Mechanisms Underlying Fetal Liver Injury Led By Free Fatty Acids Overload Resulting From Maternal Undernutrition

Intrauterine growth restriction（IUGR）,as a common phenomenon and major problem,seriously affects the fetal growth and development,and is an important incentive for stillbirth and high incidence and mortality after birth.Among them,maternal malnutrition not only leads to delayed and damaged liver development in IUGR fetuses,but also increases the risk of liver dysfunction and liver disease in offspring.However,the mechanism of fetal origin of liver disease in IUGR fetuses after birth is currently unclear,especially how maternal malnutrition is transmitted to the fetal liver through harmful fat metabolism circulating media.The mechanism of inducing fetal liver injury and increasing postnatal liver dysfunction and liver lesions is the focus of research.Therefore,this thesis selects 23Mongolian ewes,carrying single fetuses during late pregnancy（90 to 130 days of pregnancy）,were randomly divided into three treatment groups:the control group(CG,ad libitum,0.63MJ ME/BW^0.75/d),the maternal undernutrition group 1(MU1,0.33 MJ ME/BW^0.75/d),and the maternal undernutrition group 2(MU2,0.20 MJ ME/BW^0.75/d),to investigate the effects of maternal undernutrition on maternal-fetal fat metabolism during late pregnancy,detect the mechanism of abnormal fetal liver fat metabolism,and study the mechanisms underlying fetal liver injury led by abnormal fat metabolism resulting from maternal undernutrition based on transcriptomic and metabolome analysis.The results are as follows:1.Effect of maternal malnutrition during late pregnancy on fat metabolism in IUGR fetal liverMaternal undernutrition resulted in significant reductions in maternal weight,fetal weight,and liver weight in the MU1 and MU2 groups（P<0.05）.The levels of TG in maternal and fetal blood of MU1 and MU2 groups decreased sequentially,with TG in maternal and fetal blood of MU2 group significantly lower than that of CG group（P<0.05）;TC,FFA,and BHBA levels in the blood of ewes and fetuses of MU1 and MU2 groups increased sequentially.Among them,the levels of FFA,BHBA,and TC in the MU1 and MU2 groups were significantly higher than those in the CG group.TG levels in the fetal liver of MU1 and MU2 groups decreased sequentially,with MU2 group significantly lower than CG group（P<0.05）;BHBA levels in the fetal liver of MU1 and MU2 groups were significantly lower than CG group（P<0.01）,while FFA was significantly higher than CG group（P<0.05）,and MU2 group was significantly higher than MU1 group（P<0.05）.2.Fetal Liver Injury and Fatty Toxicity in the FFA-overloaded Liver of IUGR FetusesAccording to the activity score（NAS）of nonalcoholic fatty liver disease（NAFLD）based on steatosis,inflammatory infiltration and ballooning,the NAS score of MU1 group and MU2 group was significantly higher than that of CG group,and liver Reticular fiber and collagen fiber were significantly deposited（P<0.05）.The superoxide dismutase（SOD）of MU1 and MU2 groups under low nutrition treatment was significantly lower than that of CG group（P<0.05）;the total antioxidant capacity（T-AOC）of MU2 group was significantly lower than that of CG group,while malondialdehyde（MDA）was significantly higher than that of CG group（P<0.05）.At the same time,ultrastructure of fetal liver in the maternal dystrophy group showed that the endoplasmic reticulum was accompanied by moderate expansion,the shape gradually changed from strip or mesh to round ball,and some autophagosomes were visible.Regulatory factors ERAD（IRE1,SEL1L,ERDj）,UPR（ATF6B,XBP1,CHOP）and autophagy（FIP200,Beclin1,LC3B and P62）of endoplasmic reticulum stress pathways in MU1 and MU2 groups were significantly higher than those in CG group（P<0.05）.The mitochondrial structure showed swelling,cristae rupture,and deletion,and the mitochondrial copy number,complex enzyme I,and complex enzyme II activities in the fetal liver of MU1 and MU2 groups were significantly reduced（P<0.05）.3.Mechanism of FFA overload in IUGR fetal liver caused by maternal malnutritionGene Set Enrichment Analysis（GSEA）analysis showed that maternal malnutrition during late pregnancy leads to imbalanced lipid metabolism in the liver of IUGR fetuses,which is involved in liver cell lipid accumulation,lipid biosynthesis,andβ-metabolic process of oxidation is inhibited.Further gene testing of key regulatory factors in fat metabolism showed that maternal low nutrition led to key regulatory factors in the synthesis of triglycerides in IUGR fetal liverβ-oxidative regulatory genes and TCA cycle key genes were significantly lower in the CG group（P<0.05）.4.Transcriptomic Analysis Revealed Liver injury in the FFA-overloaded Liver of IUGR FetusesWe further performed transcriptome analysis of the FFA-overloaded liver of IUGR fetuses.KEGG pathway enrichment of differentially expressed genes（DEGs）shows that DEGs are mainly enriched in immune related pathways such as NET formation,Staphylococcus aureus infection,NOD like receptor signaling pathways,phagosomes,etc.Cytoscape screened and identified 10 key Hub genes related to NET formation.Quantitative real-time fluorescence PCR（q RT-PCR）showed that the