Study Of Underlying Mechanism Of The Hepatic Damage And Mitochondrial Dysfunction Of IUGR Piglets And The Protective Effects By Resveratrol

Intrauterine growth retardation(IUGR)is characterised by impaired growth and development of embryo/fetus during pregnancy.Approximately 15%-20%of newborn piglets suffer from IUGR in commercial swine industry,resulting in a huge economic loss to the producer.There is abundant evidence that liver is a target for the intrauterine programming induced by IUGR,undergoing structural and functional changes following exposure to an unfavorable intrauterine environment,leading to the long-term adverse effects on the physiological function and metabolism.Recently,focus has been more directed toward mitochondrial dysfunction as a potential mechanism underlying this phenomenon.Therefore,this study used normal birth weight(NBW)and IUGR piglets as the research subjects to investigate the effects of IUGR on their growth performance,hepatic mitochondrial function,energy metabolism,redox status,and apoptosis and analyze the related mechanism.And on this basis we evaluated the protective effects of resveratrol against the IUGR-induced hepatic damage and mitochondrial dysfunction of suckling piglets.1 Effects of IUGR on growth performance,hepatic mitochondrial biogenesis and energy metabolism of pigletsThe aim of this study was to investigate the effect of IUGR on growth performance,hepatic mitochondrial biogenesis and energy metabolism of piglets.Twenty-four normal birth weight(NBW)neonatal piglets and twenty-four IUGR littermates were selected at birth.The newborn piglets with a birth weight(BW)near the mean BW(within 0.5 SD)were defined as NBW,whereas those 2 standard deviation lower BW were identified as IUGR.Piglets were distributed into groups of two treatments(NBW and IUGR groups)×six replicates × four piglets per replicate.All piglets were allowed to suckle the dam naturally up to 21 d of age.After weaning,both IUGR and NBW piglets were fed a commercial diet for 28 d.Plasma and liver samples were collected from each piglet(one piglet per replicate)at 1,21,and 49 d of age and analyzed for the biochemical parameters.The results obtained from the present study indicated that(1)IUGR decreased the average daily gain(ADG)of piglets from 1 to 21 d of age and their ADG,average daily feed intake(ADFI),and feed efficiency(FE)from 22 to 49 d of age(P<0.05).(2)Compared with the NBW,IUGR decreased the contents of total protein(TP),albumin(ALB),free triiodothyronine(FT3),and free thyroxine(FT4)at 1 d of age in the plasma of piglets(P<0.05).IUGR piglets had reduced plasma TP,total cholesterol(TC),glucose(GLU),and FT4 whereas increased urea nitrogen(UN)and free fatty acid(FFA)contents at 21 d of age than those in the NBW piglets(P<0.05).In addition,IUGR increased plasma FFA content of weaned piglets at 49 d of age in comparison with the NBW counterparts(P<0.05).(3)Compared with the NBW piglets,IUGR piglets had a lower hepatic energy reserve and a decreased efficiency of energy metabolism,as evidenced by the decreases in hepatic glycogen concentrations at 1 and 21 d of age,as well as the reductions in the levels of adenosine triphosphate(ATP)and energy charge(EC),and the activities of citrate synthase(CS)and succinate dehydrogenase(SDH)at 21 d of age(P<0.05).(4)IUGR significantly decreased the activity of hepatic sirtuin 1(SIRT1)of piglets at 1 and 21 d of age,and reduced their mitochondrial deoxyribonucleic acid(mtDNA)copy numbers at 21 and 49 d of age(P<0.05).(5)Compared with the NBW,IUGR down-regulated the expression levels of genes related to mitochondrial biogenesis and energy metabolism,especially for peroxisome proliferation activated receptor y coactivator-la(PGC-la),nuclear respiratory factor 1(NRF1),mitochondrial transcription factor A(TFAM),peroxisome proliferator activated receptor ?