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High blood concentrations of nonesterified fatty acids (NEFA) during ketosis enhance uptake by the mammary gland and impair autophagy while causing oxidative stress. Caveolin 1 (CAV1) is closely related to autophagy and plays a role in regulating oxidative stress. The aim of this study was to explore the potential role of CAV1 on oxidative stress and autophagy during a high-NEFA challenge in the immortalized bovine mammary epithelial cell line (MAC-T). Mammary gland tissue biopsies and blood samples were collected from healthy (n = 15) and clinically ketotic (n = 15) Holstein cows at 3 to 10 (average = 6) days in milk. Compared with healthy cows, ketotic cows had lower DMI, daily milk yield, and serum glucose, and greater serum NEFA and BHB, accompanied by greater milk fat and lower milk protein. Malondialdehyde (MDA) was greater, but activities of superoxide dismutase (SOD), catalase, and glutathione peroxidase were lower in cows with clinical ketosis. A lower protein abundance of CAV1, beclin 1, autophagy relative gene 5 (ATG5), and microtubule-associated protein 1 light chain 3 (LC3) as well as greater protein abundance of sequestosome-1 (SQSTM1, also called p62) were detected in mammary tissue of cows with clinical ketosis. In vitro, the MAC-T cells were treated with 0, 0.6, and 1.2 mM NEFA for 12 h or treated with 1.2 mM NEFA for various lengths of time (0, 0.5, 1, 2, 4, 8, 12, and 24 h). Compared with 0 mM NEFA, protein abundances of CAV1, beclin 1, ATG5, and LC3 were greater in the MAC-T challenged with 0.6 mM NEFA but lower in the 1.2 mM NEFA group. Protein abundance of p62 was lower with 0.6 mM NEFA but higher with 1.2 mM NEFA. In response to increasing doses of NEFA, mRNA abundance of CAV1, total antioxidant capacity and SOD activity decreased, whereas the levels of reactive oxygen species and MDA content increased. The protein abundances of CAV1 and beclin 1 peaked at 0.5 h, the protein abundances of ATG5 and LC3 peaked at 1 h, and these proteins start to fade away at later time points under NEFA treatment, resulting in both linear and quadratic effects. The protein abundance of p62 decreased, reaching a nadir at 4 h in both a linear and quadratic manner. The silencing of CAV1 in MAC-T cells aggravated the 1.2 mM NEFA-induced decrease in beclin 1 expression, impaired autophagy, and increased severe oxidative stress, whereas the overexpression of CAV1 alleviated these effects. Pretreatment of MAC-T cells with beclin 1 small interfering RNA (si-beclin 1) and the overexpression of CAV1, followed by challenge with 1.2 mM NEFA, resulted in reversed CAV1-induced autophagy, thereby aggravating oxidative stress. Overall, these data suggest that CAV1 protects bovine mammary epithelial cells from NEFA-induced oxidative stress through enhancing the expression of beclin 1 and activating autophagy., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Hepatic oxidative injury induced by free fatty acids (FFA) and metabolic disorders of bile acids (BA) increase the risk of metabolic diseases in dairy cows during perinatal period. However, the effects of FFA on BA metabolism remained poorly understood. In present study, high concentrations of FFA caused cell impairment, oxidative stress and BA overproduction. FFA treatment increased the expression of BA synthesis-related genes [cholesterol 7a-hydroxylase (CYP7A1), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 7, sterol 12α-hydroxylase, sterol 27-hydroxylase and oxysterol 7α-hydroxylase], whereas reduced BA exportation gene (ATP binding cassette subfamily C member 1) and inhibited farnesoid X receptor/small heterodimer partner (FXR/SHP) pathway in bovine hepatocytes. Knockdown of nuclear receptor subfamily 1 group H member 4 (NR1H4) worsened FFA-caused oxidative damage and BA production, whereas overexpression NR1H4 ameliorated FFA-induced BA production and cell oxidative damage. Besides, reducing BA synthesis through knockdown of CYP7A1 can alleviate oxidative stress and hepatocytes impairment caused by FFA. In summary, these data demonstrated that regulation of FXR/SHP-mediated BA metabolism may be a promising target in improving hepatic oxidative injury of dairy cows during high levels of FFA challenges., Competing Interests: Declaration of interest The authors declare that they have no competing interests. Conflict of Interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

