The Role Of Reactive Oxygen Species-mediated Placental Barrier Dysfunction In Intrauterine Growth Restriction Induced By Bile Acid Exposure During Pregnancy

BackgroundIntrauterine growth restriction（IUGR）not only leads to miscarriage and preterm birth,but also increases the incidence of metabolic diseases in offspring in adulthood.With the development of society and economy,IUGR caused by malnutrition has been greatly reduced,but the phenomenon of placental insufficiency induced by poor drug intake and intrahepatic cholestasis during pregnancy still exists.The previous cohort study of the research group found that when the TBA of pregnant women was less than10 μmol/L（that is,the clinical diagnostic criteria for intrahepatic cholestasis of pregnancy were not met）,the incidence of IUGR increased with the increase of bile acid levels.high.At the same time,multiple studies have shown that IUGR induced by intrahepatic cholestasis of pregnancy is associated with oxidative stress.ObjectivesThis study aimed to elucidate the role of reactive oxygen species-mediated placental barrier dysfunction in intrauterine growth restriction induced by bile acid exposure during pregnancy.MethodsThe in vivo experiment was divided into two parts: Experiment 1 was to divide ICR pregnant mice into Control group,CA1 and CA2 groups.CA1 and CA2 groups were given CA（100,300 mg/kg）by gavage at 8:00 every morning,while Control group The blank solvent was administered by gavage at 8:00 every morning.When the pregnant mice were GD18,they were slaughtered,and the number of live fetuses,stillbirths,and absorbed fetuses were recorded;the fetal length,body weight,and placental diameter and weight were weighed,and the IUGR rate was calculated and compared.Maternal serum,fetal serum and placental glucocorticoids（GC）levels were detected by ELISA;11β-HSD2 expression levels were detected by immunohistochemistry and western blot;placentas were collected for the measurement of malondialdehyde（MDA）,reduced glutathione（glutathione,GSH）and activated GC levels.In experiment II,pregnant mice were divided into Control group,NAC group,CA group and NAC+CA group.GD12-17 in the Control group was administered with blank solvent vegetable oil by gavage at 8:00,and GD12-17 in the NAC and NAC+CA groups were administered with NAC（N-Acetyl-L-cysteine,200 mg/kg）by intragastric administration at 8:00.The NAC+CA group was given CA（Cholic acid,300 mg/kg）by gavage at 9 am.At 9:00 am on the 18 th day of GD,the fetus was recorded.The in vitro experiment consisted of two parts: Experiment Ⅰ was to divide the cells into four groups randomly,and respectively give CA（0,20,100,500 μM）to incubate for 24 hours to detect the level of 11β-HSD2.In experiment II,the cells were randomly divided into two groups and treated with CA（0,500 μM）respectively,and the m RNA levels of GSH-rd,GSH-px and SOD-2 were detected.In experiment III,the antioxidant NAC was given,and the cells were randomly divided into four groups（Control group,NAC group,CA group,NAC+CA group）.Control group was given normal medium,CA and NAC+CA groups were given CA（500 μM）for 24 hours,and NAC and NAC+CA groups were given NAC（4 m M）1 hour earlier.The relative fluorescence intensity of intracellular reactive oxygen species was detected by DCFHDA;the protein expression levels of 11β-HSD2,SOD1 and GPX-4 were measured by western blotting.ResultsWe used cholic aid（CA）for modeling,and found that there was no obvious IUGR in CA1 group when 100 mg/kg was administered orally,but the fetal body weight decreased,and CA2 group showed obvious IUGR when 300 mg/kg was intragastrically administered.The levels of activated glucocorticoids in serum and placental tissue of pregnant mice treated with hypercholic acid in CA2 group increased,and the protein expression level of 11βHSD2 in placental tissue decreased,which indicated that the placental barrier function was impaired.GSH and MDA levels were measured in placental tissue,and it was found that the GSH and MDA of the treatment group were significantly different from those of the control group.In order to further verify whether high bile acid exposure during pregnancy can cause oxidative stress,we used HTR-8 cells to verify,and DCFH-DA was used to detect the intracellular reactive oxygen species level.high,with a statistically significant difference.Cell RT results showed that the m RNA levels of SOD-2,GSH-px,and GSH-rd in the treatment group were significantly lower than those in the control group,with statistical differences.Pregnant mice were pretreated with classical antioxidant NAC in the later stage,and NAC pretreatment was found to alleviate CA-induced intrauterine growth restriction.ConclusionThe administration of hypercholic acid during pregnancy can induce IUGR in fetal mice,while NAC pretreatment can reduce ROS generation and up-regulate 11β-HSD2 to alleviate CA-induced IUGR.