(PPARa),and ATP synthase ? polypeptide(ATP5B)(P<0.05).In conclusion,IUGR had negative effects on the efficiency of mitochondrial biogenesis and energy metabolism,leading to the decreases in ATP production and energy supply,which may be associated with the compromised growth performance and health status during the early period after birth.2 Effects of IUGR on hepatic morphology,redox metabolism,and apoptosis of pigletsThe objective of the present study was to investigate the effect of IUGR on hepatic morphology,redox metabolism,and apoptosis of piglets.The present study shared the same details of the experiment design as that of Chapter 4.The present observations showed that(1)IUGR impaired the hepatic morphology of piglets with evidence of vacuolation of hepatocyte,the disorganization of parenchyma,as well as the increases in total bilirubin(T-Bil)concentration and alanine aminotransaminase(ALT)and aspertate aminotransferase(AST)activity in the plasma(P<0.05).These observations were most obvious at 21 d of age.(2)IUGR had a negative role on the redox metabolism of piglets,as indicated by the decreases in Mn superoxide dismutase(Mn-SOD)activity,reduced glutathione(GSH)content,and reduced/oxidized glutathione(GSH/GSSG)ratio,whereas an increase in protein carbonyls(PC)concentration at 1 d of age(P<0.05).Also,IUGR piglets showed reduced Mn-SOD and glutathione reductase(GR)activities,decreased total glutathione(T-GSH)and GSH contents,and a lower ratio of GSH to oxidized glutathione(GSSG)but increased GSSG and malondialdehyde(MDA)concentrations at 21 d of age than those in the NBW piglets(P<0.05).(3)The analysis results by flow cytometry indicated that IUGR significantly increased hepatocellular reactive oxygen species(ROS)accumulation at 21 and 49 d of age(P<0.05).Similarly,IUGR enhanced the percentage of apoptotic hepatocytes of piglets at 1,21,and 49 d of age in comparison with their NBW littermates(P<0.05).(4)Compared with the NBW,IUGR markedly increased the activities of Caspases 3 and 9 in the liver at 1 and 21 d of age(P<0.05).A similar effect was also observed for the hepatic Caspase 9 activity in the IUGR group at 49 d of age than that in the NBW group(P<0.05).(5)IUGR piglets exhibited the decreases in mRNA abundance of hepatic superoxide dismutase 2(SOD2),glutamate-cysteine ligase catalytic subunit(GCLC),and transferrin(TF),whereas an increase in expression level of second mitochondria-derived activator of caspases(Smac)at 1 d of age than those in the NBW counterparts(P<0.05).At 21 d of age,IUGR down-regulated the expression levels of glutathione peroxidase 4(GPX4),peroxidase 3(Prx3),and GCLC,but up-regulated that of Smac and apoptosis inducing factor in the liver of piglets,when compared with the NBW ones(P<0.05).In addition,IUGR group showed a reduced level of hepatic B cell lymphoma 2(BCL2)mRNA than that in the NBW group(P<0.05).In conclusion,IUGR impairs the antioxidant system of piglets,resulting in higher levels of hepatocellular ROS,and increased degree of lipid peroxidation and protein ox(?)dation,suggesting that the obvious oxidative stress occurred at early period after birth.Interestingly,IUGR stimulates the Caspase cascade and induces the up-regulated expression of pro-apoptotic genes involved in mitochondrial pathway in the liver of piglets,leading to a greater percentage of hepatocyte apoptosis.These observations may help to explain the early liver injury of IUGR piglets.3 Protective effects of resveratrol on growth performance,hepatic mitochondrial function and energy metabolism of IUGR suckling pigletsThe aim of this work was to investigate the protective effects of resveratrol on growth performance,hepatic mitochondrial biogenesis and energy metabolism of IUGR suckling piglets.Seven NBW neonatal