The Mechanism Of Liver Injury Induced By FFA-blocked Autophagy In Ketotic Cows

During the transition period,sharply increased energy expenditure and decreased feed intake causes high yielding dairy cows to go into negative energy balance(NEB).Prolonged of NEB results in cows mobilize adipose tissue and release a large amount of free fatty acids(FFA)into the blood.Subsequently,hepatic FFA uptaken were increased,resulting in a large portion of FFA in the liver are incompletely oxidized to ketone bodies,including?-hydroxy butyric acid(BHB),acetoacetic acid(AA),and acetone.High concentrations of FFA and BHB display lipotoxicity,and leading to hepatic pathological damage such as oxidative stress and apoptosis,as well as lipid metabolism dysfunction in the liver.It was reported that ketotic cows were existed in systemic inflammation.However,a link between ketosis and hepatic inflammation has not been well documented.Autophagy is a fundamental cytoprotective process involving degradation of unwanted proteins,macromolecular complexes,or dysfunctional organelles that are carried out by lysosomal mechanisms.It was reported that induction of autophagy alleviated hepatic lipid accumulation,while inhibition of autophagy further aggravated intracellular fat accumulation in human and mice with nonalcoholic fatty liver disease.In addition,impaired hepatic autophagic activity in dairy cows with severe fatty liver is associated with inflammation and hepatic dysfunction.The fact that severe fatty liver was also associated with hyperketonemia led to the suggestion that alterations in autophagy may contribute to the development of ketosis.However,the specific mechanism is still unknown.Therefore,the purpose of this experiment is to explore the role of autophagy in the development of liver injury in ketotic cows.Collected blood and liver samples from clinical ketotic(CK)cows and healthy cows to detect liver damage and liver inflammation.Results showed that compared with healthy cows,serum aspartate amino transferase(AST)and alanine amino transferase(ALT)were higher in CK cows.Concentrations of serum pro-inflammatory cytokines IL18,tumor necrosis factor(TNF)-?,and IL1B were greater and the anti-inflammatory cytokine IL10 was lower in the CK group,indicating that CK cows displayed liver damage and systemic inflammation.Moreover,CK cows displayed hepatic lipid accumulation.The expression of phosphorylated(p)-NF-?B and p-inhibitor of?B(I?B)?proteins increased,the upregulation of m RNA abundance of TNFA,nitric oxide synthase 2(NOS2),chemokine monocyte chemoattractant protein-1(MCP-1),and downregulation of IL10 m RNA abundance in cows with CK indicated that the hepatic NF-?B signaling pathway was over activated.Furthermore,the m RNA and protein abundance of NLRP3 and caspase-1(CASP1)along with CASP1 activity were greater in the liver of cows with CK,suggesting that the NLRP3 inflammasomes were over activated in ketotic cows.Collected blood and liver samples from subclinical ketotic cows(SCK),clinical ketotic(CK)cows and healthy cows to measure the status of oxidative stress,autophagy and mitophagy in the liver.Results showed that levels of oxidative stress biomarkers malondialdehyde(MDA)and hydrogen peroxide(H₂O₂)were higher in liver tissue from ketotic(SCK and CK)cows than healthy cows,indicating that ketotic(SCK and CK)cows displayed oxidative stress,and it was more severe in CK cows.Compared with cows with CK and healthy cows,the hepatic m RNA abundance of microtubule-associated protein 1 light chain 3(MAP1LC3,also called LC3),sequestosome-1(SQSTM1,also called p62),autophagy related genes(ATG)5,ATG7,ATG12,and phosphatidylinositol 3-kinase catalytic subunit type 3(PIK3C3)was upregulated in cows with SCK.Compared with healthy cows,SCK cows had a lower abundance of p62 and a greater abundance of LC3-II,indicating that the activity of autophagy was increased in SCK cows.Inversely,the expression of p62 and LC3-II protein was higher,and the SQSTM1 and MAP1LC3 m RNA abundance were lower in CK cows,which indicating the formation and degradation of autophagosome were damaged.Furthermore,the hepatic protein abundance of PTEN-induced putative kinase 1(PINK1)and Parkin was greater in cows with SCK and slightly lower in cows with CK than in healthy cows.Overall,these data demonstrated the hepatic mitophagy were enhanced in SCK cows,while,they were impaired in CK cows.Next,it was measured the effect of different concentrations of FFA,rapamycin,and chloroquine(CQ)on the oxidative stress,inflammatory signaling and the release of pro-inflammatory factors in cultured calf hepatocytes.Results revealed that 0.6 and1.2 m M FFA activated NF-?B signaling and NLRP3 inflammasome as indicated by an elevated ratio of p-NF-?B/NF-?B,protein abundance of NLRP3 and caspase-1(CASP1),activity of CASP1,and m RNA abundance of interleukin(IL)1B and IL18.The levels of MDA and H₂O₂ were higher in the 0.6 and 1.2 m M FFA treated groups than control group,suggesting that 0.6 and 1.2 m M FFA treatment induce oxidative stress in hepatocytes.In addition,greater protein abundance of p62 and LC3-II in response to 0.6 and 1.2 m M FFA indicated an impairment of autophagy.CQ plus 1.2m M FFA aggravated FFA-activated NF-?B signaling and NLRP3 inflammasome,oxidative stress,and autophagy activity.While,induction of autophagy by rapamycin ameliorated these results.Overall,high concentrations of FFA impaired the hepatocytes autophagy and triggers oxidative stress and over activation of inflammatory pathways.Activation of autophagy alleviated the hepatocytes oxidative stress and inflammatory signaling pathways induced by FFA.In conclusion,these results showed that high FFA blocked hepatic autophagy in ketotic cows,triggers oxidative stress and inflammatory pathways activation,which leading to liver damage.These results partially clarify the pathogenesis of ketosis in dairy cows,and lay a theoretical foundation for the prevention and treatment of liver damage in dairy cows with ketosis by regulating autophagy.