The Impacts Of Maternal Diabetes Combined With Obesity On Offspring And Their Related Molecular Mechanisms

Objective: To determine the impacts of pre-gestational maternal type 2 diabetes mellitus combined with obesity on placental mitochondrial function and metabolism disorders of offspring.To observe the impacts of high glucose and hyperlipidemia on the biological behavior of placental trophoblast cells.Methods:1.The women were put into the following groups(24 cases in each group): healthy women,women with pre-gestational DM(PG-DM),women with pre-gestational obesity(PG-OB)and women with both PG-DM and obesity(PG-DM + OB).The maternal age,pre-gestational weight,BMI,weight gain during pregnancy and gestational age were recorded.The placental tissue was collected and the placenta mitochondria was isolated.The reactive oxygen species(ROS)level,mitochondrial content,and the mitochondrial respiratory complex activities of the placenta were measured in the four groups.Mitochondrial DNA(mt DNA)was extracted from each placenta,and RT-PCR was used to detect the mitochondrial DNA level in the placenta.The expression of nuclear factor erythroid 2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)were detected by immunofluorescence staining and western blotting.2.The women were put into the following groups(40 cases in each group): healthy women,women with pre-gestational DM(PG-DM),women with pre-gestational obesity(PG-OB)and women with both PG-DM and obesity(PG-DM + OB).The newborn birth weight,gestational age and other parameters were recorded.Umbilical vein blood was collected.Catalase(CAT),glutathione peroxidase(GSH-PX),malondialdehyde(MDA),insulin,blood glucose,leptin,free Fatty acid,adiponectin(ADP),triglyceride of the cord blood were also measured.Linear regression analysis was performed to explore the association between oxidative stress and serum parameters of offspring.3.Placental trophoblast cells were routinely cultured and passaged,and placental trophoblast cells were harvested in logarithmic growth phase.Placental trophoblast cells were divided into 4 groups for different treatments: high glucose(HG,30 mmol/ L glucose),high lipids(HL,0.3 mmol / L palmitic acid),high glucose combined with high fat diet(HG / HL)and blank control-CON.CCK-8 reagent was used to detect the proliferation of placental trophoblast cells in each group.The levels of superoxide dismutase(SOD),MDA and lactate dehydrogenase(LDH)were measured at 24,48 and 72 hours after culture in each group.Flow cytometry was used to detect the apoptotic rate of each group after 48 hours.Transmission electron microscopy was used to observe the intracellular ultrastructural changes in each group.The mitochondrial respiratory chain complex activity were detected.Western blot and immunofluorescence were used to detect mitochondrial damage and expression of oxidative stress related proteins(Nrf2,HO-1,SMAC,Cyt-C)in each group.Results:1.There were no significant differences in maternal age and gestational age among the four groups(P>0.05).Women's weight,BMI,and weight gain during pregnancy in PG-OB and PG-DM + OB groups were all significantly higher than in the control and PG-DM groups(P<0.05).Women's weight,BMI,and weight gain during pregnancy in PG-DM groups were significantly higher than in the control group.The ROS level,NADPH oxidase(NOX)level in the PG-DM + OB group was higher than that of the control,PG-DM,and PG-OB groups(P<0.05).The SOD(anti-oxidative enzyme)level in the control,PG-DM,and PG-OB groups was higher than that in the PG-DM + OB group(P<0.05).In addition,the SOD level in the PG-DM group was lower than that in the control and PG-OB groups(P <0.05).The ATP level of the PG-DM + OB group was significantly lower than that of the control,PG-DM,and PG-OB groups(P<0.05).In addition,the mt DNA copy number in the PG-DM + OB group was evidently lower than that in the other groups(P<0.05).The mt DNA copy number in the The PG-OB group was lower than that in the control group(P<0.05).The mt DNA copy number in the PG-DM was lower than that in the control group and the PG-DM group.The activity of complex ? of the PG-DM + OB group was lower than that of the other three groups(P< 0.05).The activity of complexes ? and ? in the PG-DM + OB and PG-DM groups were all lower than that of the control group(P<0.05),but