Study On The Effect And Mechanism Of Advanced Glycation End Products On Placental Barrier And Tight Junctions In Gestational Diabetes Mellitus

The placental barrier plays a vital role maintaining and protecting the normal development of the fetus.Tight junctions(TJs)is one of important components of the placental barrier,controlling the transport of paracellular pathway.The structure and function of placental barrier are abnormal in gestational diabetes mellitus(GDM),which is related to the poor prognosis of mothers and children However,the etiology and mechanism of the abnormal placental barrier of GDM are still unclear.High levels of advanced glycaylation end products(AGEs)have been found in GDM and preeclampsia AGEs are associated with multiple barrier disfunction and TJs damage AGEs mainly work through their receptor RAGE(receptor for advanced glycation end-products.RAGE)When activated,RAGE can cause intracellular oxidative stress,which in turn activates NF-?B and regulates the expression of inflammatory cytokines,leading to vascular endothelial injury,hemodynamic and hemorheological abnormalities.We hypothesized that high levels of AGEs uprrgulation of RAGE expression in placenta and activation of NF-?B signaling pathway would lead to abnormal expression of tight junctions,which would lead to destruction of the placental barrier and increased placental permability,which would be involved in the occurrence and development of various pregnancy complications.Understanding the impact of AGEs on the placental barrier could lead to new directions for the prevention and treatment of complications during pregnancy.PART ? To investigate the alteration of placental barrier and TJs in GDM rate and their relationship with the AGEs-RAGE system by animal experimentsObjectiveThis part of the study aimed to confirm the changes in placental barrier function in GDM rats by observing the changes in placental barrier and TJs after the intervention of insulin,PFS-ZM1 and sRAGE Furthermore,the relationship between placental barrier dysfunction and AGEs-RAGE system under GDM and its possible mechanism were further explored.Methods1.To investigate the alteration of placental permeability and TJs in rats with GDMThe GDM rat model was constructed to detect placental permeability(Evans-Blue method,VEGF),to observe the ultrastructure of placental tight junction by eletron microscopy,and the expression of TJs(ZO-1,OCLN,Claudin5)gene and protein by RT-PCR,immunohistochemistry and Western Blot.2.To investigate the relationship between the alteration of placeatal permeability and TJs in rats with GDM and the AGEs-RAGE systemAfter GDM rats model constructed,insulin,RAGE receptor antagonist FPS-ZM1,sRAGE were used as intervention factors in different groups In addition to detection of blood glucose and serum level of AGEs,Evans Blue leakage and VEGF were detected to evaluate placental permeability in different groups.Then the ultrastructural alterations of TJs were observed by TEM.Further,immunohistochemical,RT-PCR and Western Blot were performed to analyze the expression of TJ factors(ZO-1,occludin,claudin5),and RAGE,NF-?B.Results1.Compared with the control group,Blood glucose,serum AGEs,EB leakage and expression of VEGF were markedly increased in GDM group rats,this was associated with reduced TJ factors(ZO-1,occludim,claudin5)expression and disruption of the structure TJs.Conversity,the expressions of RAGE and NF-?B were significantly higher than those of the control group.The results indicated that the abnormal placental barrier in GDM was accompanied by changes in AGEs-RAGE system.2.PFS-ZM1 and sRAGE attenuated this increase in placental permeability in rats with GDM and also mediated a partial recovery of TJs(ZO-1,occludin,claudin5)expression-Blood glucose and serum AGEs concentrations did not differ between the GDM and GDM+PFS-ZM1 and GDM+sRAGE groups.Furthermore,PFS-ZM1 and sRAGE treatment resulted in decreases in RAGE and NF-?B expression levels?which were upregulated in the GDM group.The results suggested that placental permeability and structure of TJs in GDM rats could be improved by blocking RAGE so AGEs-RAGE system was involved in placental barrier damage in GDM.Conclusions1.GDM was associated with increased placental permeability and this was linked with changes in TJs?2.FPS-ZM1 and sRAGE have protective effects on placental barrier damage in GDM.PART ? In vitro experiments were conducted to explore the effect of AGEs on the