The Signaling Transduction Mechanism Of Hepatocytes Apoptosis Induced By Oxidative Stress In Ketotic Dairy Cows

Due to pregnancy, parturition and lactation start, periparturient dairy cows are in the state ofhigh metabolism status which develops physiological oxidative stress. The pathologic basis ofketosis is metabolic disorders during lactation period. Ketosis is one of important common diseasesin dairy cows, characterized by glycopenia, high blood concentration of non-esterified fatty acid(NEFA) and ketone bodies which can activate oxidative stress, making ketosis more serious andcomplicated. ROS can be generated through multiple pathways by high concentrations of NEFA orβ-hydroxybutyrate (BHBA), leading to the development of oxidative stress in tissue, the damage tothe mitochondria, and cell apoptosis. Therefore, it is important for the interpretation of themechanism and prevention of ketosis that revealed the relationship between hepatocytes oxidativestress and high concentrations of NEFA and BHBA in ketotic cows and the signal transductionpathway of hepatocytes apoptosis induced by NEFA and BHBA. Therefore, blood routinebiochemical parameters, and oxidative/antioxidant indexes were detected by biology and enzymelinked immunosorbent assay (ELISA) in clinical and subclinical ketosis dairy cows and theircorrelation with high concentration of NEFA and BHBA were analysed. Meanwhile, primarycultured hepatocytes were mainly used to detemine the effect of BHBA on the oxidative status ofhepatocytes and signal transduction of apoptosis induced by NEFA and BHBA.The oxidative status of ketotic dairy cows in plasma and correlation with high concentration ofNEFA and BHBA. The indicators of lipid metabolism high density lipoprotein (HDL), very lowdensity lipoprotein (VLDL), and total cholesterol (TC) decreased, liver function aspartateaminotransferase (AST), alanine amiotransferase (ALT), and lactate dehydrogenase (LDH)increased, oxidative stress malondialdehyde (MDA), hydrogen peroxide (H2O2), oxidizedglutathione (GSSG) content increased, antioxidant activity of glutathione-peroxidase (GSH-Px),superoxide dismutase (SOD) and catalase (CAT), content of glutathione (GSH), vitamin C (Vc),vitamin E (VE), GSH/GSSG ratio, and ability of inhibiting hydroxyl radical decreased in dairy cowwith subclinical and clinical ketosis. There was positive correlation between AST, ALT, LDH,MDA, H2O2, GSSG and NEFA, BHBA in blood. TC, HDL, VLDL, SOD, CAT, GSH, GSH/GSSG,Vc, VE, inhibition of the hydroxyl radical capacity to had negative correlation with NEFA andBHBA in blood. These results suggested that oxidative stress increased in the dairy cows withsubclinical and clinical ketosis and might cause liver damage.Effect of BHBA on the oxidative status of hepatocytes cultured in vitro. Primary culturedhepatocytes were incubated with different concentrations of BHBA (BHBA:0,0.3,1.2,2.4,4.8 mM) for12,24,36h, respectively. The activity and mRNA expression levels of GSH-Px, CAT, andSOD, concentration of MDA, H2O2, and total antioxidant capacity and inhibition of the hydroxylradical capacity were measured after incubation. High concentration of BHBA may decrease bothantioxidant activity and mRNA expression levels of SOD, CAT, GSH-Px, decreased TAC andinhibition of the hydroxyl radical capacity and increased the content of H2O2and MDA. The resultsindicated that high concentration of BHBA may induce oxidative stress in bovine hepatocytes.BHBA induced hepatocytes apoptosis was mediated by mitochondrial dependentROS-JNK/ERK signaling pathway. The results of BHBA treatment showed that BHBA inducedapoptosis which was mediated by oxidative stress. The production of reactive oxygen species (ROS)is significantly increased, the protein and mRNA expression of c-Jun NH(2)-terminal kinase (JNK)were increased, while the protein and mRNA expression of extracellular signal-regulated proteinkinase (ERK) were inhibited during the process of oxidative stress. The transcription andexpression of the tumor suppressor gene p53and the pro-apoptosis genes, such as, Bcl-2-associatedX protein (Bax), Bcl-xl/Bcl-2-associated death promoter (Bad) were up-regulated. While thetranscription and expression of NF-E2-related factor2(Nrf2) and the anti-apoptosis genes,including B-cell lymphonma gene2(Bcl-2), Bcl-w, and B-cell lymphoma-extra large (Bcl-xl) weredown-regulated. The mitochondrial membrane potential (MMP) decreased, cytochrome c (cytc)and apoptosis inducing factor (AIF) were released, and lead to the activation of caspase3and polyADP-ribose polymerase (PARP). These results indicated that BHBA promotes hepatocytesapoptosis gene expression, inhibits anti-apoptosis gene expression and induces mitochondrialdysfunction through mitochondrial mediated ROS-JNK/ERK signaling pathway, eventually leadingto hepatocytes apoptosis.NEFA induced hepatocytes apoptosis was mediated by mitochondrial dependentROS-JNK/ERK signaling pathway. The results indicated that NEFA may induce oxidative stress inhepatocytes, increased level of ROS, thus contributing to JNK activation, inhibition of ERKactivation, thereby upregulated the expression of pro-apoptotic genes Bax, Bad, and downregulatedthe expression of anti-apoptotic genes Bcl-2, Bcl-w, Bcl-xl, resulting in the decreased ofmitochondrial membrane potential, release of cytc and AIF and activation of caspase3and PARP.These results indicate that NEFA may induce hepatocytes apoptosis through mitochondrialmediated ROS-JNK/ERK signaling pathway.In conclusion, ketosis dairy cows undergo oxidative stress and some extent of liver damage.There is correlation between high blood levels of NEFA, BHBA and oxidative stress. Highconcentration of NEFA, BHBA result in excessive production of ROS, which activate JNK pathway,inhibit the ERK pathway. Excessive oxidation impairs mitochondrial. These factors promote thehepatocytes apoptosis and eventually leading to liver damage.