During the periparturient period, both oxidative stress, and inflammation of adipose tissue are considered high risk factors for metabolic disorder of dairy cows. Oxidative stress can activate transcription factor nuclear factor kappa B (NF-κB), which lead to the upregulation of genes involved in inflammatory pathways. Thioredoxin-2 (TXN2) is a mitochondrial protein that regulates cellular redox by suppressing mitochondrial reactive oxygen species (ROS) generation in nonruminant, whereas the function of TXN2 in bovine adipocytes was unclear. Thus, the objective of this study was to evaluate how or by which mechanisms TXN2 regulates oxidative stress and NF-κB signaling pathway in bovine adipocytes. Bovine pre-adipocytes isolated from 5 healthy Holstein cows were differentiated and used for (1) treatment with different concentrations of hydrogen peroxide (H 2 O 2 ; 0, 25, 50, 100, 200, or 400 μM) for 2 h; (2) transfection with or without TXN2 small interfering RNA (si-TXN2) for 48 h and then treated with or without 200 μM H 2 O 2 for 2 h; (3) transfection with scrambled negative control siRNA (si-control) or si-TXN2 for 48 h, and then treatment with or without 10 mM N-acetylcysteine (NAC) for 2 h; (4) transfection with or without TXN2-overexpressing plasmid for 48 h and then treatment with or without 200 μM H 2 O 2 for 2 h. High concentrations of H 2 O 2 (200 and 400 μM) decreased protein and mRNA abundance of TXN2, reduced total antioxidant capacity (T-AOC) and ATP content in adipocytes. Moreover, 200 and 400 μM H 2 O 2 reduced protein abundance of inhibitor of kappa B α (IκBα), increased phosphorylation of NF-κB and upregulated mRNA abundance of tumor necrosis factor-α (TNFA) and interleukin-1B (IL-1B), suggesting that H 2 O 2 -induced oxidative stress and activated NF-κB signaling pathway. Silencing of TXN2 increased intracellular ROS content, phosphorylation of NF-κB and mRNA abundance of TNFA and IL-1B, decreased ATP content and protein abundance of IκBα in bovine adipocytes. Knockdown of TXN2 aggravated H 2 O 2 -induced oxidative stress and inflammation. In addition, treatment with antioxidant NAC ameliorated oxidative stress and inhibited NF-κB signaling pathway in adipocytes transfected with si-TXN2. In bovine adipocytes treated with H 2 O 2 , overexpression of TXN2 reduced the content of ROS and elevated the content of ATP and T-AOC. Overexpression of TXN2 alleviated H 2 O 2 -induced inflammatory response in adipocytes, as demonstrated by decreased expression of phosphorylated NF-κB, TNFA, IL-1B, as well as increased expression of IκBα. Furthermore, the protein and mRNA abundance of TXN2 was lower in adipose tissue of dairy cows with clinical ketosis. Overall, our studies contribute to the understanding of the role of TXN2 in adipocyte oxidative stress and inflammatory response., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is closely related to the alleviation of nonalcoholic fatty liver disease (NAFLD) by regulating oxidative stress and lipid homeostasis. Gentiopicroside (GPS), an iridoid glycoside found in the Gentianaceae, possesses anti-inflammatory and antioxidant effects. However, the protective effects of GPS on lipid accumulation and oxidative damage have not been investigated thoroughly in free fatty acid- (FFA-) induced HepG2 cells and tyloxapol- (Ty-) induced hyperlipidemia mice. Cell counting kit-8 assays, Oil Red O staining, Western blotting analysis, extraction