| Objective In the present study,fatty liver dairy cows and dairy cows fatty liver cell model in vitro will be used as the research object,and overexpression SIRT1 gene via adenovirus transfection was used to study the effect of SIRT1 on oxidative stress status,SIRT1 gene and protein expression levels in dairy cows,the expression and activity of downstream lipid metabolism related proteins and key enzymes and hepatic lipid deposition.The clarify of the mechanism of SIRT1 in the development of fatty liver disease in dairy cows will lay a theoretical and experimental basis for elucidating the pathogenesis and searching therapeutic targets of fatty liver disease in dairy cows.Methods Twelve cows were selected and blood were collected from the jugular vein for serum.After routinely liver samples were aseptically collected.According to the serum and liver tissue TG content,6 control cows and 6 fatty liver cows were selected.Fresh calf liver cells were isolated and cultured by modified collagenase two-step perfusion method.Calf liver cells were treated with various concentrations of OA(0,0.25,0.5,0.75 mM)for different time(12,24,36,48 h)to determine the optimal concentration and time of OA in inducing hepatic steatosis cell model.Dairy fatty liver cells were cultured in vitro to the optimal growth state.Liver cells were treated with NEFAs,NAC,NEFAs+NAC,and overexpressed SIRT1.Liver cells were collected at the end of co-culture.Lipid metabolism indexes were detected by automatic biochemical analyzer;oxidative and antioxidant indexes in serum and liver tissues were measured by enzyme-linked immunosorbent assay(ELISA);the optimal amount of recombinant adenovirus was determined by immunofluorescence;The mRNA expression levels of SIRT1 and its downstream transcription factors were detected by real-time fluorescence quantitative PCR(qRT-PCR).The protein expression levels of SIRT1 gene and downstream transcription factors in liver tissues and hepatocytes were detected by Western blotting.Results:1.Compared with control cows,the concentrations of ALT,ALP and GLU in fatty liver cows were significantly increased(P<0.01),and the concentration of TP was significant increased(P<0.05),and the lipid metabolism indexes TG and TC were decreased,but there were no significance(P>0.05).The ROS concentration was significantly increased(P<0.05),and the CAT and CuZn-SOD concentrations were significantly reduced(P<0.01).2.Compared with control cows,the liver enzyme activity of SIRT1 in fatty liver cows was significantly decreased(P<0.05),SREBP-1c was not significant(P>0.05),and PGC-1α was significantly reduced(P<0.01);the relative mRNA expression levels of SIRT1,PGC-1α,and ACO involved in regulating lipid oxidation were significantly reduced(P<0.01),protein expression was significantly reduced(P<0.01),and the relative mRNA and protein expression levels of PPARα,RXRα,and CPT1.The relative mRNA expression of CPT2 decreased significantly(P<0.05),and the protein expression of CPT2 decreased significantly(P<0.01).The relative mRNA expressions of Nrf1 and TFAM decreased significantly(P<0.01),the protein expressions of Nrf1 and TFAM decreased significantly(P<0.05).3.After OA modeling,compared with the control group,the contents of TG and TC in fatty liver cells of the model group increased significantly(P<0.05),and the content of VLDL did not increase significantly.The relative mRNA expressions of SIRT1 and PGC-1α were significantly reduced(P<0.05),the protein expressions of SIRT1 and PGC-1α were significantly reduced(P<0.01).The relative mRNA and protein expressions of SREBP-1c were significantly increased(P<0.01).4.Compared with normal group,the content of TG and TC in NEFAs group increased significantly(P<0.01);TG content decreased significantly(P<0.05),and TC decreased significantly(P<0.01)in NAC group.Addition of NEFAs+NAC groups did not change significantly.The relative mRNA expression of SIRT1 was significantly(P<0.05)and the protein expressions of SIRT1 and SREBP-1c were significantly decreased(P<0.05)in NEFAs group.The relative mRNA expression of SIRT1 and SREBP-1c significantly increased(P<0.05)and protein expression of SIRT1,SREBP-1c,and PGC-1α significantly increased(P<0.01)in NAC group.5.Compared with the control group,after transfection with recombinant adenovirus overexpressing SIRT1,the mRNA expression of ApoE and LDLR,which regulate lipid transport were significantly reduced(P<0.01),and SIRT1,PGC-1α,and RXRα mRNA involved in lipid oxidation were expressed significantly increased(P<0.01),and PPARα mRNA expression was significantly increased(P<0.05),SREBP-1c and ACCα genes mRNA expression involved in lipid synthesis were significantly increased(P<0.01),FAS mRNA expression was significantly increased(P<0.05);The relative protein expression of SIRT1 and the downstream target genes PPARα and RXRα in PGC-1α pathway significantly increased(P<0.05),The protein expressions of SREBP-1c、ApoE and LDLR significantly decreased(P<0.05).The protein expressions of FAS was significantly decreased(P<0.01),and p-ACC expression was significantly increased(P<0.01).Conclusion The above results show that oxidative stress in periparturient fatty liver cows interferes with lipid metabolism,induces hepatic lipid deposition mediated by oxidative stress,simultaneously promotes the accumulation of hepatic fat by modulating the SIRT1-SREBP-1c/PGC-1α pathway... |
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