Effect Of Pes1 On Mice With Cholestatic Liver Disease Via PI3K/AKT/GSK-3? Signaling Pathway

Objective: Cholestatic liver disease is very common in people and clinics.It can cause bile formation,secretion and excretion disorders caused by various causes,resulting in bile accumulation in the liver,resulting in a series of organic cells of liver cells and intrahepatic bile ducts.Hepatobiliary diseases with damage,metabolic disorders,and functional abnormalities.If this state continues to develop,it will become cirrhosis and liver cancer leading to death.It is reported in the literature that Pes1 contains a conserved BRCA1 C-terminal(BRCT)domain in its motif.Considering this domain as an integrated signal module in response to DNA damage response,Pes1 may also be involved in DNA damage and repair.This study was to investigate the expression of proteins in Pes1 and PI3K/AKT / GSK-3? signaling pathways in mice with cholestatic liver disease and their effects on diseases.Methods:(1)To investigate the effects of cholestatic liver disease on mice,mice were fed with 0.1% DDC feed and normal diet for 4 weeks,respectively,to compare the differences in body mass between cholestatic mice and normal mice.(2)In order to understand the effect of cholestasis on the liver of mice,the liver of the mice was fixed with 4% paraformaldehyde,embedded in paraffin,sectioned,and subjected to routine pathological HE staining.The mice underwent observation of cholestatic liver disease under light microscope.Histopathological changes with normal mice.(3)In order to understand the effect of cholestasis on the serum of mice,take the serum of mice and test the total bile acid(TBA)and total biliary red in the serum of mice with cholestatic liver disease and normal mice according to the instructions of the biochemical reagent test kit.Levels of TBIL,alkaline phosphatase(AKP),gamma-glutamyltranspeptidase(GGT).(4)To investigate the expression of PES1 in the liver of mice with cholestatic liver disease,RT-PCR and Western Blot were used to detect the expression of PES1 mRNA and protein in tissues.(5)To further elucidate the effect of PES1 on PI3K/AKT / GSK-3? signaling pathway,extract total mouse liver protein and detect the expression of PI3K/AKT / GSK-3?signaling pathway-related proteins.(6)In order to select the appropriate siRNA to silence the expression of PES1 in mouse hepatocytes AML12,RT-PCR and Western Blot were used to detect the mRNA and protein expression of PES1 in mouse hepatocytes AML12 by transfection reagent.(7)To investigate the effect of PES1 on value-added ability,the PES1 gene in mouse liver cell AML12 was silenced with siRNA,and the value-adding ability in cells was determined by CCK-8 assay.(8)To investigate the effect of PES1 gene on PI3K/AKT / GSK-3? signaling pathway in silencing mouse hepatocyte AML12,Western Blot was used to detect the protein expression level of PI3K/AKT / GSK-3? and its phosphorylated protein in cells.Results:(1)Compared with normal C57BL/6 mice in the control group,the weight of mice fed DDC diet,ie,cholestatic liver disease,was significantly reduced.(2)The liver tissue structure of the normal control group was basically normal,and there were almost no inflammatory cells around the bile duct.However,the DDC model mice,that is,the liver tissue of the DDC model mice,showed that the portal area wasenlarged,and there was a large amount of inflammation around the portal area and the bile duct.Cell infiltration,bile duct deformation,and bile duct hyperplasia.(3)Take the serum of mice to detect the level of biochemical indicators in serum.Compared with the normal control group C57BL/6,the total bile acid(TBA),total bilirubin(TBIL)and alkaline phosphate in the serum of mice with cholestatic liver disease The levels of enzyme(AKP)and ?-glutamyltranspeptidase(GGT)were significantly increased.(4)The expression levels of PES1 mRNA and protein were down-regulated in mice fed DDC feed,ie,cholestatic liver disease,compared with normal mouse liver.(5)Compared with normal mouse liver,the expression levels of total PI3K,AKT and GSK-3? proteins in the liver of mice fed DDC feed,ie cholestatic liver disease,did not change significantly,while p-PI3K,p-Akt,p-The expression of GSK-3? protein is significantly reduced.(6)Detect the expression of PES1 mRNA and protein after siRNA transfected into mouse liver cells AML12,and use the small interfering sequences of siRNA-2 and siRNA-3 with better transfection to knock out PES1 in mouse liver cells AML12 for subsequent experiment of.(7)In the CCK-8 experiment,the proliferation of mouse hepatocytes after 24 h of transfection,BC group,si-NC group and si-PES1 group were similar to each other;but with the extension of transfection time,si-PES1 The proliferation ability of the group was gradually decreased compared with the BC group and the si-NC group.After 72 hours of transfection,the proliferation ability of the si-PES1 group was significantly lower than that of the BC group and the si-NC group.(8)Compared with normal mouse hepatocytes,the expression levels of total PI3K,AKT and GSK-3? proteins in AML12 after siRNA transfection did not change significantly,while p-PI3K,p-Akt,p-GSK-3? protein The expression is significantly reduced.Conclusion:(1)Cholestatic liver disease has an effect on the growth and development of mice,and has organic damage to liver cells.(2)PES1 is lowly expressed in mice with cholestatic liver disease and may protect the liver in cholestatic liver disease.(3)PES1 promotes cell proliferation and can be regulated by the PI3K/AKT / GSK-3?signaling pathway.