Dairy cows with ketosis are characterized by oxidative stress, hepatic damage, and hyperketonemia. Acetoacetate (AA) is the main component of ketone bodies in ketotic cows, and is associated with the above pathological process. However, the potential mechanism was not illuminated. Therefore, the aim of this study was to investigate the mechanism of AA-induced hepatic oxidative damage in ketotic cows. Compared with healthy cows, ketotic cows exhibited severe oxidative stress and hepatic damage. Moreover, the extent of hepatic damage and oxidative stress had a positive relationship with the AA levels. In vitro, AA treatment increased reactive oxygen species (ROS) content and further induced oxidative stress and apoptosis of bovine hepatocytes. In this process, AA treatment increased the phosphorylation levels of JNK and p38MAPK and decreased the phosphorylation level of ERK, which could increase p53 and inhibit nuclear factor E2-related factor 2 (Nrf2) expression, nuclear localization, and DNA-binding affinity, thereby inducing the overexpression of pro-apoptotic molecules Bax, Caspase 3, Caspase 9, PARP and inhibition of anti-apoptotic molecule Bcl-2. Antioxidant N-acetylcysteine (NAC) treatment or interference of MAPKs pathway could attenuate the hepatocytes apoptosis induced by AA. Collectively, these results indicate that AA triggers hepatocytes apoptosis via the ROS-mediated MAPKs pathway in ketotic cows.

ABSTRACTThe transition period is a severe challenge to dairy cows. Glucose supply cannot meet demand and body fat is mobilized, potentially leading to negative energy balance (NEB), ketosis, or fatty liver. Propionate produces glucose by gluconeogenesis, which depends heavily on the number and species of microbes. In the present study, we analyzed the rumen microbiome composition of cows in the transition period, cows with ketosis, and nonperinatal cows by terminal restriction fragment length polymorphism (TRFLP) analysis of 16S rRNA genes and quantitative PCR. TRFLP analysis indicated that the quantity ofVeillonellaceaeorganisms was reduced and that ofStreptococcaceaeorganisms was increased in rumen samples from the transition period and ketosis groups, with the number ofLactobacillaceaeorganisms increased after calving. Quantitative PCR data suggested that the numbers of the main propionate-producing microbes,Megasphaera elsdeniiandSelenomonas ruminantium, were decreased, while numbers of the main lactate-producing bacterium,Streptococcus bovis, were increased in the rumen of cows from the transition period and ketosis groups, with the number ofLactobacillussp. organisms increased after calving. Volatile fatty acid (VFA) and glucose concentrations were decreased, but the lactic acid concentration was increased, in rumen samples from the transition period and ketosis groups. Our results indicate that the VFA concentration is significantly related to the numbers ofSelenomonas ruminantiumandMegasphaera elsdeniiorganisms in the rumen.

β-Hydroxybutyrate (BHB) is an important indicator for metabolic disorders in dairy cows, such as ketosis and fatty liver. Dairy cows with ketosis display oxidative stress that may be associated with high levels of BHB. The purpose of this study was to demonstrate a correlation between the high levels of BHB and oxidative stress in dairy cows with ketosis, and to investigate the molecular mechanisms underlying oxidative damage in bovine hepatocytes. The results showed that dairy cows with ketosis exhibited oxidative stress and liver damage, which was significantly correlated with plasma BHB. Similarly, high concentrations of BHB increased the oxidative stress of cow hepatocytes in vitro, resulting in the phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK), which led to increased expression, nuclear localization, and transcriptional activity of p53 and decreased Nrf2 in bovine hepatocytes. High concentrations of BHB significantly increased the expression of proapoptotic genes and significantly inhibited the expression of antiapoptotic genes. Finally, high concentrations of BHB promoted apoptosis in bovine hepatocytes. N-Acetyl-l-cysteine, glucose, and SB203580 (p38 inhibitor) significantly attenuated BHB-induced apoptotic damage in hepatocytes. These results indicate that BHB induces bovine hepatocyte apoptosis through the ROS-p38-p53/Nrf2 signaling pathway.

