Effects Of Bile Acids Metabolism In Sows During Pregnancy On Fetal Pigs Survival And Their Regulation

Imbalance of maternal bile acids(BA)homeostasis was mainly occured during middle to late gestation,studies in both human and gilts have shown that imbalance of maternal BA homeostasis during pregnancy threatened fetal survival.Unfortunately,little is known about its specific mechanism of action,predisposing factors and potential regulatory pathways.Fetal BA transplacental transport into mother plays a vital role in fetal BA homeostasis,the latter is the key for fetuses to complete pregnancy.However,it remains unclear how the mother affects placental BA transport and fetal BA homeostasis during pregnancy.Reproductive hormone is the key endocrine factor for the maintance of pregnancy.Recent studies have verified that estradiol and sulphated progesterone metabolites participated in regulating BA metabolism,it remains to be explored whether it is related to imbalance of maternal BA homeostasis during pregnancy and whether it can be used as a regulatory target.Therefore,pregnant sows were taken as the main research subjects in this study,moreover,UPLC-MS/MS,UPLC-MS,RNA-sequencing,16 Sr RNA sequencing,pig primary hepatocyte culturing and cephalic vein catheters were used to explore the metabolic and molecular mechanisms of the effect of BA metabolism in pregnant sows on fetal pig survival and its potential regulatory pathways.The results of this study are shown as follows:Experiment 1 The critical period of fetal pig death and its relationship with the bile acid levels in sows and fetal pigsImbalance of maternal BA homeostasis during pregnancy was an important factor causing fetal death.Our previous study have found that imbalance of maternal blood BA homeostasis was easy to occur in gilts during middle to late gestation,and maternal serum total bile acid(TBA)variation during middle to late gestation(expressed by the ratio of maximum maternal serum TBA to initial TBA levels)was closely related to fetal pig death.To further clarify the action pathway of maternal BA metabolism during pregnancy on fetal pig survival,we took pregnant sows as the main reaserch objects in this experiment,exploring the critical period of fetal pig death and the relationship between fetal pig death and BA levels in sows/fetal pigs.The results of this study are shown as follows:(1)The middle to late gestation was the critical period of fetal pig death,and fetal pig mortality during late gestation(day 91 of gestation till parturition)accounted for 65.40%of the total fetal pig mortality.(2)The number and mortality of fetal pig were positively correlated with the blood TBA level of sows at day 90 of gestation(G90)(P<0.05),respectively,but were not significantly correlated with the TBA levels at day 60 of gestation(G60)and the farrowing day(L0)(P>0.05),respectively.(3)Fetal pig mortality was positively correlated with the representive of fetal blood TBA-TBA levels in umbilical cord vein,on the day of parturition(L0),fetal pig mortality was significantly higher(P<0.05)in sows with higher umbilical cord vein TBA levels(TBA>13.00 ?mol/L).The average blood TBA levels of newborn piglets was 18.04 ?mol/L.The results of this study showed that middle to late gestation were both the period of imbalance of maternal blood BA homeostasis and the critical period of fetal pig death,fetal pig death were positively correlated with sows and fetal pigs blood TBA levels,maternal BA may increase the risk of fetal pig death through affecting fetal pig blood BA levels and metabolism.Experiment 2 The effect of maternal bile acids metabolism during middle to late gestation on bile acid homeostasis in fetal pigs and its mechanismsExperiment 1 have demonstrated the closely relationship between fetal pig death with its high BA levels,but it is unknown whether accumulation of BA in the fetuses is due to the immature feedback regulation of BA homeostasis via the enterohepatic circulation,impaired transplacental export of fetal pig BA,or BA detoxification capacity of fetal pigs.Therefore,we took gilts as the main reaserch objects in this experiment,comparing TBA levels,BA profiles and expression of genes and proteins involved in BA metabolism in motherplacenta-fetuses trio at G60,G90 and L0,respectivly.The results of this study are shown as follows:(1)HDCA,CDCA and HCA were the main BA profile in the blood of gilts,and their combined proportion was not less than 93.91% of TBA.Compared with G60,the blood TBA levels of gilts at G90 and L0 were increased by 3.39 times and 1.57 times,respectively,the increased blood TBA level of gilts at G90 and L0 were mainly attributed to HDCA,CDCA and HCA.As one of the most important indicators of BA detoxification ability(bile acids sulfation ability),the percentage of sulfate bile acids(BA-Sulfate)in gilts from G60 to L0 showed no significant changes(P>0.05).Analysing BA metabolism in the liver,bile,faeces and ileum showed that the increased blood TBA level at G90 was highly correlated with the decreased BA secretion in the hepatic(P<0.05)and the increased expression(P<0.05)of genes(OST?)involved in hepatic BA transported to the blood circulation system.