The Mechanism Of ROS-mediated Placental Oxidative Injury Of Sows And The Regulational Role Of N-acetylcysteine

The reproductive performances of sows directly determine the economic benefits of pig production.Sows have relatively low reproductive performance in China,which has become the limiting factor to the development of pig industry.Reproductive oxidative stress of pregnant sows can reduce its reproductive performance.The placenta is the bridge between mother and fetus.It is still unknown about the relationship and mechanism of placental oxidative stress,function and reproductive performance of sows.The present thesis is concentrated on the placenta of sows,aiming to study the mechansim of reactive oxygen species（ROS）mediating placental structure and dysfunction of sows and the regulatory role of n-acetylcysteine（NAC）.The thesis was divided into four parts:（1）Reactive oxygen species mediated placental oxidative stress,mitochondrial content,and dysfunction in intrauterine growth restricted pigsIntrauterine growth restriction（IUGR）accounts for 15-25%of newborn piglets and affects economic benefits of pig production.The mechanism regarding the mechanism between placental ROS production and dysfunction in IUGR is still unknown.Eight sows were chosen to conduct the experiment.One normal intrauterine gestation（NIUG）and IUGR fetuses were chosen from each little.The corresponding umbilical cord tissues were marked and collected at delivery（n=6）.The placental redox status,structure,gene expression,and mitogen-activated protein kinase（MAPK）pathways in IUGR were studied.Compared with the NIUG,placental ROS production,lipid peroxidation,and DNA damage were increased in IUGR.Moreover,placental cyclin E decreased and cytoplasmic vacuolization appeared in IUGR cases,which showed senescence characteristics.Placental ERK1/2 phosphorylation increased whereas the p38 and JNK phosphorylation decreased in IUGR.The genes expression of mitochondria decreased,while genes of inflammatory and negative regulator of angiogenesis increased in IUGR placenta.Correlation analysis found that the placental ROS was negatively related to mitochondrial gene expression,but positively correlated with gene expression of inflammatory cytokines and negative angiogenesis regulators.To further explore the effects of ROS on cell proliferation,the porcine trophectoderm（pTr）cells were treated by 0-2 m M H₂O₂ for 2 h.The cell viability was significantly decreased.The intracellular ROS production was firstly increased,then decreased in a dose-dependent manner.The pTr cells were treated by H₂O₂ for 2h.Then the medium was changed.Cells were cultured for another 24 h.Cell cycle distribution showed that G0/G1 phases were decreased,and S and G2/M phases were increased by flow cytometry.Our findings suggested that ROS production and oxidative injury increased in IUGR placenta,and activated ERK pathway,placental mitochondrial content decreased,cell senescence and inflammation increased.（2）ROS-induced autophagy regulates porcine trophectoderm cell proliferation and differentiationStudy showed that ROS mediated placental oxidative stress,mitochondrial content,and dysfunction in IUGR pigs.To further explore the molecular machanism of ROS regulating placental cell proliferation and differentiation,the cell experiment was conducted to elucidate the effects of ROS on pTr cell phenotypes,cell attachment and differentiation.The pTr cells were treated by different concentrations of H₂O₂ for 2 h,then treated by different concentrations of NAC for 24 h.Herein,results showed that exogenous H₂O₂ inhibited pTr cell viability,arrested cell cycle at S and G2/M phase,increased cell apoptosis and autophagy protein expression,while these effects were reversed by different concentrations of NAC post-treatment.In addition,NAC abolished H₂O₂-induced autophagic flux,inhibited intracellular and mitochondrial ROS,and restored expression of genes important for biogenesis,mitochondrial content,cell attachment and differentiation PTHLH,Cdx2 and Maspin,and inflammatory cytokines.NAC reversed H₂O₂-activated MAPK and Akt/m TOR pathways in dose-dependent manners.Furthermore,analyses with 3-methyladenine and RNA interference suggested that autophagy regulated gene expression of Cdx2 and IL1β.Collectively,these results suggested that ROS-induced autophagy regulated pTr cell proliferation,attachment and differentiation.（3）Regulation of NAC supplementation on maternal-placental redox status,placental NLRP3 inflammasome,fecal microbial communities and metabolites in sows during late pregnancy（1）and（2）chapters reported oxidative stress increased in IUGR placenta,and NAC alleviate the H₂O₂-induced pTr cells proliferation and differentiation in vitro,whether dietary NAC supplementation can improve placental oxidative stress,structure and function in sows is not known.Thus,sows（n=16）were divided into either the control group（n=7）or the NAC group（n=9）which received 500 mg NAC per kg basal diet from day 85 of gestation to delivery.Redox status,placental structure and function,and fecal microbiota were evaluated.Although NAC did not ameliorate reproductive performance of sows,it significantly improved maternal plasma and placental health,which was accompanied by increased activities of antioxidant enzyme,decreased oxidative injury and lowered nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3（NLRP3）inflammasome.Additionally,NAC significantly increased placental growth factors contents,elevated the expression of genes involved in angiogenesis and amino acids transporters,and decreased cell autophagy.Furthermore,NAC increased the relative abundances of fecal Prevotella,Clostridium cluster XIVa and Roseburial/Eubacterium rectale,which were negatively correlated with placental NLRP3 and positively with solute carrier family 7,member 8（Slc7a8）.In conclusion,NAC supplementation during late gestation alleviated maternal-placental oxidative stress and inflammatory response,improved placental function and altered fecal microbial communities and metabolites.（4）Metabolomics analysis the effects of dietary NAC on placental metabolites in sowsNAC could improve the placental structure and function,as placenta is an important nutrients metabolic organ during pregnancy,to further explore the effects of NAC on the placental metabolites and pathways,the metabolomic profiles of the placeta were investigated.The experimental design was the same as（3）,placental samples were collected ar term when they were deliveried.Then the samples were used for UHPLC-MS analysis.PLS-DA and OPLS-DA analysis showed that NAC could change the composition of placental metabolites.Compared with the control group,NAC supplementation significantly increased the relative concentrations of placental Arg-Gln,N-ω-hydroxyarginine,1-stearoyl-2-oleoyl-sn-glycerol 3-phosphocholine and citrulline,significantly decreased the relative concentrations of placental branched chain and aromatic amino acids,uracil,uridine,pipecolic acid,carnitine,S-methyl-5’-thioadenosine,indoleacetic acid,epsilon-（gamma-glutamyl）-lysine,indole-3-lacticacid,asparagine,pyridoxal5’-phosphate1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine,methionine,proline,and uridine.It is noteworthy that microbial metabolites indole-3-lactic acid and indoleacetic acid,were significantly decreased.In conclusion,the results suggested that NAC increased arginine family and Arg-Gln synthesis,decreased branched amino acids,aromatic amino acids,fatty acids,vitamin B6,and indoles,promoting the efficiency of placental accumulation of protein nitrogen and angiogenesis.In conclusion,IUGR placental ROS production increased,placental mitochondrial content reduced,cell sencecense and inflammatory cytokines increased.ROS-induced autophagy is involved in regulating pTr cells proliferation,attachment and differentiation.Dietary NAC supplementation during late gestation altered fecal microbial metabolism,placental metabolism and metabolites,inhibited placental inflammatory response and improved placental function.