Mechanisms Of Lipid Metabolism Disorder And Oxidative Stress In Maternal And Fetal Livers Of Hu-sheep Induced By Severe Feed Restriction During Late Gestation

Inadequate nutrient intake during late gestation of Hu-sheep severely threatens maternal and fetal health.However,the alterations of metabolic homeostasis and oxidative stress in maternal and fetal livers under this condition and its molecular regulatory mechanisms are poorly understood.This dissertation used severe feed restriction(SFR)to induce negative energy balance model on Hu-sheep and studied the effect of SFR during late gestation on the metabolic homeostasis and oxidative stress in maternal and fetal livers of Hu-sheep based on the integrated analysis of transcriptomics and metabolomics.The specific contents are as follows:1.Study on the effect of SFR on metabolic homeostasis in maternal livers of Hu-sheep during late gestation.To study the effect of SFR on metabolic homeostasis in maternal livers of Hu-sheep during late gestation,twenty purebred Hu-sheep with similar body condition,pregnancy for 115 days,and gestating for twins or multiple fetuses were enrolled in this study and randomly assigned to 2 groups.Hu-sheep in the control(CON)group were fed normally with total mixed ration according to the feed intake calculated in preliminary feeding period while Husheep in the treated(TR)group were restricted to 30%feeding level.Blood samples were collected every 5 days to investigate the biochemical indicators.After 15-day treatment,we slaughtered all the Hu-sheep and collected maternal liver samples for morphological and metabolome analysis.Results showed that the levels of blood beta-hydroxybutyrate(BHB A),non-esterified fatty acids(NEFAs),and cholesterol increased(P<0.05)while blood glucose level decreased(P<0.05)in the TR group when compared with the CON group.Hepatic morphological analysis displayed that,maternal livers of Hu-sheep upon SFR were enlarged,deep yellow,and fragile;Lots of lipid droplets were distributed inside and outside of hepatocytes;Nuclei,mitochondria,and endoplasmic reticulum were damaged.Besides,in maternal livers of Hu-sheep upon SFR,the contents of NEFAs,BHBA,cholesterol,and triglyceride increased(P<0.05)while glycerol content decreased(P<0.05)when compared with the CON group.Metabolome results displayed that the overview of metabolic profiles was changed in maternal livers of Hu-sheep upon SFR.Differential metabolites(DMs)were mainly classified into fatty acids and lipids,which were higher(P<0.05)in the TR group than the CON group.Furthermore,pathway and enrichment analysis of DMs revealed the marked alterations of fatty acid metabolism and phospholipid metabolism.In general.SFR during late gestation changed metabolic homeostasis in maternal livers of Hu-sheep,increasing the contents of fatty acids and lipids.Histomorphology of maternal liver was changed in response to SFR and the inner structures of hepatocytes were damaged.2.Study on the molecular mechanisms of lipid metabolism disorder in maternal livers of Husheep induced by SFR.The objective of this chapter was to study the mechanisms of lipid metabolism disorder in maternal livers of Hu-sheep induced by SFR.Animal experiment was the same as part 1,and we collected maternal liver samples and extracted RNA to perform transcriptome and quantitative real-time PCR(qRT-PCR)analysis.Compared with the CON group,the transcriptional profiles in maternal livers of Hu-sheep upon SFR were changed.KOG functional classification showed that differential expressed genes(DEGs)were mainly classified into lipid transport and metabolism.Compared with the CON group,DEGs involved in fatty acid transport,binding,and oxidation and triglyceride degradation were upregulated(P<0.05)in maternal livers of Hu-sheep upon SFR while DEGs related to fatty acid and cholesterol synthesis and phospholipid degradation and synthesis were downregulated(P<0.05).Results of qRT-PCR demonstrated,when compared with the CON group,key enzyme genes involved in fatty acid oxidation and triglyceride genesis were upregulated(P<0.05)while key enzyme genes related to cholesterol synthesis and ketogenesis were downregulated(P<0.05)in maternal livers of Hu-sheep upon SFR.KEGG pathway analysis demonstrated that retinol metabolism and PPAR signaling were enriched by DEGs(P<0.05).Correlation analysis showed that DEGs enriched in PPAR signaling were positively correlated with NEFAs content in maternal liver(P<0.05)while DEGs enriched in retinol metabolism were negatively correlated with BHBA level(P<0.05)。In general,SFR during late gestation changed the expression of genes involved in lipid metabolism which enhanced fatty acid oxidation but inhibited cholesterol synthesis and ketogenesis and finally increased triglyceride genesis.PPARA/RXRA signaling pathway was changed upon SFR.3.Study on the regulatory mechanisms of PPARA/RXRA signaling pathway in lipid metabolism in Hu-sheep hepatocytesThe objective of this chapter was to study the regulatory function of PPARA/RXRA signaling pathway in lipid metabolism in Hu-sheep hepatocytes.Primary hepatocytes were isolated from newborn lambs and treated with different concentrations of NEFAs and BHBA as well as PPARA agonist WY14643(WY)and RXRA agonist 9-cis retinoic acid(RA)to detect the changes of PPARA/RXRA signaling pathway and the expression of key enzyme genes and the contents of metabolites involved in lipid metabolism.Primary hepatocyte experiments showed that high concentrated NEFAs could upregulate(P<0.05)the expression of PPARA and genes involved in fatty