| Objective In this study,the differences of plasma cytokines(IL-1、IL-10、TNF-a) and protein molecules(TLR4、NF-κB) was detected in the experiment before and after the invervention between control and GDM groups.On this basis,1μg/ml LPS was induced by peripheral blood mononuclear cells to establish activated model in GDM group,then modeled different concentrations of rMBL(0.1、0.5、1.0、2.0μg/ml) to discover the expression level of cytokines and protein molecules,exploring the mononuclear cells lipopolysaccharide Toll-like receptors(TLR4) /regulation of nuclear transcription factor-κB signal pathway activity and the role of MBL in the mechanism of GDM inflammatory injury.Methods and groups After strict disinfection,fasting venous blood of pregnant women was taken 15 ml.The plasma and mononuclear cells were collected by density gradient centrifugation separation. Mononuclear cells were pre-incubated at 6*106/L in the absence of LPS for 48h.The next 24h,samples were divided into seven groups according to different treatments,including untreated group A which compared with control group.The other six groups were incubated with LPS and four different concentrations of rMBL(0.1、0.5、1.0、2.0μg/ml).Signs were labeled with different groups:group B(only LPS treatment group)、group C(LPS and 0.1μg/ml rMBL treatment group)、group D(LPS and 0.5μg/ml rMBL treatment group)、group E(LPS and 1.0μg/ml rMBL treatment group)、group F(LPS and 2.0μg/ml rMBL treatment group).Enzyme-linked immunosorbent(ELISA) and Western-Blot measures were adopted to detect the changes of expression of plasma cytokines(IL-1、IL-10、TNF-a) and level of cell total protein molecules(TLR4、NF-κB).Data were analyzed using independent sample t-test,one-way anova analysis,LSD test and Pearson correlation analysis test.Results (1)The levels of plasma LPS in GDM group were much higher than that in control group([(0.92±0.41)VS(0.57±0.22) EU/ml,t=3.21]).While the MBL level were opposite[(254.4±123.4)VS(424.8±150.4)μg/ml,t=-1.96].The expression of cytokines IL-1、TNF-a in GDM group were much higher than that in control group [respectively(14.5±2.1)VS(10.5±4.0) pg/ml, (5.72±0.99)VS(2.79±0.87) pg/ml, respectively t=-3.46 and 3.77],the differences were statistically significant (P<0.05).There was no siginificant change in the level of IL-10[(4.37±0.88)VS(3.00 ±1.01)pg/ml,t=-4.37] (P>0.05) (Table1).(2)The level of inflammatory cytokines(IL-1、IL-10、TNF-a) in GDM group were positively correlated with lipopolysaccharide(r1=0.78,r2=0.69,r3=0.73,P<0.05),while negatively correlated with MBL(r1=-0.58,r2=-0.41,r3=-0.74,P<0.05).What is more,LPS level was negatively correlated with MBL(r=-0.51,P<0.05).(3)Cell culture results showed that the expression of TLR4 and NF-κ B protein molecules of peripheral blood mononuclear cells in GDM group were higher than that in control group [respectively (1.85±0.41) VS (1.27±0.72),(1.20±0.62)VS(0.84± 0.31),respectively t=-13.57 and -7.19, P<0.05].(4)Mononuclear cells stimulated with LPS reached a maximal response in the secretion of inflammatory cytokines.As shown in Figure2,MBL could directly affect cytokines production of LPS-stimulated mononuclear cells. The level of cytokines were significantly downregulated in the presence of a high concentration of MBL.Compared with group B,the expression of cytokines and protein molecules in group C [respectively (15.44±2.29)、(4.77±1.84)、(5.83±1.08) pg/ml and 2.01± 0.15、1.47±0.04];group D[respectively (13.37±1.34)、(3.13±1.71)、(4.23 ±1.71) pg/ml and 1.58±0.69、1.20±0.16];group E[respectively (12.18±1.38)、(2.72 ±0.69)、(3.15±0.78) pg/ml and 0.85±0.02、0.89±0.02];group F[respectively(10.59 ±1.20)、(2.03±0.92)、(2.71±0.96) pg/ml and 0.56±0.44、0.61±0.21](Figure1 and Table2).We also observed MBL inhibited the expression and the activity of pathway in a dose-independent manner.Conclusions Taken together,these data suggest that low level of plasma MBL may affect the expression of cytokines and protein molecules through modulation of LPS/TLR4/NF-κB signaling pathways. These findings suggest that MBL may play an important role in immune regulation and the signaling pathways involved in cytokines networks may be correlated to the pathogenesis of GDM. |
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