Study On The Role Of Ferroptosis In Liver Injury In Glucose Metabolic-disordered Mice Induced By Gestational DEHP Exposure

Objective: Di-(2-ethylhexyl)phthalate(DEHP)is a commonly used phthalate substance,widely used in the manufacture of polyvinyl chloride plastics and various household products.Studies have shown that DEHP exposure during pregnancy causes glucose metabolism disorders,but its mechanism of action is not clear.This project mainly studies the effect of gestational DEHP exposure on the mice liver and explores whether ferroptosis play an important role in liver injury and its potential mechanism in glucose metabolism disorders.Methods: This research is divided into two parts: in the vivo and in the vitro experiments.In the vivo experiment 1): To investigate the role and potential mechanism of liver injury in glucose metabolism disorders caused by pregnant DEHP exposure.ICR mice were divided randomly into three groups,respectively control group,500 mg/kg DEHP group,and 1000 mg/kg DEHP group.Gavage was performed on the 4th day of pregnancy(Gestational day 4,GD4),and on the 18 th day of pregnancy,biological samples such as liver and serum were collected.Fasting blood glucose levels were measured during the first,second and third trimesters;assessment of glucose tolerance and insulin tolerance;Serum liver function indicators AST and ALT were simultaneously detected.Observation of the histopathological condition of the liver;the iron content and oxidative stress level in liver were measured.RT-q PCR and Western-blotting detected the expression of Gpx4,Slc7a11,Fth1,Ftl,ptgs2 related genes and proteins in the liver tissues of pregnant mice.In the vivo experiment 2): To investigate whether ferrostatin-1,a ferroptosis inhibitor,can alleviate liver damage and glucose metabolism disorder in pregnant mice caused by DEHP exposure.ICR pregnant mice were randomly divided into control group,DEHP exposure group(1000 mg/kg),DEHP exposure group(1000 mg/kg)+Fer-1(5 mg/kg)group,and pregnant mice in the control group and DEHP exposure group were given corn oil and 1000 mg/kg DEHP by gavage from GD4 days to GD17 days,respectively.Pregnant mice in the DEHP+Fer-1 group were injected intraperitoneally with Fer-1 from GD4,DEHP gavage was performed after one hour,and relevant biological samples were collected at GD18.Relevant experimental contents were consistent with experiment(1).In the vitro experiment: To further explore the effect of cellular-level MEHP exposure on hepatic damage.Hep G2 cells were selected for in vitro experiments,and Hep G2 was cultured serum-free for 6 hours and then treated with MEHP(0.1% DMSO,100 μM,200μM)for 24 hours.The method of CCK8 was used to detect the cell activity,The kit determined the change of glucose content after MEHP exposure.To verify the role of ferroptosis in Hep G2 cell and glucose metabolism disorders caused by MEHP exposure.Cell samples were retained by pretreatment with GPX4 activity inhibitor(RSL3,1 μM),Fer-1(10 μM)for 1 hour followed by MEHP(200 μM)for 24 h.Transmission electron microscopy to observe cell microstructure;Immunofluorescence experiments and flow cytometry to determine the level of intracellular reactive oxygen species;RT-q PCR and Western blotting assays were used to detect whether the inhibitor Fer-1 treatment reversed the expression of Gpx4,Nrf2,Slc7a11 genes and their proteins in the ferroptosis pathway.Results: DEHP exposure causes disorders of glucose metabolism and an increased risk of insulin resistance in pregnant mice.Through in the vivo and in the vitro experiments,it was found that the degree of liver injury of pregnant mice in the high-dose group was significantly higher than that in the control group.There were pathological changes such as cell hyperemia and inflammatory cell infiltration in tissues,and at the same time,the cell proliferative activity was significantly reduced,and the glucose uptake rate was significantly decreased.In order to further explore the mechanism of ferroptosis in DEHP-induced liver injury,the level of oxidative stress of liver cells was evaluated,the level of liver oxidative stress in the high-dose DEHP group was significantly increased,the iron content was significantly increased,and the levels of ferrous pathway-related proteins GPX4,SLC7A11 and NRF2 were significantly changed,the intervention of ferroptosis inhibitor Fer-1 alleviated the degree of liver damage in pregnant mice,reduced the fasting blood glucose level,and reversed the expression level of GPX4 and SLC7A11 protein in liver,confirming that ferroptosis mediates liver injury and affects the process of glucose metabolism disorders during pregnancy.We observed the micropathological structure of Hep G2 cells by transmission electron microscopy,and found that the mitochondrial crest shrinkage,membrane density increased,and mitochondrial outer membrane damage in the high-dose MEHP-exposed group,and subsequently,we used RSL3 for intervention,and found that the level of GPX4 protein in Hep G2 cells in the RSL3 and MEHP combined exposure group decreased significantly.Compared with MEHP exposure alone,the level of GPX4 protein in the ferroptosis pathway was significantly alleviated in the Fer-1 and MEHP combined exposure group.These results further confirm that gestational DEHP exposure induces ferroptosis by inhibiting GPX4 enzyme activity,causing liver injury,which in turn adversely affects maternal glucose metabolism regulation homeostasis.Conclusion: Gestational DEHP exposure can cause glucose metabolism disorders and liver injury in pregnant mice.GPX4 mediates ferroptosis was involved in liver damage caused by gestational DEHP exposure and has a certain regulatory effect on glucose metabolism.