of nuclear and cytosolic proteins, and biochemical index assay were employed to explore the mechanisms by which GPS exerts a protective effect on FFA-induced HepG2 cells and Ty-induced hyperlipidemia mouse model. This paper demonstrates that GPS could effectively alleviate NAFLD by elevating cell viability, reducing fatty deposition, downregulating TG, and activating nucleus Nrf2 in FFA-induced HepG2 cells. Meanwhile, GPS significantly regulated the activation of phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, Nrf2 antioxidant pathway, peroxisome proliferator-activated receptor α (PPAR α ), and GPS-inhibited sterol regulatory element-binding protein-1c (SREBP-1c) expression in FFA-stimulated lipid accumulation of HepG2 cells and Ty-treated mice. Interestingly, we highlight that PI3K/AKT inhibitor (LY294002) markedly increased the expression of Nrf2 antioxidant pathway, PPAR α , and downregulated SREBP-1c in FFA-stimulated HepG2 cells. For these reasons, we found that the deletion of Nrf2 could lose the protective effects of GPS on the Nrf2 antioxidant pathway and PPAR α activation and SREBP-1c inactivation in FFA-stimulated HepG2 cells and Ty-treated mice. GPS treatment had no effect on abnormal lipogenesis and antioxidant enzymes in Ty-induced Nrf2 -/- mice. This work gives a new explanation that GPS may be a useful therapeutic strategy for NAFLD through upregulation of the Nrf2 antioxidant pathway, which can alleviate oxidative damage and lipid accumulation., Competing Interests: Meiyu Jin, Haihua Feng, Yue Wang, Siru Yan, Bingyu Shen, Zheng Li, Haiyan Qin, Qi Wang, Jinxia Li, and Guowen Liu declare that they have no conflicts of interest., (Copyright © 2020 Meiyu Jin et al.)

Chicoric acid is polyphenol of natural plant and has a variety of bioactivity. Caused by various kinds of stimulating factors, acute liver injury has high fatality rate. The effect of chicoric acid in acute liver injury induced by Lipopolysaccharide (LPS) and d-galactosamine (d-GalN) was investigated in this study. The results showed that CA decreased the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum and reduced the mortality induced by LPS/d-GalN. CA can restrain mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) to alleviate inflammation. Meanwhile, the results indicated CA can active nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway with increasing the level of AMP-activated protein kinase (AMPK). And with the treatment of CA, protein levels of autophagy genes were obvious improved. The results of experiments indicate that CA has protective effect in liver injury, and the activation of AMPK and autophagy may make sense., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Aims: Nuclear factor E2-related factor 2 (Nrf2) is a regulator of cellular oxidative stress and is also involved in lipid metabolism in adipocytes. However, it remains unknown as to whether Nrf2 is the link between oxidative stress and the induction of lipid accumulation in adipocytes. Results: Here, we show that oxidative stress is markedly increased in white adipose tissue from mice with high-fat diet-induced or genetically (ob/ob)-induced obesity and from human subjects with obesity. Notably, in response to oxidative stress, Nrf2 expression and activity were induced, further promoting lipid accumulation in adipocytes and exacerbating the development of obesity. In contrast, Nrf2 ablation alleviated oxidative stress-induced lipid accumulation. Mechanistically, oxidative stress promoted Nrf2 recruitment to the sterol regulatory element binding protein 1 promoter, inducing target gene transcription and subsequent lipogenesis. In addition, Nrf2 mediated oxidative stress-inhibited lipolysis in adipocytes via the protein kinase A pathway. Innovation and Conclusion: Our data provide a novel insight that Nrf2, as a critical signaling node, links oxidative stress to the induction of fat accumulation in adipocytes.