(2)The BA transport direction of placental was from gilts to fetuses from G60 to G90.BA of maternal origin accounted for the increased(P<0.05)TBA concentration in placentas at G90,but failed to directly affect fetal BA levels.RNA-sequencing of placentas showed that,compared with G60,the continuously decreased expression of genes(CA2 and SLC4A4)significantly(P<0.05)enrich in KEGG pathway "bile secretion" and participated in bicarbonate production and uptake at G90 and L0 may accounted for impaired the transplacental export fetal pig BA.(3)BA exchange between gilts and fetal pigs was gradually decreased.On the one hand,The changes of fetal pigs BA profiles was inconsistent with gilts,on the other hand,secondary BA of maternal origin in fetal pigs was gradually decreased with the advance of pregnancy.Simutaneously,fetal pig hepatic BA synthesis was gradually increased,whereas the development of hepatic biliary BA secretion was not paralled with BA synthesis.Under these conditions,TBA levels in the fetal pigs kept in a tight range from G60 to G90,the constant TBA levels was highly correlated with increased BA sulfation capacity,as indicated by the elevated percentage(P<0.05)of BA-Sulfate and the paralled expression of hepatic CYP7A1 and SULT2A1.On the contrary,the significantly elevated TBA(P<0.05)from G90 to L0 accompanied with the constant percentage of BA-Sulfate(P>0.05)and the unparalled expression of hepatic CYP7A1 and SULT2A1.The results of this study showed that imbalance of maternal BA homeostasis during middle to late gestation was highly correlated with impaired hepatic BA secretion and increased BA transport from liver to blood circulation system.BA of maternal origin accounted for the elevated placental TBA levels,which further leading to dysregulated expression of genes involved in BA transport and thus impairing transplacental export of fetus-derived BA.BA homeostasis in fetal pigs was regulated by BA sulfation capacity,whereas BA sulfation capacity failed to maintain BA homeostasis of fetal pigs during late gestation.Therefore,the BA metabolism of gilts was the key factor for fetal pig BA homeostasis.Experiment 3 The effect of sulphated progesterone metabolites on bile acids metabolism during middle to late gestation and its mechanismsResults from experiment 1 and 2 suggested maternal BA homeostasis during pregnancy have important implications for fetal pig BA homeostasis and survival,but it was unknown whether the imbalance of BA homeostasis in gilts was result from reproductive hormone metabolism,which plays an vital role in maintaing pregnancy,therefore,we took gilts as the main reaserch objects in this experiment,using UPLC-MS/MS,oral gavage of pregnant mice and pig primary hepatocytes culturing technology to explore the effect of reproductive hormones(estradiol,progesterone and sulphated progesterone metabolites)variation in gilts and fetal pigs on the BA metabolism and the potential regulation role of FXR signaling pathways during middle to late gestation.The results of this study are shown as follows:(1)FXR target protein FGF19 levels during middle to late gestation was decreased(P<0.05)in gilts with imbalance of BA homeostasis,indicated by the ratio of maximum maternal serum TBA to initial TBA levels >3.0,which was consistent with decreased maternal hepatic BA secretion during the middle to late gestation reported in experiment 2.In addtion,differ from estradiol and progesterone,the PM4 S and PM5 S in both gilts and fetal pig were showd consistent change trend and positively correlated with(P<0.05)their corresponding TBA levels from G60 till L0.(2)Pregnant mice have higher(P<0.05)gallbladder weight,hepatic TBA concentrations and contents and blood TBA levels than their non-pregnant counterparts,blood total cholesterol levels,the synthesis precursors of BA,showed opposite change trend(P>0.05).Compared with control group,the gallbladder weight,total gallbladder BA content and TBA pool of pregnant mice gavaged with PM5 S during late gestation were significantly increased(P<0.05),respectively,whereas the total cholesterol levels was significantly decreased(P<0.05).