Estrogen Receptor α Participation In The Pathogenesis Of Gestational Diabetes Mellitus Through Ferroptosis Pathway

Objective:To study the correlation between oestrogen receptorsαon ferroptosis pathway and gestational diabetes mellitus.Methods:24 pregnant SD rats were randomly divided into 4 groups,including normal pregnancy control group（control）,gestational diabetes mellitus model group（GDM）,gestational diabetes mellitus model group+low-dose estrogen receptorα（ERα）antagonist treatment group（GDM+LERa）and gestational diabetes mellitus model group+high-dose ERαantagonist treatment group（GDM+HERa）,with six rats in each group.GDM model was established by intraperitoneal injection of streptozotocin.GDM rats were treated with different doses of ER antagonist tamoxifen（2.5 mg/kg/d and 5 mg/kg/d）for 14 days.The m RNA and protein expression levels of ERαgene and derroptosis pathway related genes,including long-chain acyl-Co A Synthase 4（ACSL4）,glutathione peroxidase 4（GPX4）and ferritin heavy chain 1（FTH1）were detected by q RT-PCR and Western blot.The concentrations of insulin,glucose and glycated hemoglobin in serum,and the activities of antioxidant enzymes,including catalase（CAT）,superoxide dismutase（SOD）and glutathione peroxidase（GSH-Px）,and the content of antioxidant glutathione（GSH）,oxidant hydrogen peroxide（H₂O₂）and oxidative stress product malondialdehyde（MDA）,as well as tissue iron content,in pancreatic tissues of rats were detected by biochemical kits and enzyme plate analyzer.Pancreatic histomorphology changes were detected by hematoxylin-eosin（HE）staining.The ultrastructural changes of islet cells were observed by transmission electron microscopy（TEM）.Results:1.The m RNA of ERαgenes and ferroptosis pathway related genes ACSL4,GPX4and FTH1 in rat pancreatic tissue detected by q RT-PCR:（1）Compared with normal control group,the m RNA expression of ERα[（3.67±0.31）vs（1.01±0.17）]and ACSL4[（3.05±0.42）vs（0.98±0.10）]was obviously increased in GDM group,while the expression of GPX4[（0.32±0.09）vs（1.09±0.12）]and FTH1[（0.28±0.06）vs（1.07±0.18）]was obviously reduced.The differences were statistically significant（P<0.01）.（2）Compared with GDM group,the m RNA expression of ERα[（2.71±0.36,1.93±0.30）vs（3.67±0.31）]and ACSL4[（2.22±0.21,1.66±0.14）vs（3.05±0.42）]was obviously reduced in the low-dose or high-dose group respectively,while the expression of GPX4[（0.47±0.09,0.81±0.11）vs（0.32±0.09）]and FTH1[（0.48±0.06,0.84±0.10）vs（0.28±0.06）]was obviously increased（P<0.01）.The decrease of ERα、ACSL4 and the increase of GPX4、FTH1 in HERa group were more obviously than those in LERa group.The differences were statistically significant（P<0.05）.2.The detection of the ERαprotein and related protein of ferroptosis pathway（ACSL4、GPX4、FTH1）in rat pancreatic by western blot:（1）Compared with normal control group,the protein expression of ERα[（0.31±0.07）vs（0.05±0.01）]and ACSL4[（0.56±0.05）vs（0.12±0.02）]was obviously increased in GDM group,while the expression of GPX4[（0.06±0.02）vs（0.42±0.05）]and FTH1[（0.06±0.01）vs（0.45±0.06）]was obviously reduced.The differences were statistically significant（P<0.01）.（2）Compared with GDM group,the protein expression of ERα[（0.16±0.05,0.07±0.02）vs（0.31±0.07）]and ACSL4[（0.49±0.03,0.24±0.05）vs（0.56±0.05）)]was obviously reduced in the low-dose or high-dose group respectively,while the expression of GPX4[（0.13±0.04,0.35±0.08）vs（0.06±0.02）]and FTH1[（0.14±0.01,0.40±0.04）vs（0.06±0.01）]was obviously increased（P<0.01）.The decrease of ERα、ACSL4 and the increase of GPX4、FTH1 in HERa group were more obviously than those in LERa group.The differences were statistically significant（P<0.05）.3.Results of serum biochemical analysis in rats:（1）Compared with normal control group,the insulin concentration[（4.98±0.63）vs（2.03±0.32）],glucose concentration[（13.20±0.87）vs（4.71±0.49）],and glycosylated hemoglobin concentration[（45.82±3.89）vs（19.21±3.57）]in the serum were obviously increased in GDM group.The differences were statistically significant（P<0.01）（2）Compared with GDM group,the insulin concentration[（4.09±0.29,2.94±0.35）vs（4.98±0.63）],glucose concentration[（10.94±0.66,6.74±0.86）vs（13.20±0.87）],and glycosylated hemoglobin concentration[（34.23±3.63,26.89±2.53）vs（45.82±3.89）]in the serum were obviously reduced in the low-dose or high-dose group respectively（P<0.01）.The decrease in HERa group were more obviously than that in LERa group.The differences were statistically significant（P<0.05）.4.Test results of redox index and iron content in rat pancreatic tissue:（1）Compared with normal control group,the antioxidant enzyme activity in pancreas,such as CAT[（3.34±0.49）vs（9.38±0.70）],GSH-PX[（44.98±4.70）vs（90.47±3.07）],SOD[（9.27±1.18）vs（21.09±1.27）]and antioxidant GSH content[（1.59±0.13）vs（4.69±0.35）]were significantly decreased in GDM group,while the oxidant H₂O₂[（10.72±0.83）vs（3.84±0.45）],oxidative stress product MDA[（4.04±0.28）vs（1.67±0.15）]and tissue iron content[（0.96±0.08）vs（0.21±0.04）]were significantly increased.The differences were statistically significant（P<0.01）.（2）Compared with GDM group,the antioxidant enzyme activity in pancreatic tissue,such as CAT[（5.36±0.64,7.89±0.52）vs（3.34±0.49）]、GSH-PX[（61.08±3.78,82.74±2.21）vs（44.98±4.70）]、SOD[（12.66±0.57,17.29±0.71）vs（9.27±1.18）]and antioxidants GSH content[（2.85±0.14,3.77±0.13）vs（1.59±0.13）]were obviously elevated in the low-dose or high-dose group respectively,while the oxidant H₂O₂[（7.51±0.39,5.15±0.25）vs（10.72±0.83）]and the oxidative stress product MDA[（2.98±0.16,2.08±0.14）vs（4.04±0.28）]and tissue iron content[（0.69±0.05,0.43±0.03）vs（0.96±0.08）]were obviously decreased（P<0.01）.The increase of CAT、GSH-PX、SOD and GSH and the decrease of H₂O₂、MDA and tissues iron content in HERa group were more obviously than those in LERa group.The differences were statistically significant（P<0.05）.5.HE staining and transmission electron microscopy revealed that GDM rats performed obvious pathomorphological changes of pancreas.The ultra microstructure of pancreatic islet had obvious ferroptosis characteristics.After different doses of ERαantagonists,the damnification of pancreas was obviously ameliorated.The ferroptosis degree of islet ultra microstructure was effectively alleviated.The high dose group was more effective.Conclusion:1.The increased ERαexpression level may be involved in the pathogenesis of GDM by affecting oxidative stress and ferroptosis in pancreatic tissue.2.Inhibiting the expression level of estrogen receptorαthus inhibiting the ferroptosis pathway,may be beneficial to alleviate GDM-induced pathological injury.