Dairy cows with ketosis display excessive lipolysis in adipose tissue. Heat-shock protein B7 (HSPB7), a small heat-shock protein, plays important roles in mediating cytoprotective responses to oxidative stress in rodent adipose tissue. Accordingly, it is assumed that HSPB7 may also play important roles in the antioxidant response in adipose tissue of ketotic cows. Therefore, the aim of this study is to investigate (1) the redox state of adipose tissue in ketotic cows and (2) the role and mechanism of HSPB7 on the regulation of oxidative stress in adipocytes from preruminant calves. An in vivo study consisting of 15 healthy and 15 clinically ketotic cows was performed to harvest subcutaneous adipose tissue and blood samples. In addition, adipocytes isolated from calves were treated with different concentrations of H 2 O 2 (0, 12.5, 25, 50, 100, or 200 μM) for 2 h, transfected with adenovirus-mediated overexpression of HSPB7 for 48 h, or transfected with small interfering RNA of HSPB7 for 48 h followed by exposure to H 2 O 2 (200 μM) for 2 h. Serum concentrations of nonesterified fatty acids and β-hydroxybutyrate were greater in cows with clinical ketosis, whereas serum concentration of glucose was lower. Compared with healthy cows, the malondialdehyde content was greater but the activity of glutathione peroxidase and superoxide dismutase was lower in adipose tissue of clinically ketotic cows. The abundance of HSPB7 and nuclear factor, erythroid 2 like 2 (NFE2L2) was greater in adipose tissue of clinically ketotic cows. In vitro, H 2 O 2 treatment induced the overproduction of reactive oxygen species and malondialdehyde, and inhibited the activity of antioxidant enzymes glutathione peroxidase and superoxide dismutase in adipocytes from preruminant calves. The low concentration of H 2 O 2 (12.5, 25, and 50 μM) increased the abundance of HSPB7 and NFE2L2, but high concentrations of H 2 O 2 (100 or 200 μM) reduced the abundance of HSPB7 and NFE2L2. The overexpression of HSPB7 improved the H 2 O 2 -induced oxidative stress in adipocytes via increasing the abundance of NFE2L2 and its downstream target genes heme oxygenase-1 (HMOX1) and NADH quinone oxidoreductase 1 (NQO1). Knockdown of HSPB7 markedly inhibited the expression of NFE2L2, HMOX1, and NQO1 and further exacerbated H 2 O 2 -induced oxidative stress. Overall, these results indicate that activation of the HSPB7-NFE2L2 pathway increases cellular antioxidant capacity, thereby alleviating oxidative stress in bovine adipocytes., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Dairy cows with fatty liver or ketosis exhibit hyperketonemia, oxidative stress, and a low rate of very low density lipoprotein (VLDL) assembly, and there may be a potential link among these characteristics. Therefore, the objective of this study was to determine the effect of acetoacetic acid (AcAc) on the assembly of VLDL in cow hepatocytes. Cultured cow hepatocytes were treated with different concentrations of AcAc with or without N-acetylcysteine (NAC, an antioxidant). AcAc treatment decreased the mRNA expression and activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), and significantly increased malondialdehyde (MDA) content, indicative of oxidative stress. Furthermore, AcAc treatment significantly down-regulated the mRNA expression of apolipoprotein B100 (ApoB100), apolipoprotein E (ApoE), and low density lipoprotein receptor (LDLR), which thus decreased VLDL assembly and increased triglyceride (TG) accumulation in these bovine hepatocytes. Importantly, NAC relieved AcAc-induced oxidative stress and increased VLDL assembly. In summary, these results suggest that AcAc-induced oxidative stress affects the assembly of VLDL, which increases TG accumulation in bovine hepatocytes.

