspring.At the same time,the effect of SEB exposure on the Notch signaling pathway exists cellular difference between CD4~+T and CD8~+T cells.