Studies On ANGPTLs Promoting Goat Pregnancy Toxemia And Drugs Regulation

Recently, a family of a secretory glycoprotein, sharing the signature structure of the angiogenic regulating factors angiopoietins, was identified and designated "angiopoietin-like proteins"(Angptls). At the same time, data abound connecting Angptls, especially Angptl3, Angptl4and Angptl6, with regulation of lipid, glucose and energy metabolism. Pregnancy toxemia, also known as ketosis, is a metabolic disorder in goats that usually develops during the last gestation. It is seen more often in goats with multiple fetuses and the syndrome is associated with a mortality rate of60%-70%. However, a complete understanding of the underlying mechanism leading to pregnancy toxemia remains to be elucidated.To date, the data about Angptls focus on human and mice, and no study on Angptls in ruminant has been reported. In the present study, to observe the effects of three members of Angiopoietin-like protein (Angptl3, Angptl4, Angptl6) on pregnant toxemia and whether natural antioxidants can improve lipid metabolic disorder by regulating the expression of Angptl3, Angptl4, Angptl6, we cloned the cDNA encoding Angptl3, Angptl4, Angptl6, and the key enzyme of ketogenesis HMGCS2for the first time in Liuyang Black goat. Then the change of hepatic Angptl3, Angptl4, Angptl6, and HMGCS2expression was investigated during goat pregnancy toxemia induced by fasting for72h. Subsequently, we analyzed the effects of natural antioxidants including magnolol, honokiol, resveratrol,(-)-epigallocatechin gallate (EGCG), apple polyphenol, and baicalin on expression of primary goat hepatocellular Angptl3, Angptl4, Angptl6, and HMGCS2.I. Cloning of goat Angptl3,Angptl4,Angptl6,and HMGCS2cDNART-PCR,3'-RACE, and5'-RACE technology were used to obtain the full length of Angptl3cDNA. The goat Angptl3cDNA contained74bp of3'-UTR,1380bp of coding region which encoded a459-amino acid polypeptide, and54bp of5'-UTR. Angptl4whose cDNA was obtained partially, contained861bp of coding region which encoded a286-amino acid polypeptide, Angptl6obtained partially contained819bp of coding region which encoded a272-amino acid polypeptide. The full length of HMGCS2contained2040bp,1527bp of open reading frame, coding508-amino acid polypeptide. The goat Angptl3, Angptl4,Angptl6and HMGCS2cDNA was submitted to GenBank (accession no.GQ293360, HQ008534, HQ008535, and GU586190) Ⅱ. Induction of goat pregnancy toxemia and recoveryPregnancy toxemia was induced in twelve goats by allowing access only to water for72h at130d pregnancy. Feed deprivation resulted in significant increases in serum B-hydroxybutyric acid (BHBA) and FFA concentrations (P<0.001), while a significant decrease in serum glucose level (P<0.001). There were no significant changes in the concentrations of serum TG between pregnant toxaemic and control goats. Histopathological findings showed numerours lipid deposits in the cytoplasm of hepatocytes. Additionally, there was a marked decrease in plasma LPL activity and insulin level (P<0.05), a significant increase in plasma cortisone concentration (P<0.05). Meanwhile, the clinical symptoms of hypoglycaemic pregnancy toxaemia in goats pregnant with twins appeared after72hours of starvation. The results suggested that the fasted goats in the current study had mild pregnancy toxemia. And all goats recovered after refeeding for5days with reversion of the above index.Ⅲ. Expression of hepatic Angptl3, Angptl4, Angptl6and HMGCS2mRNAPrimers for real-time PCR were designed based on cloning of Angptl3, Angptl4, Angptl6and HMGCS2cDNA. And the expression of hepatic Angptl3, Angptl4, Angptl6and HMGCS2mRNA was assayed by real-time PCR. The expression of Angptl3, Angptl6mRNA in pregnant toxemic goats was significantly down-regulated compared with that of the control does (P<0.01), while the expression level of Angptl4and HNGCS2mRNA in pregnant toxemic goats was significantly up-regulated compared with that of the control does (P<0.01or P<0.001). After recovered, the mRNA expression was reversed. The results suggested that Angptl3,Angptl4,Angptl6may play a certain role in development of pregnancy toxemia in goats.Ⅳ. Isolation and primary culture of goat hepatocyteCollagenase perfusion method was used to isolate the goat hepatocytes. Total cell count and survival rate of freshly isolated hepatocytes were determined by trypan blue exclusion. Hepatocytes were seeded onto a culture flask at a density of5×105cells/mL. Morphological assessment of hepatocyte monolayers and determination of lactate dehydrogenase activity, urea and albumin levels in the culture supernatant were carried out. The results showed that the cell yield was (8.65±0.43)×106hepatocytes per gram liver tissue. The survival rate of freshly isolated hepatocytes was (92.86±4.42)%. The optimal growth state of hepatocyte monolayers was observed during the4-5d culture period, which was applicable in studies relating to hepatocytes gene expression. V. Effect of natural antioxidants on mRNA expression of Angptl3, Angptl4, Angptl6and HMGCS2in goat primary hepatocytesRT-PCR technique was used to determine mRNA expression of Angptl3, Angptl4, Angptl6and HMGCS2in goat primary hepatocytes following exposured to natural antioxidants i.e. magnolol, honokiol, resveratrol, EGCG, apple polyphenol, and baicalin. Expression of Angptl3, Angptl4, Angptl6and HMGCS2mRNA was down-regulated after exposured to magnolol(10,20,40μmol/L), honokiol(10,20,40μmol/L), resveratrol(10,20,40μmol/L), apple polyphenol(15,30,60mg/L), and baicalin(20,40,80mmol/L). Expression of Angptl4mRNA was up-regulated following exposured to EGCG (40,20,10μmol/L), while down-regulating expression of Angptl3, Angptl6and HMGCS2mRNA. The results suggested these natural antioxidants decreased serum lipid by suppression expression of Angptl3,Angptl4,Angptl6and inhibited ketogenesis by suppression expression of the key enzyme HMGCS2.