| Objective:Lipopolysaccharide activates TLR4-NF-κB signaling pathway in peripheral blood mononuclear cells of GDM pregnant women in vitro, in this condition we apply trigalacturonic acid and lipopolysaccharide to stimulate the TLR4 signaling pathway, to observe the inhibition.Materials:According to the diagnostic criteria of International Association of Diabetes and Pregnancy Study Group( IADPSG), select 30 GDM pregnant women and collect 15 ml peripheral venous blood to obtain the mononuclear cells, dividing into 6 groups to culture and treatment. The 6 groups respectively are the blank control group which without any treatment, the LPS group which stimulated by the LPS(1μg/ml) only, the TGA group which stimulated by the TGA(1.0 mg/ml) only, the other three groups which stimulated by TGA(0.25,0.5,1.0 mg/ml) except the LPS. Test the expression products of the downstream of TLR4 signaling pathway, Western blot and ELISA were used to detect the expression of TLR4, NF-κB protein and the level of TNF-α,IL-1, IL-10.Results:(1) the level of TNF-α in each group Blank control group(55.53±2.86) pg/ml and LPS group(74.85±4.38) pg/ml were compared, TGA group(73.60±4.38) pg/ml and 0.25mg/ml TGA group(71.22±4.14) pg/ml, 0.5 mg/ml TGA group(67.30±4.24) pg/ml, 1 mg/ml TGA group(57.68±3.20) pg/ml were compared with the blank control group, LPS group respectively. Results indicate: the level of TNF-α of the LPS group is higher than the control group, the difference was statistically significant(P < 0.05).LPS group, TGA group, 0.25 mg / ml TGA group, 0.5mg/ml TGA group, 1.0mg/ml TGA group compared with the control group respectively, there were both statistically significant differences. However, there was no statistically significant differences between the TGA group and LPS group(P > 0.05).0.25mg/ml TGA group, 0.5mg/ml TGA group, 1.0mg/ml TGA group compared with LPS group respectively, and the level of the TNF-α decreased gradually with the TGA concentration increasing, furthermore the difference is statistically significant.(2) the level of IL-1 in each group Comparison among the groups using LSD-t test, the method is same as above.Results indicate: there is statistically significant difference(P < 0.05) between control group(70.42±3.94) pg/ml and LPS group(87.45±2.49) pg/ml. TGA group(86.17±2.51) pg/ml, 0.25mg/ml TGA group(83.24±2.56) pg/ml, 0.5mg/ml TGA group(79.51±2.76) pg/ml compared with the control group respectively, and the difference is statistically significant.Nevertheless there was no statistically significant difference(P > 0.05) between 1.0mg/ml TGA group(70.83±3.81) pg/ml and the control group. So is the same between LPS group and TGA group. 0.25mg/ml TGA group, 0.5mg/ml TGA group, 1.0mg/ml TGA group compared with the LPS group respectively, and the level of the IL-1 decreased gradually with the TGA concentration increasing, furthermore the difference is statistically significant.(3) the level of IL-10 in each group The comparison of IL-10 among the groups indicate: there is statistically significant difference(P < 0.05) between control group(20.92±2.47) pg/ml and LPS group(37.46±2.59) pg/ml. TGA group(36.07±2.47) pg/ml, 0.25mg/ml TGA group(33.88±2.54) pg/ml, 0.5mg/ml TGA group(29.98±2.52) pg/ml compared with the control group respectively, and the difference is statistically significant.However there was no statistically significant difference(P > 0.05) between 1.0mg/ml TGA group(21.64±2.51) pg/ml and the control group. TGA group, 0.25 mg / ml TGA group, 0.5mg/ml TGA group, 1.0mg/ml TGA group compared with the LPS group respectively, and the level of the IL-10 decreased gradually with the TGA concentration increasing, furthermore the difference is statistically significant.(4) the expression of TLR4 and NF-κB p65 protein TLR4 and NF-κB p65 protein bands of LPS group and TGA group was darker compared with the control group, and the protein bands of 0.25mg/ml TGA group, 0.5mg/ml TGA group and 1.0mg/ml TGA group fade gradually,especially the 1.0mg/ml TGA group. The expression of TLR4 and NF-κB p65 protein of LPS group and TGA group were higher than the control group, while the protein expression of the 0.25mg/ml TGA group, 0.5mg/ml TGA group, 1.0mg/ml TGA group was decreasing gradually.(5) The correlation between TLR4 protein and NF-κB p65 protein in each group using the Pearson correlation analysis,they were positively correlated,and the correlation was statistically significant(r=0.882,0.838,0.939,0.868,0.846,0.853; P﹤0.01).Conclusions: TGA inhibits the TLR4-NF-κB signaling pathway of peripheral blood mononuclear cells of the GDM pregnant women which induced by the LPS,reducing the release of inflammatory factors in the downstream of the pathway,which may be a new way for the prevention and treatment of GDM. |
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