| Objective:The expression of transmembrane protein TLR4 and nuclear transcription factor NF-КB on the immune inflammatory pathway LPS-TLR4-NF-κB was regulated to study the relationship between immune inflammation and the development of GDM.Methods:1.15 ml venous blood samples were collected from 20 normal pregnant women at the morning wake-up time.Peripheral blood mononuclear cells(PBMCs)were obtained by Ficoll-hypaque density gradient centrifugation.At the culture time of 4h,24 h,48h and 72 h,counstar(IC1000)cell counter was used to count the total PBMC concentration in culture media,living cell concentration,dead cell concentration,survival rate of PBMC,average cell diameter,average cell roundness and average cell cluster rate,and a continuous data variance analysis was applied to compare the difference of data at different time points.The levels of LPS,inflammatory factors and TNF-α in the culture media were tested respectively in four phases including in the sera,before the culture,after the culture,and +LPS after culture.The t test was applied to compare the concentrations of inflammatory factors in culture plates at different time.Lipopolysaccharide(LPS)was tested through the limulus reagent chromogenic substrate method.And LAL,IL-1,IL-10 and TNF-α were tested through enzyme-linked immunosorbent assay(ELISA).2.30 cases of pregnant women were included respectively in the GDM group and the control group and 15 ml venous blood was collected respectively at the morning wake-up time.PMBCs were obtained after leaving sera and grown for 48 h.LPS levels,TNF-α and HOMA-IR values in the sera were tested,and after the culture TLR4 acetylation expressions,NF-κB expressions and the levels of TNF-α of both groups were tested.After the culture the GDM group was divided into four subgroups which were cultured under conditions of no reagents,adding LPS,adding LPS+MBL,and adding LPS+PDTC respectively for another 24 h to test TLR4 expressions,NF-κB expressions,and the levels of TNF-α.The t test was applied to compare LPS levels,TNF-α levels and HOMA-IR values in the sera between the GDM group and the control group;and meanwhile the acetylated TLR4 expressions,NF-κB expressions and TNF-α levels were also compared.The methods of single factor variance analysis and SNK-Q test were applied to compare in pairs the TLR4 acetylation expressions,NF-κB expressions and TNF-α levels in the non-reagent subgroup,LPS subgroup,LPS+MBL subgroup,LPS+PDTC subgroup in the GDM group;and the method of Pearson correlation analysis was applied to compare the relativity of TLR4 acetylation expressions,NF-κB expressions and TNF-α levels in the LPS subgroup,LPS+MBL subgroup and LPS+PDTC subgroup.The limulus reagent chromogenic substrate method(microplate test method)was applied to test the levels of LPS in the culture media;the immunoprecipitation(IP)and western blot(WB)methods were applied to test the TLR4 acetylation expressions;the WB method was applied to test NF-κB expressions(especially NF-κB-P65);and the levels of TNF-αin the culture supernatant liquid were tested through the ELISA method.3.20 cases of pregnant women were enrolled respectively in the GDM and normal groups.15 ml blood was collected respectively at the morning wake-up time during pregnancy(24-28 weeks of pregnancy)and since the 42 th day after delivery.LPS levels,TNF-α levels and HOMA-IR values in the sera were tested,and at the same time the PMBCs obtained were grown.The TLR4 acetylation expressions,NF-κB expressions and TNF-α levels were tested respectively after the culture and the addition of LPS after the culture,and the methods of single factor variance analysis and SNK-Q test were applied to compare in pairs the TLR4 acetylation expressions,NF-κB expressions and TNF-α levels after the culture and the addition of LPS after the culture.TLP4 acetylation expressions and NF-κB expressions were tested through the IP and WB methods;and the levels of TNF-α in the culture supernatant liquid were tested through the ELISA method.Results:1.With the increase of the culture time of PMBCs,the total concentration of cells increased continuously,the concentration of living cells and survival rate of cells decreased gradually,and the concentration of dead cells and the average cluster rate increased continuously,the deviation of which had a significance in statistics(see Table 3 for the value of F,P<0.05).Values of the average diameter and average roundness of cells at each culturing time were compared and there was no obvious difference among different time points(P>0.05).The appropriate culture time is considered by this research to be 48 h.2.LPS in the culture media could only be tested in the sera.A sequence of TNF-α levels in different phases from high to low were phases of +LPS after the culture,in the sera,after the culture and before the culture,the deviation of which had a significance in statistics(P<0.05).3.Ages and pregnant weeks