Non-esterified fatty acids (NEFAs) are important induction factors of inflammatory responses in some metabolic diseases. High plasma levels of NEFAs and oxidative stress exist in the dairy cows with ketosis. The aim of this study was to investigate whether high levels of NEFAs can induce inflammatory response and the specific molecular mechanism in the hepatocytes of dairy cow. In vitro, primary cultured bovine hepatocytes were treated with different concentrations of NEFAs, PDTC (an NF-κB inhibitor) and NAC (an antioxidant). NEFAs significantly activated NF-κB pathway. Activated NF-κB upregulated the release of pro-inflammatory cytokines, thereby inducing inflammatory response in bovine hepatocytes. When PDTC was added, activation of NF-κB-mediated inflammatory response induced by NEFAs was inhibited. NEFAs treatment results in the overproduction of the markers of oxidative stress, reactive oxygen species (ROS) and malondialdehyde (MDA), which were ameliorated by NAC treatment. These increased ROS and MDA were caused by decreasing activity of antioxidant system, including glutathione peroxidase, superoxide dismutase and catalase, in bovine hepatocytes treated with NEFAs. NAC also ameliorated NEFAs-mediated NF-κB activation and the release of pro-inflammatory cytokines. These results indicate that high concentrations of NEFAs can induce cattle hepatocytes inflammatory response through activating the oxidative stress-mediated NF-κB signaling pathway., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Acetaminophen (APAP) overdose is the predominant cause of drug-induced liver injury worldwide. The macroautophagy/autophagy-lysosomal pathway (ALP) is involved in the APAP hepatotoxicity. TFEB (transcription factor EB) promotes the expression of genes related to autophagy and lysosomal biogenesis, thus, pharmacological activation of TFEB-mediated ALP may be an effective therapeutic approach for treating APAP-induced liver injury. We aimed to reveal the effects of narirutin (NR), the main bioactive constituents isolated from citrus peels, on APAP hepatotoxicity and to explore its underlying mechanism. Administration of NR enhanced activities of antioxidant enzymes, improved mitochondrial dysfunction and alleviated liver injury in APAP-treated mice, whereas NR did not affect APAP metabolism and MAPK/JNK activation. NR enhanced TFEB transcriptional activity and activated ALP in an MTOR complex 1 (MTORC1)-independent but PPP3/calcineurin-dependent manner. Moreover, knockout of Tfeb or knockdown of PPP3CB/CNA2 (protein phosphatase 3, catalytic subunit, beta isoform) in the liver abolished the beneficial effects of NR on APAP overdose. Mechanistically, NR bound to PPP3CB via PRO31, LYS61 and PRO347 residues and enhanced PPP3/calcineurin activity, thereby eliciting dephosphorylation of TFEB and promoting ALP, which alleviated APAP-induced oxidative stress and liver injury. Together, NR protects against APAP-induced liver injury by activating a PPP3/calcineurin-TFEB-ALP axis, indicating NR may be a potential agent for treating APAP overdose. Abbreviations: ALP: autophagy-lysosomal pathway; APAP: acetaminophen; APAP-AD: APAP-protein adducts; APAP-Cys: acetaminophen-cysteine adducts; CAT: catalase; CETSA: cellular thermal shift assay; CQ: chloroquine; CYP2E1: cytochrome P450, family 2, subfamily e, polypeptide 1; CYCS/Cyt c: cytochrome c, somatic; DARTS: drug affinity responsive target stability assay; ENGASE/NAG: endo-beta-N-acetylglucosaminidase; GOT1/AST: glutamic-oxaloacetic transaminase 1, soluble; GPT/ALT: glutamic pyruvic transaminase, soluble; GSH: glutathione; GPX/GSH-Px: glutathione peroxidase; KD: dissociation constant; Leu: leupeptin; MCOLN1: mucolipin 1; MTORC1: MTOR complex 1; NAC: N-acetylcysteine; NAPQI: N-acetyl-p-benzoquinoneimine; NFAT: nuclear factor of activated T cells; NR: narirutin; OA: okadaic acid; RRAG: Ras related GTP binding; ROS: reactive oxygen species; PPP3CB/CNA2: protein phosphatase 3, catalytic subunit, beta isoform; PPP3R1/CNB1: protein phosphatase 3, regulatory subunit B, alpha isoform (calcineurin B, type I); SOD: superoxide dismutase; SPR: surface plasmon resonance analysis; TFEB: transcription factor EB. [ABSTRACT FROM AUTHOR]

