Effect Of Sex Hormone-binding Globulin On The Metabolism Of Gestational Diabetes Mellitus Via RAS/RAF/ERK Pathway

Objective:Gestational diabetes mellitus(GDM)refers to different degrees of abnormal glucose tolerance found in any period of pregnancy.At present,the diagnostic standard of gestational diabetes mellitus in China is the 75 g glucose tolerance test(OGTT)in IADPSG and ADA guidelines.The incidence rate of gestational diabetes mellitus is increasing year by year,which has adverse effects on both mother and child,and is prone to be associated with hypertensive disorder complicating pregnancy,fetal malformation,excessive amniotic fluid,premature delivery,and so on,leading to adverse outcomes.Its pathogenesis has not yet been fully elucidated.It is generally believed that it is similar to the pathogenesis of type 2 diabetes and is related to insulin resistance and abnormal glucose metabolism.Due to the influence of placental factors during pregnancy,the pathophysiological changes of gestational diabetes mellitus are more complex.Sex hormone-binding globulin(SHBG)is a kind of globulin that can be synthesized and secreted by the liver.It is mainly combined with steroid hormones,and its research focuses on diabetes and metabolic syndrome.SHBG is an important marker of insulin resistance and a useful index for early prediction of gestational diabetes mellitus.Low SHBG level is an independent risk factor for insulin resistance in pregnant women with gestational diabetes mellitus.Some studies have shown that SHBG can also be secreted by placenta and participate in the occurrence of GDM,but the mechanism of SHBG signal transduction pathway is not clear.In insulin pathway,MAPK pathway is more classic,MAPK regulates adipocyte differentiation and proliferation.RAS-RAF-MEK-ERK is a classic pathway of MAPK family,which is involved in the regulation of a large number of cell biological behaviors,including cell adhesion,cycle,migration,survival,differentiation,metabolism,proliferation and transcription,causing cell proliferation and tissue growth,participating in the signal pathway of promoting cell division,playing the role of transmission,amplification and regulation of insulin signal,and playing a role in insulin resistance,It mediates the occurrence of diabetes.Studies have shown that enhanced ERK is associated with increased glucose and lipid metabolism and dysregulation of adipocytokine expression.Diabetic growth factor affects insulin resistance by activating ERK pathway.Labeling ERK pathway is a potential treatment for insulin resistance and type 2 diabetes.In this study,the relationship between SHBG and RAS/RAF/ERK pathway in placental trophoblast was studied.Real time PCR and Western blot were used to detect the expression of SHBG and various indicators of RAS/RAF/ERK pathway in placental tissue.In order to further understand the mechanism of SHBG,SHBG was transfected into trophoblast cells and silenced to detect the changes of each pathway and observe its effect on the biological behavior of trophoblast cells.Objective to evaluate the relationship between SHBG and RAS/RAF/ERK pathway at the whole animal level and further verify its correlation,so as to provide a new target and possibility for the treatment of gestational diabetes mellitus.Methods: 1.According to the diagnostic criteria recommended by ADA,30 pregnant women with gestational diabetes who delivered in Shengjing Hospital in 2017 were selected.The placentas were collected from full-term cesarean section,and the normal pregnant women with OGTT without obstetric complications in the same period were selected as the control group.Real-time PCR and Western blot were used to detect the expression of SHBG and RAS/RAF/ERK pathway related factors in placenta of pregnant women with gestational diabetes mellitus and normal pregnant women,and their correlation was detected.2.SHBG silencing and overexpression plasmids were constructed.HTR8/SV neo cell lines were transfected with SHBG silencing and overexpression plasmids.The expression of RAS/ RAF/ ERK pathway factors was detected by real-time PCR and Western blot.Cell proliferation,cycle and apoptosis were detected in different SHBG expressing trophoblast cell lines.3.The expression of SHBG,RAS,RAF1 and P-ERK in placentas of SHBG knockout mice and wild-type mice were detected by immunohistochemistry.The relationship between SHBG and RAS/RAF/ERK signaling pathway was evaluated at the whole animal level.Result: 1.Changes in the expression levels of SHBG and ERK pathway markers in placental tissues of patients with GDM: The expression levels of SHBG,RAS,RAF,and ERK1/2 in the placental tissues of puerperae in the GDM group and the control group were determined via PCR and WB assays.We found that the GDM group had lower m RNA and protein levels of SHBG than the control group.In contrast,the expression levels of RAS,RAF,and ERK in the GDM group were significantly higher than those in the control group,and were negatively correlated with the expression of SHBG.2.Changes in the expression of ERK pathway markers after altering the expression level of SHBG in HTR-8/SVneo trophoblasts: The plasmid-transfection-mediated regulation of SHBG expression levels revealed that the m RNA and protein levels of RAS,RAF,and ERK were downregulated in the high-SHBG-expression group,whereas the expression of all ERK pathway markers was upregulated in the low-SHBGexpression group.3.The changes of biological behavior of trophoblast after changing the expression of SHBG: the proliferation ability of trophoblast in SHBG high expression group was decreased,on the contrary,there was difference in 24 hours and 48 hours in SHBG silence group;the apoptosis of trophoblast in SHBG high expression group was increased,while that in SHBG silence group was decreased,there was significantly difference between two groups;the S phase of cell cycle in SHBG high expression group was decreased.There was difference between the two groups.4.Changes of SHBG and RAS/RAF/ERK pathway related indicators in SHBG knockout mice: SHBG was negative in the placenta of SHBG knockout mice.The expression of RAS/RAF/ERK pathway in the placenta of gene knockout mice was higher than that of wild-type mice.Conclusion:1.The expression of SHBG in the placenta of gestational diabetes decreased,but the expression of RAS,RAF and ERK increased,which were negatively correlated with SHBG.It is considered that SHBG may be negatively correlated with RAS/RAF/ERK pathway in the placenta.2.Overexpression of SHBG in trophoblast resulted in decreased expression of RAS/RAF/ERK pathway,but increased expression of each pathway when SHBG was silenced.SHBG may play a role by inhibiting RAS /RAF/ ERK pathway in trophoblast.3.Overexpression of SHBG can reduce the proliferation of trophoblast,but increase the apoptosis.On the contrary,the S phase of SHBG overexpression group decreased.SHBG can reduce the proliferation and increase the apoptosis of trophoblast,and may regulate the biological behavior of trophoblast through RAS/ RAF/ ERK pathway.4.SHBG was negatively correlated with RAS/RAF/ERK pathway in the whole knockout animal level,which was consistent with the results in placental tissue and cell lines.