piglets and fourteen IUGR littermates were selected at birth according to the same selection as Chapter 4.Piglets were allowed to suckle the dam naturally up to 7 d of age and then allocated to receive the control milk supplemented with 0(NBW-CON),0(IUGR-CON),and 0.1%(IUGR-RSV)resveratrol of milk dry matter from 7 to 21 d of age.Each group consisted of seven replicates with one piglet per replicate.Plasma and liver samples were collected from each piglet at the end of this trial and analyzed for the biochemical parameters.The results indicated that(1)IUGR substantially decreased the ADG and ADFI from 7 to 21 d of age in the IUGR-CON group than those in the NBW-CON group(P<0.05).Resveratrol treatment effectively improved the FE of IUGR-RSV piglets during this period(P<0.05).(2)IUGR-CON group exhibited the reductions in the levels of hepatic ATP and nicotinamide adenine dinucleotid(NAD+),and the ratio of NAD+ to reduced form of nicotinamide adenine dinucleotid(NADH)than those in the NBW-CON group(P<0.05).Compared with the IUGR piglets fed the control milk,resveratrol feeding significantly increased the contents of ATP and NAD+,the ratio of NAD+ to NADH,and the level of energy charge(EC),whereas decreased adenosine monophosphate(AMP)/ATP ratio and NADH concentration in the liver of IUGR-RSV piglets(P<0.05).(3)IUGR-CON group showed decreased activities of mitochondrial citrate synthase(CS),complex ?,and ATP synthase in the liver than those in the NBW-CON group(P<0.05).In contrast,resveratrol intervention improved the activities of CS,complexes ? and ?,and ATP synthase in the hepatic mitochondria of IUGR-RSV group,when compared with the IUGR-CON group(P<0.05).(4)Compared with the NBW,IUGR decreased the mitochondrial numbers,mtDNA content,SIRT1 activity,and the relative phosphorylation levels of AMP-activated protein kinase alpha(AMPKa)at Thr172 and liver kinase B1(LKB1)at Ser428 in the liver of control piglets(P<0.05).However,IUGR-RSV group showed the increases in mitochondrial numbers,mtDNA copy number,SIRT1 activity,PGC-la protein expression in the liver than those in the IUGR-CON group,along with the elevated relative phosphorylation levels of AMPKa at Thr12and LKB1 at Ser428(P<0.05).(5)The mRNA expression levels of hepatic estrogen-related receptor ?(ERR?)and acyl-CoA synthetase medium-chain family member 4(ACSM4)were reduced more in the IUGR piglets fed either a control or resveratrol supplemented diet than in the heavier ones(P<0.05).Compared with NBW,IUGR decreased mRNA abundance of nuclear respiratory factor 2(NRF2),TFAM,polymerase gamma(POLG),mitochondrial DNA single-strand binding protein(SSBP1),cytochrome c oxidase I(COX I),PPARa,and acyl-CoA synthetase long-chain family member 1(ACSL1)in the liver of the control piglets(P<0.05).IUGR-RSV piglets exhibited increased expression levels of hepatic NRF1 and PPARa compared with other two groups(P<0.05).Resveratrol diet up-regulated the mRNA expression levels of PGC-la,sirtuin 3(SIRT3),NADH dehydrogenase 1?subcomplex 1(NDUFA1),ATP synthase alpha subunit(ATP5A1),ATP5B,and carnitine palmitoyltransferase 1A(CPT1A)in the liver of IUGR piglets(P<0.05).In addition,resveratrol treatment attenuated the IUGR-induced decreases in the expression of TFAM,COX I,and ACSL1 at the transcriptional level(P<0.05).