permeability and TJs of BeWo cells and its mechanismObjectiveThe effects of AGES at different concentrations on the permeability and TJs of trophoblast cells were observed,and anti-RAGE blocking RAGE and PDTC blocking NF-?B were used to explore whether AGEs played their role in the damage of trophoblast cells through RAGE/NF-?B pathway.Methods1.To investigate the effects of AGEs at different concentrations on the permeability and TJs of BeWo cellsTo investigate how permeability changes undergoing different concentrations of AGEs-BSA in vitro,transepithelial electrical resistance(TEER)and paracellular permeability(CPP)were detected.Then the ultrastructural alterations of TJs were observed by TEM.Further,immunofluorescence,RT-PCR and Western Blot were performed to analyze the expression of TJ factors(ZO-1,occludin,claudin5),and RAGE,NF-?B.2.To observe the changes of permeability and TJs of BeWo cells after intervention of RAGE antibody and NF-?B inhibitor PDTC accompanied with AGEsThe changes of cell permeability(TEER,CPP)were observed after co-cultured with AGEs-BSA and RAGE antibody or PDTC in vitro,the changes of TJs ultrastructure were observed by TEM,and the expressions of TJs(ZO-1,OCLN,Claudin5),RAGE,NF-?B genes and proteins were detected by RT-PCR,immunohistochemistry and Western Blot.Results1.AGEs significantly decreased TEER,while increased CPP in a time and dose dependent manner,suggesting that AGEs could lead to increased permeability of trophoblast cells TEM showed that AGEs made cell junction loose.ACEs inhibited TJ factors(ZO-1,occhidin,claudin5)expressions,while increased RAGE and NF-?B expressions.2.Anti-RAGE or PDTC partially restored the permeability and the levels of TJ factors(ZO-1,occludin,claudin5),which was down-regulated by AGEs he expressions of RAGE and NF-?B in BeWo cells were also reversed by anti-RAGE and PDTC.Conclusions1.In vitro.,AGEs can increase trophoblast permeability,down-regulate the expression of TJs,and TJs structure of cells.2.AGEs can damage the permeability and TJs of trophoblast cells through RAGE/NF-?B signaling pathway.PART ? In vivo experiments verified the effect of AGEs on placental barrier function and TJs in pregnant rats and its mechanismObjectiveIn this study,a pregnant rat model with AGEs intervention was established to observe the changes of placental permeability and tight connection in the third trimester after AGEs intervention,so as to clarify the influence of AGEs on placental permeability and tight connection structure and its possible mechanism.Methods1.Toinvestigate the alteration of AGEs on placental permeability and TJs in pregnant ratsEvans Blue leakage and VEGF were detected to reveal the placental permeability in rats after treated with AGEs.The ultrastructural alterations of TJs were observed by TEM.Western blot and RT-PCR were adopted to determine the protein and mRNAlevels of TJ factors(ZO-1,occludin,claudin5)and RAGE,NF-?B.2.Changes of placental permeability and TJs in pregnant rats after co-intervention by RAGE receptor antagonists PES-ZM1 and sRAGE with AGEsAfter co-intervention by PFS-ZMlor sRAGE with AGEs,placental permeability changes(Evans-Blue method,VEGF)in late pregnancy were observed,ultrastructure of placental TJ was observed by electron microscope,and expression of placental TI factors(ZO-1,OCLN,Claudin5),RAGE,NF-?B genes and proteins were detected by RT-PCR and Western Blot.Results1.AGEs were found to dys-regulation of Evans Blue leakage and VEGF in rats.TEM results showed that ultrastructure of TJs were destroyed by AGEs.Western blot and RT-PCR showed that the expressions of TI factors(ZO-1,occludin,claudin5)were down-regulated by AGEs,which was opposite with the expressions of RAGE and NF-?B.2.Increased placental permeability(EB leakage and VEGF),as well as changes in TJ structures and factors,and increased expression of RAGE and NF-?B in pregnant rats intervene by AGS-RSA,were all reversed after treated by PFS-ZM1 or sRAGE,without affecting blood glucose.The results suggested that AGEs could impair the placental barrier function by activating RAGE and upregulation of NF-?B expression in vivo.Conclusions1.In vivo,AGEs can reduce the expressions of TI factors and destroy the structure of tight junctions,thus increasing placental vascular permeability.2.AGEs disturb the placental barrier through RAGE/NF-?B signaling pathway in vivo.3.The specific inhibitors of RAGE,PFS-ZM1 and sRAGE,have protective effects against placental barrier damage.