Background/Aims: Dairy cows with ketosis are characterized by oxidative stress and hepatic damage. The aim of this study was to investigate hepatic oxidative stress and the apoptotic status of ketotic cows, as well as the underlying apoptosis pathway. Methods: The blood aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamate dehydrogenase (GLDH) and gamma-glutamyl transferase (GGT) activities and the haptoglobin (HP), serum amyloid A (SAA) and serum apoptotic cytokeratin 18 neo-epitope M30 (CK18 M30) concentrations were determined by commercially available kits and ELISA kits, respectively. Liver histology, TUNEL and Oil red O staining were performed in liver tissue samples. TG contents were measured using an enzymatic kit; Caspase 3 assays were carried out using the Caspase 3 activity assay kit; oxidation and antioxidant markers were measured using biochemical kits; apoptosis pathway were determined by qRT-PCR and western blot. Results: Ketotic cows displayed hepatic fat accumulation. The hepatic malondialdehyde (MDA) content was significantly increased, but the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were markedly decreased in ketotic cows compared with control cows, indicating that ketotic cows displayed severe oxidative stress. Significantly higher serum levels of the hepatic damage markers AST, ALT, GGT and GLDH were observed in ketotic cows than in control cows. The blood concentration of the apoptotic marker CK18 M30 and the number of TUNEL-positive cells in the liver of ketotic cows were 1.19- and 2.61-fold, respectively, higher than the values observed in control cows. Besides, Caspase 3 activity was significantly increased in the liver of ketosis cows. Importantly, the levels of phosphorylated c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK) were significantly increased but the level of phosphorylated extracellular signal-regulated kinase1/2 (ERK1/2) was markedly decreased, which further promoted tumor protein 53 (p53) expression and inhibited nuclear factor E2-related factor 2 (Nrf2) expression. The apoptosis-related molecules p21, MDM2, Caspase 3, Caspase 9 and Bax were expressed at significantly higher levels in ketotic cows than in healthy cows, whereas the anti-apoptosis molecule Bcl-2 was expressed at significantly lower levels. Conclusions: Based on these results, ketotic cows display severe hepatic oxidative stress. The hepatic MAPK-p53-Nrf2 apoptotic pathway is over induced and partially mediated apoptotic damage in the liver.

BackgroundSubacute ruminal acidosis (SARA) is a metabolic disease in high-producing dairy cattle, and is accompanied by rumenitis. However, the mechanism of rumenitis remains unclear. Therefore, the aim of this study was to investigate the molecular mechanism of rumenitis in dairy cows with SARA.ResultsThe results showed that SARA cows displayed high concentrations of ruminal volatile fatty acids, lactic acid and lipopolysaccharide (LPS). Furthermore, the blood concentrations of LPS and acute phase proteins haptoglobin, serum amyloid-A, and LPS binding protein were significantly higher in SARA cows than in control cows. Importantly, the phosphorylation levels of nuclear factor-kappaB (NF-κB) p65, inhibitor of NF-κB (IκB), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2) were significantly higher in the rumen epithelium of SARA cows than those of control cows. The ruminal mRNA and protein levels of NF-κB- and mitogen-activated protein kinase (MAPK)s -regulated inflammatory cytokines, tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and interleukin 1β (IL-1β), were markedly higher in SARA cows than in control cows. Similarly, serum concentrations of TNF-α and IL-6 were also significantly higher in SARA cows.ConclusionsThese results indicate that SARA results in high concentration of ruminal LPS, which over activates the NF-κB and MAPKs inflammatory pathways and then significantly increases the expression and synthesis of pro-inflammation cytokines in the rumen epithelium, thereby partly inducing rumenitis.

Aucubin ( AU ) is the main active ingredient of Aucuba japonica which has showed many positive effects such as anti‐inflammation and liver protection. Non‐alcoholic fatty liver disease ( NAFLD ) is the most common cause of chronic liver disease. In this research, we explored the effects of AU on the tyloxapol‐induced NAFLD in mice and apolipoprotein C‐ III (apoC‐ III ) induced‐3T3L1 cells. Tyloxapol (300 mg/kg) was injected to C57 BL /6 mice with aucubin. The differentiated 3T3‐L1 cells were treated with or without aucubin after stimulation of apoC‐ III (100 μg/mL). In results, aucubin inhibited hyperlipidaemia, oxidative stress and inflammation by influencing the content of total cholesterol ( TC ), triglyceride ( TG ), low density lipoprotein ( LDL ), very low density lipoprotein ( VLDL ), myeloperoxidase ( MPO ), superoxide dismutase ( SOD ), tumour necrosis factor receptor‐α ( TNF ‐α), interleukin‐1β ( IL ‐1β), and IL ‐6 in blood. AU activated NF ‐E2‐related factor 2 (Nrf2), peroxisome proliferator‐activated receptor α ( PPAR α), PPAR γ and hemeoxygenase‐1 ( HO ‐1) and promoted the phosphorylation of adenosine 5′‐monophosphate‐activated protein kinase ( AMPK α), AMPK β, acetyl‐CoA carboxylase ( ACC ) and protein kinase B ( AKT ). In conclusion, AU performed the function of hypolipidaemic by its obvious anti‐inflammation and antioxidant activity, which may become a kind of new drug targeting at NAFLD .