(6)Compared with the NBW-CON piglets,IUGR-CON piglets had greater CpG methyaltion levels of the sites-62,-52,and-41 of PGC-la promoter(P<0.05).Resveratrol alleviated the IUGR-induced decreased methylation level of PGC-1? promoter at the site of-41(P<0.05).In conclusion,these observations indicate that resveratrol effectively stimulates the SIRT1/PGC-la signaling pathway,induces the transcription expression of its down-stream targets,and improves the efficiency of mitochondrial biogenesis and oxidative metabolism,resulting in a greater FE during the suckling period in the IUGR-RSV piglets.4 Protective effects of resveratrol on hepatic morphology,redox metabolism,and apoptosis of IUGR suckling pigletsThe present study investigated the protective effects of resveratrol on hepatic morphology,redox metabolism,and apoptosis of IUGR suckling piglets.The present study shared the same details of the experiment design as that of Chapter 6.The present observations showed that(1)the hepatocytes in:the IUGR-CON group arrayed loosely with swollen sizes than those in the NBW-CON group.Also,IUGR induced the cytoplasmic vacuolation and parenchymal disorganization in the hepatocytes of control piglets compared with the NBW.These histopathological alternations were obviously attenuated by resveratrol administration.(2)The swollen and irregular-shaped mitochondria with disrupted and poorly defined cristae were observed in some hepatocytes of IUGR control piglets,which were mostly abrogated by RSV administration.(3)Compared with the NBW,IUGR significantly increased ROS accumulation,PC and MDA contents,and mitochondrial superoxide(·02-)generation,whereas obviously decreased GR activity and GSH/GSSG ratio in the liver of control piglets(P<0.05).In addition,the contents of T-GSH and GSH were decreased in the liver of both the IUGR-CON and IUGR-RSV groups,when compared with the NBW-CON group(P<0.05).However,resveratrol treatment significantly inhibited the increases in hepatic ROS accumulation,PC and MDA contents,and mitochondrial ·O2-generation of suckling piglets induced by IUGR,as well as enhanced the activity of Mn-SOD and the ratio of GSH to GSSG(P<0.05).(4)IUGR caused increased percentage of hepatocyte apoptosis and elevated activities of hepatic Caspases 3 and 9 in the IUGR-CON piglets than those in the NBW-CON ones(P<0.05).The results of qRT-PCR analysis showed that IUGR-CON piglets had up-regulated heme oxygenase 1(HO-1)and SOD2 but down-regulated Smac and B cell lymphoma 2 associated X protein(BAX)mRNA abundance in the liver compared with their heavier counterparts(P<0.05).Feeding IUGR piglets with the resveratrol diet alleviated the IUGR-induced decrease in the expression level of hepatic Prx3(P<0.05).(6)Compared with the IUGR-CON group,resveratrol administration induced an increased protein content of intranuclear NRF2 in the liver of IUGR-RSV group(P<0.05).In conclusion,resveratrol treatment effectively decreases ROS accumulation,oxidative damage,and apoptosis in the liver of IUGR suckling piglets by reducing mitochondrial ·O2-production,activating NRF2 signaling pathway,and up-regulating Mn-SOD at the transcriptional expression and activity levels,which may contribute to the improvement of hepatic morphology.Based on the aforementioned results,it can be therefore concluded that:(1)IUGR decreases the efficiency of mitochondrial biogenesis and energy metabolism in the liver of piglets,which may acccount for their reduced energy supply and compromised growth performance during early period after birth.(2)IUGR reduces the hepatic anitoxidant ability of piglets,resulting in the increases in the degree of oxidative damage and the numbers of apoptotic cells,which may contribute to the impairment of liver morphology.This phenomenon is especially obvious at 21 d of age.(3)Resveratrol effectively activates the SIRT1/PGC-la signaling pathway and improves the efficiency of mitochondrial biogenesis and energy metabolism in the liver of IUGR-RSV piglets,which may responsible for their increased FE from 7 to 21 d of age.(4)Resveratrol obviously decreases the oxidative damage and apoptosis,and improves the morphology in the liver of IUGR suckling piglets by reducing mitochondrial ·02-production,activating NRF2 signaling pathway,and up-regulating Mn-SOD at the transcriptional expression and activity levels.