Non‐alcohol fatty liver disease (NAFLD) is a common disease which causes serious liver damage. Geniposide (GEN), a kind of iridoid glycoside extracted from Gardenia jasminoides fruit, has many biological effects, such as resistance to cell damage and anti‐neurodegenerative disorder. Lipid accumulation was obvious in tyloxapol‐induced liver and oil acid (OA) with palmitic acid (PA)‐induced HepG2 cells compared with the control groups while GEN improved the increasing conditions. GEN significantly lessened the total cholesterol (TC), the triglyceride (TG), low‐density lipoprotein (LDL), very low‐density lipoprotein (VLDL), myeloperoxidase (MPO), reactive oxygen species (ROS) and increased high‐density lipoprotein (HDL), superoxide dismutase (SOD) to response the oxidative stress via activating nuclear factor erythroid‐2–related factor 2 (Nrf2), haeme oxygenase (HO)‐1 and peroxisome proliferator‐activated receptor (PPAR)α which may influence the phosphorylation of adenosine 5'‐monophosphate–activated protein kinase (AMPK) signalling pathway in mice and cells. Additionally, GEN evidently decreased the contents of sterol regulatory element‐binding proteins (SREBP)‐1c, phosphorylation (P)‐mechanistic target of rapamycin complex (mTORC), P‐S6K, P‐S6 and high mobility group protein (HMGB) 1 via inhibiting the expression of phosphoinositide 3‐kinase (PI3K), and these were totally abrogated in Nrf2−/− mice. Our study firstly proved the protective effect of GEN on lipid accumulation via enhancing the ability of antioxidative stress and anti‐inflammation which were mostly depend on up‐regulating the protein expression of Nrf2/HO‐1 and AMPK signalling pathways, thereby suppressed the phosphorylation of mTORC and its related protein. [ABSTRACT FROM AUTHOR]

Dairy cows with ketosis display severe oxidative stress as well as high blood concentrations of non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHB). Cytochrome P4502E1 (CYP2E1) plays an important role in the induction of oxidative stress. The aim of this study was to investigate CYP2E1 expression and activity in the liver of clinically ketotic cows (in vivo) and the effects of NEFA and BHB on CYP2E1 expression and activity in hepatocytes (in vitro). Dairy cows with clinical ketosis exhibited a low blood concentration of glucose but high concentrations of NEFA and BHB. Hepatic mRNA, protein expression, and activity of CYP2E1 were significantly higher in cows with clinical ketosis than in control cows. In vitro, both NEFA and BHB treatment markedly up-regulated the mRNA and protein expressions as well as activity of CYP2E1 in cow hepatocytes. Taken together, these results indicate that high levels of NEFA and BHB significantly up-regulate the expression and activity of hepatic CYP2E1, and may be influential in the induction of oxidative stress in cows with clinical ketosis. [ABSTRACT FROM AUTHOR]

Acetaminophen (APAP) overdose-induced fatal hepatotoxicity is majorly characterized by overwhelmingly increased oxidative stress while enhanced nuclear factor-erythroid 2- related factor 2 (Nrf2) is involved in prevention of hepatotoxicity. Although Licochalcone A (Lico A) upregulates Nrf2 signaling pathway against oxidative stress-triggered cell injury, whether it could protect from APAP-induced hepatotoxicity by directly inducing Nrf2 activation is still poorly elucidated. This study aims to explore the protective effect of Lico A against APAP-induced hepatotoxicity and its underlying molecular mechanisms. Our findings indicated that Lico A effectively decreased tert-butyl hydroperoxide (t-BHP)- and APAP-stimulated cell apoptosis, mitochondrial dysfunction and reactive oxygen species generation and increased various anti-oxidative enzymes expression, which is largely dependent on upregulating Nrf2 nuclear translocation, reducing the Keap1 protein expression, and strengthening the antioxidant response element promoter activity. Meanwhile, Lico A dramatically protected against APAP-induced acute liver failure by lessening the lethality; alleviating histopathological liver changes; decreasing the alanine transaminase and aspartate aminotransferase levels, malondialdehyde formation, myeloperoxidase level and superoxide dismutase depletion, and increasing the GSH-to-GSSG ratio. Furthermore, Lico A not only significantly modulated apoptosisrelated protein by increasing Bcl-2 expression, and decreasing Bax and caspase-3 cleavage expression, but also efficiently alleviated mitochondrial dysfunction by reducing c-jun N-terminal kinase phosphorylation and translocation, inhibiting Bax mitochondrial translocation, apoptosis-inducing factor and cytochrome c release. However, Lico A-inhibited APAP-induced the lethality, histopathological changes, hepatic apoptosis, and mitochondrial dysfunction in WT mice were evidently abrogated in Nrf2-/- mice. These investigations firstly implicated that Lico A has protective potential against APAPinduced hepatotoxicity which may be strongly associated with the Nrf2-mediated defense mechanisms. [ABSTRACT FROM AUTHOR]