The inevitable deficiency in nutrients and energy at the onset of lactation requires an optimal adaptation of the hepatic metabolism to overcome metabolic stress. Fatty liver is one of the main health disorders after parturition. Therefore, to investigate changes in hepatic lipid metabolic status and mitochondria in dairy cows with mild fatty liver, liver and blood samples were collected from healthy cows (n = 15) and cows with mild fatty liver (n = 15). To determine the effects of palmitic acids (PA), one of the major component of fatty acids, on lipid metabolism and mitochondria in vitro, calf hepatocytes were isolated from healthy calves and treated with various concentrations of PA (0, 50, 100, and 200 μM). Dairy cows with mild fatty liver displayed hepatic lipid accumulation. The protein levels of sterol regulatory element-binding protein 1c (SREBP-1c) and peroxisome proliferator-activated receptor-α (PPARα) and mRNA levels of acetyl CoA carboxylase 1 (ACC1), fatty acid synthase (FAS), acyl-CoA oxidase (ACO), and carnitine palmitoyltransferase 1A (CPT1A) were significantly higher in dairy cows with mild fatty liver than in control cows. The hepatic mitochondrial DNA content, mRNA levels of oxidative phosphorylation complexes I to V (CO 1-V), protein levels of cytochrome c oxidase subunit IV (COX IV), voltage dependent anion channel 1 (VDAC1), peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α) and nuclear respiratory factor 1 (NRF1), and adenosine triphosphate (ATP) content were all markedly increased in the liver of dairy cows with mild fatty liver compared with healthy cows. The PA treatment significantly increased lipid accumulation; protein levels of SREBP-1c and PPARα; and mRNA levels of ACC1, FAS, ACO, and CPT1A in calf hepatocytes. Moreover, the mitochondrial DNA content, mRNA levels of CO 1-V, protein levels of COX IV, VDAC1, PGC-1α, NRF1, mitochondrial transcription factor A, and ATP content were significantly increased in PA-treated hepatocytes compared with control hepatocytes. The protein level of mitofusin-2 was significantly decreased in PA-treated groups. In conclusion, lipid synthesis and oxidation, number of mitochondria, and ATP production were increased in the liver of dairy cows with mild fatty liver and PA-treated calf hepatocytes. These changes in hepatic mitochondria and lipid metabolism may be the adaptive mechanism of dairy cows with mild fatty liver.

With the aim to discuss the similarities and differences of phytochemicals in Moringa oleifera leaves collected from China (CML) and India (IML) in mind, comparative ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC-QTOF-MS) analysis was performed in this study. A screening analysis based on a UNIFI platform was first carried out to discuss the similarities. Next, untargeted metabolomic analysis based on multivariate statistical analysis was performed to discover the differences. As a result, a total of 122 components, containing 118 shared constituents, were characterized from CML and IML. The structure types included flavonoids, alkaloids, glyosides, organic acids and organic acid esters, iridoids, lignans, and steroids, etc. For CML, 121 compounds were characterized; among these, 18 potential biomarkers with higher contents enabled differentiation from IML. For IML, 119 compounds were characterized; among these, 12 potential biomarkers with higher contents enabled differentiation from CML. It could be concluded that both CML and IML are rich in phytochemicals and that CML is similar to IML in the kinds of the compounds it contains, except for the significant differences in the contents of some compounds. This comprehensive phytochemical profile study provides a basis for explaining the effect of different growth environments on secondary metabolites and exists as a reference for further research into or applications of CML in China.