of pregnant women in the control group and GDM group did not have a significance in statistics(P>0.05).The LPS levels,TNF-α levels and HOMA-IR values in the GDM group were all higher than those in normal group and the deviations had a significance in statistics(P<0.05).The TLR4 acetylation expressions,NF-κB expressions and TNF-α levels in the GDM group after the culture in vitro were all higher than those in normal group and the deviations had a significance in statistics(P<0.05).4.The TLR4 acetylation expressions in the LPS subgroup,LPS+MBL subgroup and LRS+PDTC subgroup in the GDM group after the culture in vitro were all higher than those in the non-reagent group,the TLR4 acetylation expressions in the LPS subgroup and LPS+PDTC subgroup were higher than those in the LPS+MBL group,and the deviations had a significance in statistics(P<0.05).There was no deviation between the TLR4 acetylation expressions in the LPS subgroup and LPS+PDTC subgroup,and the deviations did not have a significance in statistics(P>0.05).The NF-κB expressions in the LPS subgroup was higher than those in the LPS+MBL subgroup,LPS+PDTC subgroup and the non-reagent subgroup,and the deviations had a significance in statistics(P<0.05).There was no deviation between the NF-κB expressions in the LPS+MBL subgroup,LPS+PDTC subgroup and the non-reagent subgroup,and the deviations did not have a significance in statistics(P>0.05).The TNF-α expressions in the LPS subgroup was higher than those in the LPS+MBL subgroup,LPS+PDTC subgroup and the non-reagent subgroup,and the deviations had a significance in statistics(P<0.05).There was no deviation between the TNF-α expressions in the LPS+MBL subgroup,LPS+PDTC subgroup and the non-reagent subgroup,and the deviations did not have a significance in statistics(P>0.05).5.In the LPS subgroup,the TLR4 expressions,the NF-κB expressions and the levels of TNF-α were correlative,(P<0.05);in the LPS+MBL subgroup,the TLR4 expressions,the NF-κB expressions and the levels of TNF-α were correlative,(P<0.05);in the LPS+PDTC subgroup,the TLR4 expressions were not correlative with the NF-κB expressions and the levels of TNF-α,(P<0.05),while the NF-κB expressions and the levels of TNF-α were correlative,(P>0.05).6.The pregnancy subgroup in the GDM group,the LPS levels,TNF-α levels and HOMA-IR values in the sera were higher than those in the postpartum subgroup in the GDM group,normal pregnancy subgroup and normal postpartum subgroup,and the deviations had a significance in statistics(P<0.05).There was no deviation between the LPS levels,TNF-α levels and HOMA-IR values in the postpartum subgroup in the GDM group,normal pregnancy subgroup and normal postpartum subgroup,and the deviations did not have a significance in statistics(P>0.05).After the culture of four subgroups of PMBCs,the TLR4 expressions,the NF-κB expressions and the TNF-α levels in the pregnancy subgroup of the GDM group were higher than those in the other three subgroups,and the deviations had a significance in statistics(P<0.05).There was no deviation between the TLR4 acetylation expressions,the NF-κB expressions and the TNF-α levels in the postpartum subgroup of the GDM group,the normal pregnancy subgroup and the normal postpartum subgroup,and the deviations did not have a significance in statistics(P>0.05).After the addition of LPS in the culture of PMBCs,the TLR4 acetylation expressions,the NF-κB expressions and the TNF-α levels in the pregnancy subgroup of the GDM group were higher than those in the other three subgroups,and the deviations had a significance in statistics(P<0.05).The TLR4 acetylation expressions,the NF-κB expressions and the TNF-α levels in the postpartum subgroup of the GDM group were higher than those in the normal pregnancy subgroup and the normal postpartum subgroup,and the deviations had a significance in statistics(P<0.05).There was no deviation between the TLR4 acetylation expressions,the NF-κB expressions and the TNF-α levels in the normal pregnancy subgroup and the normal postpartum subgroup,and the deviations did not have a significance in statistics(P>0.05).Conclusions:The Ficoll-hypaque density gradient centrifugation method was applied to obtain PMBCs and after the culture in vitro,an LPS-induced PMBC immune inflammation model in vitro could be constructed.The pathway of LPS-TLR4-NF-κB in pregnant women with GDM was in the state of activation,which contributed to the regulation of the acetylation of transmembrane protein TLR4 and the activity of nuclear transcription factor NF-κB in the LPS-TLR4-NF-κB pathway to regulate the TNF-α level in the downstream of the pathway.By regulating the level of TNF-α,the LPS-TLR4-NF-κB inflammatory pathway could be involved in the development of IR and GDM.The decrease of LPS-TLR4-NF-κB activity in postpartum women with GDM suggested that GDM may be in a “transient inflammation” state. |
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