| Objective To explore the effect of circulating exosomes on the relative expression levels of key proteins and m RNA of AMPKα-SIRT1 pathway in dairy cow liver cells.Methods 15 perinatal non-ketosis,subclinical ketosis and clinical ketosis cows were screened,blood samples were collected to detect liver function,kidney function and lipid metabolism and other indicators,purified exosomes for miRNA analysis,screening differential miRNA.Meanwhile,primary liver cells of dairy cows were isolated in vitro,exosomes were used to stimulate 1.2 m M and 2.4 m M NEFA-induced hepatocytes.The relative m RNA expression levels and protein expression levels of p AMPK,AMPK,SIRT1,PGC-1 and SREBP-1C in liver cells were detected by realtime fluorescence quantitative PCR(QRT-PCR)and western blot,respectively.The overexpression of SIRT1 adenovirus was then transfected into NEFA-induced hepatocytes,and compared with the optimal concentration of circulating exosomes,the above methods were used to detect changes in the expression level of key genes and enzymes in the AMPKα-SIRT1 pathway.Results:1.The saucer-like structure of exosomes was observed by TEM,and the expressions of marker proteins Alix,CD9,CD63 and HSP70 were detected by western blotting.NTA traced the particle size distribution to 30-150 nm.2.The results of miRNA omics analysis showed that differential miRNAs are mainly involved in metabolic pathways,and a total of miRNAs were screened for up 10 and down 10.3.The treatment of 40 μg/mL circulating exosomes stimulated for 12 h is the best concentration and time to improve primary hepatocyte activity induced by NEFA in dairy cows.4.In 1.2 mM and 2.4 mM NEFA-induced hepatocytes,compared with the control group,the relative m RNA and protein expression levels of AMPK,PGC-1 and SIRT1 were increased,the relative m RNA and protein expression levels of SREBP-1C were decreased in 40 μg/m L circulating exosomes of non-ketosis and subclinical ketosis cows.5.After induction of liver cells with 1.2 m M NEFA,relative m RNA and protein expression levels of SIRT1 and PGC-1 increased and SREBP-1C decreased in the SIRT1 overexpression group when compared with the blank control group.Conclusion Liver lipid metabolism disorder exists in cows with ketosis,and miRNA sequencing results indicate that circulating exosomes in perinatal cows are intrinsically related to liver lipid metabolism disorder.In vitro experiments show that circulating exosomes in non-ketogenic and subclinical ketogenic cows can increase the expression of AMPK,SIRT1 and PGC-1α in NEFA-induced hepatocytes,and decrease the expression of SREBP-1C,that is to say,the circulating exosomes mediated AMPKα-SIRT1 pathway to regulate NEFA-induced liver lipid metabolism disorder and repair liver cell injury,which was consistent with the transfection effect of overexpressed SIRT1 adenovirus,and the effect of subclinical ketosis cows on circulating exosomes was superior to that of non-ketosis cows. |
