The Role And Mechanism Of NEDD4 In Hepatocyte Injury In Ketotic Cows

Ketosis is a major metabolic disorder of high-producing dairy cows during the transition period,whose pathological basis is hyper-free fatty academia and its hallmark is hyperketonemia.High concentrations of free fatty acid(FFA)and its metabolic products [β-hydroxybutyrate(BHB),reactive oxygen species(ROS),etc.] induce oxidative stress,mitochondrial damage,lipid accumulation,apoptosis and inflammation in calf hepatocytes.These pathological events are mutually promoted and thus lead to hepatocyte injury in ketotic cows.Hepatocyte injury affects milk production and quality,increases susceptibility to other diseases,and leads to poor outcomes of conventional treatment strategies.Thus,a better understanding of the molecular mechanisms controlling hepatocyte injury in ketotic cows and identification of feasible therapeutic targets are important to ensure profitability in the dairy industry.Neural precursor cell expressed developmentally downregulated 4(NEDD4)is an E3 ubiquitin ligase,whose aberrant expression is associated with multiple tissue injury.However,to the best of our knowledge,no prior study has reported the role of NEDD4 in hepatocyte injury in ketotic cows.Thus,this study aimed to investigate the potential role of NEDD4 in the progression of hepatocyte injury in ketotic cows and identify the ubiquitination substrate of NEDD4.For in vivo experiments,twenty dairy cows were selected into a healthy group(n= 10;serum BHB concentration < 0.6 m M)and a clinical ketosis group(n = 10;serum BHB concentration > 3.0 m M)according to their serum BHB concentration.Blood samples were collected for determing liver function.Compared with healthy cows,cows with clinical ketosis had higher activities of serum alanine aminotransferase,aspartate aminotransferase and lactate dehydrogenase,and lower albumin concentration,indicating abnormal liver function in dairy cows with clinical ketosis.Liver biopsies were used to detect the status of oxidative stress,mitochondrial damage and the protein and m RNA abundances of NEDD4.Cows with clinical ketosis had higher levels of hepatic ROS,malondialdehyde and 8-hydroxy-2-deoxyguanosine,lower activities of superoxide dismutase and glutathione peroxidase,and exhibited hepatic adenosine triphosphate depletion,mitochondrial swelling and cytosolic leakage of mitochondrial intermembrane proteins(apoptosis-inducing factor,cytochrome c and endonuclease G),indicating they suffered more pronounced oxidative stress and mitochondrial damage.Besides,the protein and m RNA abundance of NEDD4 was lower in the liver of cows with clinical ketosis than in healthy cows.The low abundance of NEDD4 may be a key node in the progression of hepatocyte injury in ketotic cows.High concentrations of FFA are the main inducer of hepatocyte injury in ketosis cows.Palmitic acid(PA)is a representative saturated fatty acid released during lipolysis.Thus,the present study used PA as a stimulator for in vitro experiments to construct a lipid-derived cell injury model in calf primary hepatocytes,and detected the status of oxidative stress,mitochondrial damage and cell injury,and the expression of NEDD4.Results showed that high concentration of PA induced oxidative stress,mitochondrial damage and cell death,and downregulated the expression of NEDD4 in calf hepatocytes.To further confirm the function of NEDD4 in hepatocyte injury in ketotic cows,we performed gain-and loss-of function experiments by transfecting adenovirus delivering cattle NEDD4 gene sequence(Ad-NEDD4)or small interfering RNA targeting cattle NEDD4(si NEDD4),in the setting of PA treatment.Compared to AdControl group,Ad-NEDD4 group showed alleviation of oxidative stress,mitochondrial damage and cell injury in response to PA challenge,as determined by the hallmarks of oxidative stress(mitochondrial ROS,malondialdehyde and 8-hydroxy-2-deoxyguanosine levels),mitochondrial damage(mitochondrial membrane potential,mitochondrial swelling,cytosolic leakage of mitochondrial intermembrane proteins,and mitochondrial respiratory function)and cell injury(cell viability,lactate dehydrogenase release rate,and the activities of alanine aminotransferase and aspartate aminotransferase in the cell culture medium).Consistent with gain-of-function experiment,knockdown of NEDD4 exacerbated PA-induced oxidative stress,mitochondrial damage and cell injury.Taken together,these in vitro results reveal that NEDD4 protects calf hepatocytes against the hepatotoxicity of PA by regulating oxidative stress and mitochondrial damage,foreshadowing the protective effect of NEDD4 against hepatocyte injury in dairy cows with clinical ketosis.The striking effect of NEDD4 on mitochondrial intermembrane protein leakage in the setting of PA treatment prompted us to investigate whether the protection of NEDD4 against the hepatotoxicity of PA is involved in voltage-dependent anion channel 1(VDAC1),which is the most abundant gatekeeper in the mitochondrial outer membrane and regulates mitochondrial outer membrane permeabilization.The present study showed that the protein abundance of VDAC1 in dairy cows with clinical ketosis was greater than in healthy cows.PA significantly increased the protein abundance of VDAC1.Moreover,overexpression of NEDD4 significantly decreased the protein abundance of VDAC1 in the setting of PA hepatotoxicity.However,m RNA expression of VDAC1 did not differ significantly,suggesting that the changes in the VDAC1 protein abundance was not due to changes in transcription.Functional studies showed that knockdown of VDAC1 significantly alleviated PA-induced oxidative stress,mitochondrial damage and cell injury.More importantly,silencing VDAC1 significantly alleviated the enhanced hepatotoxicity of PA caused by NEDD4 knockdown,indicating that NEDD4 protects calf hepatocytes against PA hepatotoxicity by regulating VDAC1.Mechanistically,the present study demonstrated that the decrease of VDAC1 protein abundance caused by NEDD4 overexpression could be blocked by the proteasome inhibitor MG132 but not by the lysosome inhibitor chloroquine,emphasizing that NEDD4 regulates the proteasomal degradation of VDAC1.Subsequent co-immunoprecipitation assay demonstrated the interaction of NEDD4 with VDAC1.Ubiquitination assay demonstrated that NEDD4 significantly facilitated the ubiquitination of VDAC1 in calf primary hepatocytes.These data indicate that NEDD4 interacts with VDAC1 and promotes the ubiquitination of VDAC1,eventually resulting in its degradation.In summary,the present data indicated that the E3 ubiquitin ligase NEDD4 protects calf hepatocytes against PA-induced oxidative stress,mitochondrial damage and cell injury by targeting VDAC1 for proteasomal degradation,thus plays a protective role in the progression of hepatocyte injury in ketotic cows.