Nefas Activate NF-?B-Orai1 Signaling Pathway To Mediate The Regulation Mechanism Of De Novo Lipogenesis In Calf Hepatocytes

Transitional dairy cow fatty liver disease is an important nutritional metabolic disease of dairy cows.It is often found in high-yield dairy cows in transitional period.The cause of the disease is that during the transitional period,the feed intake of dairy cows decreases,the demand for lactation rises,and the peak of lactation is earlier than the peak of feeding,which leads to negative energy balance in the body and produces fat mobilization to generate energy to supply the body with lactation.A large number of non-esterified fatty acids(NEFAs)produced by fat mobilization are transported to the liver,and a large amount of triglycerides(TG)are synthesized in the liver,far exceeding the amount that the liver can output in the form of very low-density lipoprotein(VLDL).Thus,A large amount of TG accumulates in the liver.Transitional dairy cow fatty liver disease which reduces the feed intake and milk yield of dairy cows,damages the reproductive capacity and immune function of dairy cows,reduces the quality of dairy cows' milk production,increases the risk of various postpartum diseases in dairy cows,and increases the elimination rate of dairy cows,will causes a huge economic loss to the dairy industry every year.In order to explore the effects of NEFAs on de novo lipogenesis in dairy cows,the role of NEFAs in the development of fatty liver disease in dairy cows,and to explore whether Ca2+-associated NF-?B-Orai1 signaling pathway is regulated by NEFAs-mediated de novo lipogenesis in dairy cows,we cultured primary calf hepatocytes in vitro,added NEFAs to stimulate cells,and added NF-?B inhibitor Wogonin and Orai1 inhibitor 2-APB to block the pathway.By detecting intracellular concentration changes of Ca2+,the expression regulation of the de novo lipogenesis related regulatory factor,SREBP-1c,ACC1 and FAS,and the observation of cell lipid droplet enrichment,we comprehensively evaluated the effects of NEFAs in the development of fatty liver disease in dairy cows.In addition,we verified the role of Orai1 signaling pathway on de novo lipogenesis of transitional dairy cows by cell silencing experiments.Intracellular Ca2+ flow cytometry showed that intracellular Ca2+ concentration increased with the prolongation of NEFAs stimulation time.There was a significant difference between 0 h and 3 h,9 h in intracellular Ca2+(p<0.05),and there was an extremely difference between 0 h and 6 h,9 h in intracellular Ca2+(p<0.01).The results of QRT-PCR detection of m RNA expression levels of NF-?B p65,Orai1,Srebp-1c,Fas and Acc1 in different treatments of primary calf hepatocytes showed that the m RNA expression levels of NF-?B p65,Orai1,Srebp-1c,Fas and Acc1 in NEFA group were extremely significantly higher than those in controlgroup(p<0.01).The m RNA expression levels of NF-?B p65 and Srebp-1c in NEFA group pretreated with 2-APB reagent were significantly lower than those in NEFA group(p<0.05),and the m RNA expression levels of Orai1,Fas and Acc1 were extremely significantly lower than NEFA Group(p<0.01).The m RNA expression level of Fas in NEFA group pretreated with Wogonin reagent was significantly lower than that in NEFA group(p<0.05).The m RNA expression levels of NF-?B p65,Orai1,Srebp-1c and Acc1 were extremely significantly lower than those in NEFA group(p<0.01).The results of Western Blotting detection of protein expression levels of NF-?B p65,ORAI1,SREBP-1,FAS and ACC1 in different treatments of primary calf hepatocytes showed that The protein expression levels of ORAI1 and ACC1 in NEFA group were significantly higher than those in control group(p<0.05),and the protein expression levels of NF-?B p65,SREBP-1 and FAS were extremely significantly higher than that of control group(p<0.01).The protein expression levels of ORAI1 and SREBP-1 in NEFA group pretreated with 2-APB reagent were significantly lower than those in NEFA group(p<0.05),and the protein expression levels of NF-?B p65,FAS and ACC1 were extremely significantly lower than NEFA group(p<0.01).The protein expression levels of FAS and ACC1 in NEFA group pretreated with Wogonin reagent were significantly lower than those in NEFA group(p<0.05),and the protein expression level of NF-?B p65 was extremely significantly lower than that in NEFA group(p<0.01).The lipid droplet enrichment levels of NF-?B p65,Orai1,SREBP-1c,FAS and ACC1 have differences in different treatments of primary calf liver cells.The lipid droplet enrichment level in NEFA group was extremely significantly higher than that in control group(p<0.01),the lipid droplet enrichment level of NEFA group pretreated with2-APB and pretreated with Wogonin reagent was significantly lower than that of NEFA group(p<0.05).After silencing the Orai1 gene,the m RNA and protein expression levels of Orai1,SREBP-1c,FAS and ACC1 were significantly reduced(p<0.05).The results showed that NF-?B can activate Orai1 signaling pathway in dairy cows,stimulate cellular Ca2+influx,regulate the up-regulation of hepatic de novo lipogenesis regulators,increase the synthesis of NEFAs and increase the risk of fatty liver disease in transitional dairy cows.This experiment is important for the research,prevention,diagnosis and treatment of fatty liver in transitional dairy cows.The research on Orai1 pathway may open a new way for drug development of transitional dairy cows fatty liver disease.