Study On The Effect Of P53-SIRT6-Foxo1 Sub-axis Niacin And Non-esterified Fatty Acids On The Gluconeogenesis Of Sheep Liver Cells In Perinatal Peri

The perinatal period is the most critical period in the ruminant female production cycle.The biggest feature of this period is the negative energy balance.At this stage,the dry matter intake of females and the level of non-lipidized fatty acids(NEFA)in plasma will change greatly.Niacin can regulate the process of liver gluconeogenesis through the NEFA and p53-SIRT6-Foxo1 axis,thereby promoting liver gluconeogenesis to relieve energy negative balance.This study is based on the in vitro culture of sheep hepatocytes,and explores the related effects of NEFA and niacin on sheep liver cell proliferation,gluconeogenesis rate-limiting enzymes,and p53-SIRT6-Foxo1 axis-related gene mRNA and protein.This test is mainly divided into the following four parts:Experiment 1 Sheep liver cells were cultured in vitro using a two-step perfusion method.Subsequently,the morphology and function of the cultured cells were identified.The cell morphology was detected by glycogen staining,and the function identification was detected by cellular immunofluorescence chemical staining.Morphological identification results showed that the cells stained under the microscope were mostly round or oval,with different shapes and sizes.The adherent growth is evenly distributed,the cell body is full and full,and the nucleus is divided into single or multiple nuclei.The observed cell characteristics are consistent with the morphological characteristics of liver cells.The function test result of sheep liver cells showed that the expression of CK18 in liver cells was positive,that is,the test cells had the functional characteristics of liver cells,and the test cells were liver cells.In addition,the cell cycle and cell survival rate were tested.The cell cycle is in accordance with the normal growth cycle of liver cells,and the cell survival rate is high,which can meet the test needs.Experiment 2 NEFA was added to hepatocytes,and the proliferation of sheep hepatocytes,gluconeogenesis rate-limiting enzymes,and p53-SIRT6-Foxo1 axis-related gene mRNA expression were measured,and the NEFA addition concentration was screened.The concentration of NEFA was 0 mmol/L,0.3 mmol/L,0.6 mmol/L,1.2 mmol/L,2.4 mmol/L,4,8 mmol/L.The results show that the addition of NEFA can significantly inhibit the proliferation of sheep liver cells,and the strongest inhibitory effect on cell proliferation is in the 1.2 mmol/l concentration group.With the increase of NEFA concentration,the activities of phosphoenolpyruvate carboxykinase(PEPCK)and pyruvate carboxylase(PC)in sheep liver cells gradually decreased,and NEFA inhibited the activities of PEPCK and PC enzymes.The addition of different concentrations of NEFA significantly reduced the mRNA expression of the genes G6P,Foxo1,SIRT6,PEPCK,P53,and PC,and increased the mRNA expression of the AKT and PI3K genes.The mRNA expression of the GLU2 and INSR genes did not change significantly.Experiment 3 Niacin was added to hepatocytes to investigate the effect of niacin on liver cells.The concentration of niacin was(0mol/L,10-3mol/L,10-4mol/L,10-5mol/L,10-6mol/L,10-7mol/L),to study the effect of different concentrations of niacin on the proliferation of sheep liver cells,gluconeogenesis rate-limiting enzymes and p53-SIRT6-Foxo1 axis related gene mRNA expression.The test results show that the addition of niacin can promote the proliferation of sheep liver cells,and can significantly increase the activity of hepatic cell gluconeogenesis key enzymes PEPCK and PC.Niacin at a concentration of 10-4mol/L has the strongest effect on promoting the activity of PEPCK and PC.Niacin can increase the mRNA expression of G6P,Foxo1,SIRT6,GLU2,PEPCK,P53,PC genes,and reduce the mRNA expression of AKT,PI3K,INSR genes.Experiment 4 NEFA and niacin were added to hepatocytes to explore the effects of NEFA and niacin on hepatocyte proliferation,gluconeogenesis rate-limiting enzymes,and p53-SIRT6-Foxol axis-related gene mRNA and protein expression.The test results show that niacin can alleviate the inhibitory effect of NEFA on hepatocyte proliferation.Compared with the NEFA group,the niacin+NEFA group significantly increased the mRNA expression levels of PEPCK,PC,G6P,Foxo1,P53,AKT,SIRT6,INSR,GLU2 genes.Compared with the NEFA group,the niacin+NEFA group significantly increased the protein expression of the PEPCK,PC,G6P,Foxol,P53,SIRT6,and GLU2 genes.