expression level of the low nutrient treatment group was significantly increased compared to the control group（P<0.05）.In addition,the detection of NETs markers MPO and NE in fetal liver by immunofluorescence staining showed that the positive rates of MPO and NE in the MU1 and MU2 groups were significantly higher than those in the CG group（P<0.05）,and MPO in the low nutrient group was significantly increased in maternal blood and fetal liver（P<0.05）,while it was significantly decreased in fetal blood（P<0.05）.Trend analysis of the unique significant difference gene combination showed that of the 16 trended expression model maps obtained,5 had significant trends,and 30 Hub genes were selected from the 5 significant change trends,including 27 upregulated genes and 3 downregulated genes.Most of these genes are G2/M regulatory factors.Furthermore,TCGA database retrieval found that TOP30 gene expression in hepatocellular carcinoma（LIHC）was significantly higher than that in normal tissues（P<0.05）.The number of G2/M phase cells in the low nutrient treatment groups MU1 and MU2 was significantly higher than that in the CG group（P<0.05）.q RT-PCR verification found that compared with CG group,the expression of the first 10 Hub genes and the expression of representative genes such as cytokinin regulator（PRC1）,maternal Leucine zipper protein kinase（MELK）,and topoisomeraseⅡ（TOP2A）protein in MU1 and MU2groups in the low nutrition treatment group significantly increased（P<0.05）.5.Correlation Analysis of Fat Metabolites in IUGR Sheep Fetal Liver with Neutrophil Extracellular Bactericidal Networks（NETs）Pathways and G2/M Phase Related Pathogenic GenesFurther analysis by Mantel test revealed that with IUGR fetal liver fat metabolism disorder,especially abnormal TG and FFA metabolism,there is a strong correlation with the occurrence of NETs in IUGR fetal liver,while FFA overload in fetal liver is more strongly correlated with G2/M phase pathogenic genes than abnormal TG metabolism.In addition,there is a strong correlation between FFA overload IUGR fetal liver NETs pathway and G2/M phase pathogenic genes.Further analysis by Mantel test revealed that with IUGR fetal liver fat metabolism disorder,especially abnormal TG and FFA metabolism,there is a strong correlation with the occurrence of NETs in IUGR fetal liver,while FFA overload in fetal liver is more strongly correlated with G2/M phase pathogenic genes than abnormal TG metabolism.In addition,there is a strong correlation between FFA overload IUGR fetal liver NETs pathway and G2/M phase pathogenic genes.6.Metabolomics Analysis Fetal Liver Dysfunction with FFA Overload IUGRThe albumin content in fetal blood of the MU1 and MU2 groups was lower than that of the CG group（P<0.05）,whileγ-Glutamine was significantly higher than CG group（P<0.05）,and the content of cholinesterase in MU2 group was significantly higher than CG group（P<0.05）;The peripheral blood granulocytes,total number of red blood cells,intermediate cells,and platelet count in the MU1 and MU2 groups were significantly lower than those in the CG group（P<0.05）,while the lymphocyte and granulocyte ratios in the MU2 group were significantly lower than those in the CG group（P<0.05）.Metabolomic analysis showed metabolic disorders of alkaloids and their derivatives,organic nitrogen compounds,lipids and lipid molecules,and organic acids and their derivatives in fetal liver tissues of the MU1and MU2 groups,with reduced levels of tumor suppressor metabolites and abnormal detoxification functions of P450 and glutathione metabolic pathways.In summary,maternal undernutrition during late pregnancy leads to excessive FFA by maternal fat mobilization.FFAs act as a harmful circulating metabolic medium that transmits to the fetal liver,disrupting fetal liver fat homeostasis and causing FFAs overload which leads to liver lipotoxicity and damage.With the reduction of maternal malnutrition,NETs were induced and took the central stage in hepatic lipotoxicity and injury in FFA overload IUGR fetal liver,in which FFA activation of neutrophils via PGLYRP1-PPBP signaling may be the main pathway for NETs release.Further research found that IUGR fetal hepatocyte proliferation was retarded in G2/M phase,and a few of disease-promoting regulators genes of G2/M cell cycle were upregulated,which may be an important molecular cause of high incidence of fetal liver disease after birth.Ultimately,liver dysfunction,reduced hematopoietic capacity and metabolic disorders including alkaloids and derivatives,organic nitrogen compounds,lipids and lipid molecules and organic acids and derivatives are observed in IUGR fetal liver.All the findings offer scientific evidence for elucidating the pathophysiology of maternal undernutrition-induced IUGR fetal liver injury,revealing the molecular cause of fetal liver innate immunity initiated and hepatocyte proliferation blocked by maternal fetal toxic metabolic cycle,which led to fetal liver dysfunction and high incidence and mortality of postnatal liver disease.Furthermore,the study provide insights into preventing fetal growth restriction in the north China,disaster prevention and animal protection,and related medical research.