acid oxidation,ketogenesis,and triglyceride synthesis and increase(P<0.05)the contents of BHBA,cholesterol,and triglyceride;High concentrated BHBA could downregulate(P<0.05)the expression of RXRA and genes related to fatty acid oxidation and ketogenesis,upregulate(P<0.05)the expression of genes related to triglyceride synthesis,and increase(P<0.05)the contents of NEFAs,cholesterol,and triglyceride;WY and RA could upregulate(P<0.05)the expression of PPARA and RXRA and genes related linked to fatty acid oxidation and ketogenesis,downregulated(P<0.05)the expression of genes involved in triglyceride synthesis,decrease(P<0.05)NEFAs level,and increase(P<0.05)BHBA level.In general,NEFAs could activate PPARA signaling and facilitate fatty acid oxidation and ketogenesis;BHBA could inhibit RXRA signaling and repress fatty acid oxidation and ketogenesis;Activating PPARA/RXRA signaling pathway could enhance fatty acid oxidation and ketogenesis and decrease triglyceride synthesis.4.Study on the effect of SFR on metabolic homeostasis in fetal livers of Hu-sheep during late gestation and its mechanismsIn this chapter,we studied the effect of SFR on lipid metabolism in fetal livers of Husheep during late gestation and its mechanism based on integrated transcriptome and metabolome analysis.Animal experiment was the same as part 1,and we collected fetal liver samples to perform morphological,transcriptome,metabolome,qRT-PCR analysis.Results showed that fetal weight and fetal liver weight upon SFR were lower(P<0.05)than the CON group.When compared with the CON group,in fetal livers of Hu-sheep upon SFR,NEFAs content increased(P<0.05)while cholesterol content decreased(P<0.05);Many lipid droplets were accumulated and the nuclei of hepatocytes were extruded and out of shape.Transcriptome analysis showed that the overview of transcriptional profiles was changed obviously in fetal livers of Hu-sheep upon SFR when compared with the CON group.KOG functional classification showed that,among substrate metabolism pathways,lipid transport and metabolism displayed the most enriched DEGs.Compared with the CON group,the gene and protein levels of key enzymes involved in fatty acid oxidation and ketogenesis were upregulated(P<0.05)in fetal livers of Hu-sheep upon SFR,while key enzyme genes related to the synthesis of fatty acid,cholesterol,and steroid were downregulated(P<0.05);Key enzyme genes related to triglyceride degradation were downregulated(P<0.05)while genes related to triglyceride genesis were upregulated(P<0.05);Key enzyme genes involved in phospholipid degradation and synthesis were downregulated(P<0.05).Metabolome results demonstrated that DMs were mainly classified into fatty acids and lipids.Pathway analysis of DMs displayed glycerophospholipid metabolism was significantly enriched(P<0.05).In general.SFR during late gestation induced lipid metabolism disorder in fetal liver;Fatty acid oxidation,ketogenesis,and triglyceride synthesis were enhanced while fatty acid and steroid synthesis as well as phospholipid degradation and synthesis were inhibited.5.Study on SFR induced oxidative stress in maternal and fetal livers of Hu-sheep during late gestation and its mechanismsBased on assays in vivo and in vitro,we studied the effect of SFR on oxidative stress response and its mechanism in maternal and fetal livers of Hu-sheep during late gestation.Animal experiment was the same as part 1,we collected blood samples every 5 days.After 15-day treatment,we slaughtered all the Hu-sheep and collected maternal and fetal liver samples to detect oxidant and antioxidant indicators.Results showed that oxidant indicators including hydrogen peroxide(H2O2)and malondialdehyde(MDA)contents were increased(P<0.05)while antioxidant indicators including catalase(CAT)activity,total antioxidant capacity(TAC),and superoxide dismutase(SOD)activity were decreased(P<0.05)in blood,maternal liver,and fetal liver in response to SFR.Most DEGs related to antioxidation were downregulated in maternal and fetal livers upon SFR.Furthermore,oxidative indicators were positively correlated(P<0.05)with NEFAs and BHBA levels,while antioxidant genes were negatively correlated(P<0.05)with NEFAs and BHBA levels in maternal and fetal livers.Based on this,we isolated primary hepatocytes from newborn lambs and treated hepatocytes with different concentrations of NEFAs and BHBA as well as PPARA agonist(WY)and RXRA agonist(RA)to detect the changes of oxidant and antioxidant indicators.Primary hepatocyte experiments showed high concentrated NEFAs increased H2O2 and MDA contents(P<0.05)and decreased TAC,CAT activity,and SOD activity(P<0.05);High concentrated BHBA increased H2O2 and MDA contents(P<0.05)and decreased TAC and SOD activity(P<0.05);Activating PPARA/RXRA signaling pathway using WY and RA decreased(P<0.05)H2O2 and MDA contents(P<0.05)and increased TAC.In general,SFR induced lipid metabolism disorder in maternal and fetal livers during the late gestation increased the contents of NEFAs and BHBA,which decreased antioxidant capacity and induced oxidative stress.Activating PPARA/RXRA signaling pathway could enhance antioxidant capacity and relieve oxidative stress.Taken together,SFR during late gestation of Hu-sheep induced severe lipid metabolism disorder in maternal and fetal livers,which triggered oxidative stress in maternal and fetal livers.PPARA/RXRA signaling pathway was positively regulated by NEF As and was negatively regulated by BHBA,further,activating PPARA/RXRA signaling pathway could mitigate lipid metabolism disorder and relieve oxidative stress.