| Objective: Non-alcoholic steatohepatitis(NASH)is a very common chronic liver disease worldwide.Its pathogenesis is lipid accumulation in the liver,fatty degeneration of hepatocytes,and lipid peroxidation of hepatocytes.And oxidative stress and hepatic inflammatory cell infiltration are the main features.Mammalian target of rapamycin(mTOR)is an important regulatory factor for cell proliferation,differentiation and apoptosis,and protein synthesis.It is also involved in the pathogenesis of NASH.The presence or absence of P18/LAMTOR1 affects the ability of mTORC1 to localize on the surface of lysosomal membranes and is activated to exert physiological and pathological effects.This study focuses on the effect of specific knockout of liver P18/LAMTOR1 gene on lipid metabolism,inflammation and key signaling factors related to ER stress signaling pathway in liver tissue of NASH,and further explores the mechanism of P18/LAMTOR1 affecting NASH through mTORC1.Methods: 1.Construction and detection of P18/LAMTOR1 liver-specific knockout mice: Using a mouse strain with background C57BL/6,using a targeting vector homologous recombination technique,a loxp sequence was inserted on each side of the hepatocyte genome P18/LAMTOR1 exon.The specific gene was knocked out by the Cre-Loxp recombinase system because the Cre protein recognizes the loxp sequence and excise the gene fragment between the two loxp sequences.The Cre protein was specifically expressed in the liver by inserting a sequence linked to the albumin promoter and Cre gene in the Albumin-Cre(Alb-Cre)mouse genome.The two mice were crossed a number of times to obtain the progeny mice with genotypes of P18/LAMTOR1 flox/flox,Alb-Cre,and the purpose of liver-specific knockout of the P18/LAMTOR1 gene was achieved.That is P18/LAMTOR1 LKO(liver knockout)mice in the experimental group.2.Animal rearing and animal handing: All mice were housed in an SPF barrier environment.In this study,21-day-old mice were used for the initial hybrid breeding.Each litter of offspring male mice was subjected to genetic identification.When P18/LAMTOR1 gene was targeted for knockout,it was raised to 8-10 weeks of age.They were sacrificed and male littermates were used as experimental controls(P18/LAMTOR1 flox/flox group).Blood was collected from both groups of mice for liver extraction.3.Measurement indicators and methods: 3.1Determination of serum ALT and AST levels in two isolated mice using an automated biochemical analyzer;3.2 HE staining of liver tissues of two groups of mice;3.3 Western Blotting assay detected the expression of mTOR,P-mTOR,P-S6 K and P-JNK protein in liver tissue of two groups;3.4 RT-PCR detection of liver tissue inflammation related factors IL-1? and mi NOS,endoplasmic reticulum stress related factors BIP,CHOP and ERDJ4,lipid synthesis related factors SREBP1 c,SCD1,FASN,ACC1 and DGAT1,lipids The mRNA expression levels of the relevant factors PPAR?,PPAR?,ACOX1,PGC1?,and FGF21 were decomposed.Results: 1.Changes of serum ALT and AST in two groups: The level of ALT and AST in serum of P18/LAMTOR1 LKO experimental group(ALT:55.786±11.131 U/L,AST:83.740±14.729 U/L)was significantly higher than that in the P18/LAMTOR1 flox/flox group(ALT:22.412±6.592 U/L,AST:30.898±14.831 U/L),and the difference was statistically significant(P<0.05).2.Comparison of HE staining of liver tissue in two groups: Under the light microscope,the liver sinus in the P18/LAMTOR1 flox/flox control group was clearly visible in the liver tissue,the hepatic cord was arranged neatly,and the hepatic lobule was borderlined.The liver cells were normal in size and arranged in order,with round and central nucleus,even cytoplasm staining,no accumulation of lipid droplets,and no changes in steatosis and inflammation.In the P18/LAMTOR1 LKO experimental group,the hepatic sinus in the liver tissue was narrowed or even disappeared,and the hepatic cord was arranged disorderly in some areas,and the boundary of the hepatic lobule was unclear.Hepatocytes become swollen and their boundaries blur.In the cytoplasm,lipid droplets of different sizes were observed,and the steatosis of the cells was spot-shaped.Inflammatory cell infiltration was also observed.3.Liver inflammatory changes in two groups: The results of RT-PCR showed that the expression of IL-1? and mi NOS mRNA of the inflammation-related factors in the liver of P18/LAMTOR1 LKO mice(IL-1? : 0.013±0.005,mi NOS : 0.002±0.006)was significantly higher than that in the P18/LAMTOR1 flox/flox control group(IL-1?:0.0007±0.0004,mi NOS : 0.0002±0.00009),and the difference was statistically significant(P<0.05).P-JNK Protein express obviously.4.Changes of mTOR signaling pathway-related factor protein expression in liver of two groups: P18/LAMTOR1 knockout inhibited mTOR and S6 K activation,p-mTOR protein expression was slightly decreased,and p-S6 K protein expression was significantly reduced without effect.Expression of total mTOR protein was not affected.5.mRNA expression changes of endoplasmic reticulum stress-related factors in the liver of two groups of mice: RT-PCR results showed that the expression levels of endoplasmic reticulum stress-related factors BIP,CHOP and ERDJ4 mRNA in the P18/LAMTOR1 LKO mice(BIP:0.246±0.002,CHOP:0.003±0.0003,ERDJ4:0.046±0.005)were significantly higher than that in the P18/LAMTOR1 flox/flox group(BIP:0.188±0.025,CHOP:0.002±0.0007,ERDJ4:0.035±0.003),and the difference was statistically significant(P<0.05).6.mRNA expression changes of hepatic lipid metabolism factors in two groups of mice: The results of RT-PCR showed that compared with the P18/LAMTOR1 flox/flox control group,the lipid metabolism related factors SREBP1c(experimental group: 0.002±0.0001 vs control group: 0.053±0.008),SCD1 in the liver tissue of the P18/LAMTOR1 LKO mice were significantly reduced,FASN,ACC1,DGAT1,PPAR?(experimental group: 0.011±0.0002 vs control group: 0.004±0.002),PPAR?,ACOX1,PGC1? and FGF21 were significantly increased,and the difference was statistically significant(P<0.05).Conclusion: 1.Liver-specific knockout of P18/LAMTOR1 gene can inhibit the phosphorylation of S6 K downstream of mTORC1 and aggravate the deterioration of liver function and inflammation in NASH mice;2.Liver-specific knockout of P18/LAMTOR1 gene can enhance endoplasmic reticulum stress in NASH mice;3.Liver-specific knockout of P18/LAMTOR1 gene can significantly affect lipid metabolism in NASH mice,including lipid synthesis and lipolysis. |
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