(3)PM4S inhibited(P<0.05)the expression of FXR and its targeted genes involved in BA sulfation,reabsorption and secretion,and increased(P<0.05)TBA levels in cell culture medium in dose-dependent manner;PM5S treatment alone activated(P<0.05)the genes involved in FXR signaling pathway;PM5S treatment alleviated the stimulation role of CDCA on the expression of FXR and its targeted genes involved in BA synthesis and secretion(P<0.05),increased TBA levels(P<0.05)of cell culture medium in dose-dependent manner.The results of this study showed pregnancy was an important factor affecting maternal BA homeostasis,gilts and fetal pig BA metabolism during middle to late gestation were highly correalted with sulphated progesterone metabolites PM4 S and PM5 S,whereas were not correlated with estradiol and progesterone.PM4 S and PM5 S directly or compectively inhibited FXR signaling pathway and then stimulating hepatic BA synthesis and inhibiting BA secretion,the dynamic change of sulphated progesterone metabolites PM4 S and PM5 S may be the important factors causing hepatic BA synthesis or BA secretion dysregulation in gilts and fetal pigs during middle to late gestation.Experiment 4 The effects of vancomycin and probiotics supplemented in diet on bile acids metabolism of gilts during middle to late gestation and its mechanismsResults from experiment 3 suggested sulphated progesterone metabolites PM4 S and PM5 S were important causing factors for imbalance of maternal BA homeostasis during middle to late gestation.In combination with the regulation role of gut microbiota on BA metabolism and sulfated steroids and bile salt hydrolase(BSH)of gut microbiota on BA metabolism,we took gilts as the main reaserch objects in this experiment,exploring the effect of antibiotics(Vancomycin,VAN)and BSH expressing probiotic(Lactobacillus plantarum,PRO)on the gut microbiota composition,sulphated progesterone metabolites and BA metabolism of gilts and its mechanisms during middle to late gestaion.The results of this study are shown as follows:(1)In contrast to the continuously increased maternal blood TBA levels in CON group from middle to late gestation(P<0.05),maternal blood TBA levels in VAN group remained relative constant(P>0.05).In addition,compared with CON group,TBA levels in VAN group have decreased trend(0.05<P<0.10)at G90,though the TBA levels was higher in VAN than CON group(P<0.05)at G60.The reduction role of VAN treatment on maternal blood TBA levels was mainly attributed to primary BA.(2)In contrast to the continuously decreased FXR-FGF19 signaling pathway and fecal TBA of gilts with imbalance of BA homeostasis in CON group(P<0.05),VAN treatment maintained FXR-FGF19 signaling pathway and fecal TBA levels,namely VAN treatment maintaining normal function of hepatic BA secretion and thus BA homeostasis via hepatic FXR signaling pathway.(3)VAN treatment alleviated maternal blood PM4 S and PM5 S accumulation accompanied with significantly increased(P<0.05)fetal PM4 S and PM5 S excretion.(4)PRO treatment have no significant(P>0.05)effect on maternal BA metabolism during middle to late gestation.The results of this study showed that VAN treatment alleviated imbalance of BA homeostasis during middle to late gestaion was highly correlated with alleviated maternal blood PM4 S and PM5 S accumulation and thus maintaing normal function of FXR signaling pathway,whereas no correlation with BA deconjugation mediated by BSH.The regulation role of VAN on maternal blood PM4 S and PM5 S was highly correlated with the elevated fecal PM4 S and PM5 S excretion through decreasing the intestinal microbiota potentially involved in sulfated steroid desulfation.PRO treatment failed to alter BA metabilsim during middle to late gestation through BSH pathway.To sum up,the main results in this study showed that imbalance of maternal BA homeostasis was an important factor causing fetal pig death;The decreased hepatic BA secretion and increased hepatic BA transport to the blood were main meabolic factors causing imbalance of maternal BA homeostasis,the latter impaired transplacental function of fetal pig BA through the placentas and thus impairing BA homeostasis of fetal pigs during late gestation.Sulphated progesterone metabolites PM4 S and PM5 S were the key endocrine factors causing gilts and fetal pigs abnormally BA synthesis or secretion during middle to late gestation,PM4 S and PM5 S participated in BA metabolism through FXR signaling pathway.VAN treatment maintained relative stable of maternal blood PM4 S,PM5S and thus BA homeostasis through regulating intestinal microbiota potentially involved in sulfated steroid desulfation.