Abstract:

Objective： To investigate the expressions of Apelin and its receptor angtiotensin Ⅱ type 1 receptor associated protein （APJ） in placenta tissue of the patients with gestational diabetes mellitus （GDM），and to explore its effect on the insulin resistance（IR）， proliferation and invasion of trophoblast cells. Methods The placenta tissue of patients with GDM （GDM group，30 cases） and pregnant women with normal glucose tolerance （NGT） （NGT group，30 cases） were collected. Immunohistochemistry was used to detect the rates of positive cells of Apelin and APJ in placenta tissue of the subjects in two groups； Western blotting method was used to detect the expression levels of Apelin and APJ proteins in placenta tissue； real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the expression levels of Apelin， APJ and adenosine monophosphate-activated protein kinase （AMPK）mRNA； Pearson correlation analysis method was used to analyze the correlation between the expression levels of Apelin and APJ mRNA and the expression level of AMPK mRNA. The trophoblast cells HTR-8/SVneo （HTR-8） were cultured in vitro and divided into control group， Apelin group， high glucose group（HG group）， HG+Apelin group，and HG+Apelin+anti-APJ group.The expression levels of Apelin， APJ， insulin receptor substrate 1 （IRS1）， insulin receptor substrate 2 （IRS2），and glucose transporter 4 （GLUT4） proteins and the phosphorylated levels of phosphatidylinositol 3-kinase （PI3K）（p-PI3K/PI3K） and AMPK （p-AMPK/AMPK） in the cells in various groups were detected by Western blotting method； 5-bromo-2-deoxyuracil（EdU）method was used to detect EdU positive expression rates in the cells in various groups； the number of invasion cells in various groups was detected by Transwell chamber assay. Results Apelin and APJ were widely expressed in placental villus tissue； compared with NGT group， the expression levels of Apelin and APJ mRNA and protein in placenta tissue of the patients in GDM group were significantly decreased （P<0.05）. The correlation analysis results showed that the expression levels of Apelin and APJ mRNA in placenta tissue of the patients in GDM group was positively correlated with the expression level of AMPK mRNA （R²=0.148， P<0.05；R²=0.275， P<0.05）.The Western blotting results showed that compared with control group， the expression levels of Apelin， APJ， IRS1， IRS2，and GLUT4 proteins and the ratios of p-PI3K/PI3K and p-AMPK/AMPK in Apelin group were significantly increased （P<0.05）， the expression levels of Apelin，APJ，IRS1，IRS2，and GLUT4 proteins and the ratios of p-PI3K/PI3K and p-AMPK/AMPK in HG group， HG+Apelin group and HG+Apelin+anti-APJ group were significantly decreased （P<0.05）； compared with HG group， the expression levels of IRS1， IRS2， and GLUT4 proteins and the ratios of p-PI3K/PI3K and p-AMPK/AMPK in HG+Apelin group were significantly increased（P<0.05）， and the differences in the indicators mentioned above in HG+Apelin+anti-APJ group were not statistically significant （P>0.05）.The EdU and Transwell assay results showed compared with control group， the EdU positive expression rate and the number of invasion cells in Apelin group were significantly increased （P<0.05）， while those in HG group， HG+Apelin group and HG+Apelin+anti-APJ group were significantly decreased （P<0.05）； compared with HG group， the EdU positve expression rate and the number of invasion cells in HG+Apelin group were significantly increased （P<0.05）， while the differences between HG group and HG+Apelin+anti-APJ group were not statistically significant （P>0.05）. Conclusion The expressions of Apelin and APJ in placenta tissue of the GDM patients are decreased， and exogenous administration of Apelin can improve IR of trophoblast cells and promote the abilities of proliferation and invasion of trophoblast cells under HG environment，and its mechanism may be related to the up-regulation of AMPK signaling pathway proteins by Apelin.

Key words: Gestational diabetes mellitus, Apelin, Angtiotensin Ⅱ type 1 receptor associated protein, Insulin resistance, Trophoblast cells