there was no difference between the PG-DM + OB and PG-DM groups,and there was also no difference between the control and PG-OB groups(P>0.05).The normalised activity of complexes ?,?,and ? had no significant difference among the four groups(P>0.05).Western blot analysis of Nrf2 and HO-1 protein expression levels confirmed that Nrf2 and HO-1 protein levels in PG-DM + OB and PG-DM groups decreased dramatically compared with those in the Control and PG-OB groups(P< 0.05).2.The levels of CAT and GSH-PX in the umbilical cord blood of the PG-DM+OB group were significantly lower than those of other groups(P<0.05).The level of MDA in umbilical cord blood of the PG-DM+OB group was significantly higher than that of the other groups(P<0.05).In the PG-DM + OB group,the level of leptin was correlated with CAT,GSH-PX,MDA(P<0.05),and adiponectin levels were correlated with CAT,GSH-PX(P<0.05).Moreover,linear regression analysis showed a negative correlation with leptin and CAT,GSH-PX levels in PG-DM + OB groups(P<0.05),and showed a positive correlation with leptin and MDA levels in PG-DM +OB groups(P< 0.05).In addition,it showed a negative correlation with adiponectin and CAT,GSH-PX levels in PG-DM + OB groups(P<0.05).Insulin and NEFA were not correlated with CAT,GSH-PX and MDA among the four groups(P>0.05).3.There were significant differences in the number of cells in all four groups at three time points of 24,48 and 72 hours.The number of cells in the control group was significantly higher than that in the HL group and the HG / HL(P<0.05),while the number of cells in the HG and HG/ HL group have no significant difference.The leakage of LDH in HG,HL group and HG/ HL group cells was significantly higher than that in control group,and the HL group was also higher than HG group(P<0.05).LDH leakage in HG/ HL group cells was the most significant.At three time points,the level of SOD in the control group was significantly higher than the other three groups(P<0.05),while the level of SOD in the HG/ HL group was significantly lower than the other groups(P<0.05).The level of MDA in the blank control group was significantly lower than the other three groups(P<0.05),while the level of SOD in the HG/ HL group was significantly higher than the other groups(P<0.05).The apoptosis rate of the control group was only 1.1%,while that of the HG/ HL group was 35.2%,which was significantly higher than the other groups(P<0.05).The activities of mitochondrial complex ? and ? in HG/ HL diet group were significantly lower than those in the other three groups(P<0.05),while there was no significant difference between HG and HL group(P>0.05).The activities of mitochondrial complex ? and ? in HG/ HL group were significantly lower than those in the other three groups(P<0.05),while there was no significant difference between HG and HL(P>0.05).Among the total proteins,Nrf2 and HO-1 protein in HG/ HL group were significantly lower than those in the other three groups(P<0.05),while the relative expression of Nrf2 and HO-1 in HG group and HL group also decreased,both lower than the control group(P<0.05).The relative expression of SMAC and Cyt-C in the mitochondria of HG/ HL group was the lowest(P<0.05),while the SMAC and Cyt-C of mitochondria in HG and HL diet group were also significantly decreased compared with the control group,and higher than HG/ HL group(P<0.05).The mitochondrial damage was more obvious in HG/ HL group,with mitochondrial swelling,local atrophy and incomplete mitochondrial membrane.Conclusion:1.Pre-pregnancy diabetes can damage placental oxidative stress through the Nrf2/ARE pathway(antioxidant response element),and detrimentally alter placental mitochondrial dysfunction.Pre-pregnancy obesity further aggravated the oxidative stress of placenta in pregnant women.2.Maternal pre-pregnancy diabetes combined with obesity indeed affect the sugar metabolism and lipid metabolism of their offspring.And the oxidative stress in the offspring of pre-pregnancy diabetes combined with obesity is related to its lipid metabolism.3.Both high glucose and high lipid can cause oxidative stress injury to trophoblast placental trophoblast cells,through the Nrf2/ARE signaling pathway.And the injury of placental trophoblast cells is more obvious when both high glucose and high lipid are simultaneously stimulated.