Background/Aims: β-hydroxybutyrate (BHBA) is the major component of ketone bodies in ketosis. Dairy cows with ketosis often undergo oxidative stress. BHBA is related to the inflammation involved in other diseases of dairy cattle. However, whether BHBA can induce inflammatory injury in dairy cow hepatocytes and the potential mechanism of this induction are not clear. The NF-κB pathway plays a vital role in the inflammatory response. Methods: Therefore, this study evaluated the oxidative stress, pro-inflammatory factors and NF-κB pathway in cultured calf hepatocytes treated with different concentrations of BHBA, pyrrolidine dithiocarbamate (PDTC, an NF-κB pathway inhibitor) and N-acetylcysteine (NAC, antioxidant). Results: The results showed that BHBA could significantly increase the levels of oxidation indicators (MDA, NO and iNOS), whereas the levels of antioxidation indicators (GSH-Px, CAT and SOD) were markedly decreased in hepatocytes. The IKKβ activity and phospho-IκBα (p-IκBα) contents were increased in BHBA-treated hepatocytes. This increase was accompanied by the increased expression level and transcription activity of p65. The expression levels of NF-κB-regulated inflammatory cytokines, namely TNF-α, IL-6 and IL-1β, were markedly increased after BHBA treatment, while significantly decreased after NAC treatment. However, the p-IκBα level and the expression and activity of p65 and its target genes were markedly decreased in the PDTC + BHBA group compared with the BHBA (1.8 mM) group. Moreover, immunocytofluorescence of p65 showed a similar trend. Conclusion: The present data indicate that higher concentrations of BHBA can induce cattle hepatocyte inflammatory injury through the NF-κB signaling pathway, which may be activated by oxidative stress. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Excessive free fatty acid (FFA) oxidation and related metabolism are the major cause of oxidative stress and liver injury in dairy cows during the early postpartum period. In nonruminants, activation of transcription factor EB (TFEB) can improve cell damage and reduce the overproduction of mitochondrial reactive oxygen species. As a downstream target of TFEB, peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α, gene name PPARGC1A) is a critical regulator of oxidative metabolism. Nuciferine (Nuc), a major bioactive compound isolated from the lotus leaf, has been reported to possess hepatoprotective activity. Therefore, the objective of this study was to investigate whether Nuc could protect bovine hepatocytes from FFA-induced lipotoxicity and the underlying mechanisms. A mixture of FFA was diluted in RPMI-1640 basic medium containing 2% low fatty acid bovine serum albumin to treat hepatocytes. Bovine hepatocytes were isolated from newborn calves and treated with various concentrations of FFA mixture (0, 0.3, 0.6, or 1.2 m M) or Nuc (0, 25, 50, or 100 μ M), as well as co-treated with 1.2 m M FFA and different concentrations of Nuc. For the experiments of gene silencing, bovine hepatocytes were transfected with small interfering RNA targeted against TFEB or PPARGC1A for 36 h followed by treatment with 1.2 m M FFA for 12 h in presence or absence of 100 μ Μ Nuc. The results revealed that FFA treatment decreased protein abundance of nuclear TFEB, cytosolic TFEB, total (t)-TFEB, lysosome-associated membrane protein 1 (LAMP1) and PGC-1α and mRNA abundance of LAMP1 , but increased phosphorylated (p)-TFEB. In addition, FFA treatment increased the content of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2) and decreased the activities of catalase (CAT) and glutathione peroxidase (GSH-Px) in bovine hepatocytes. Moreover, FFA administration enhanced the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactose dehydrogenase (LDH) in the medium of FFA-treated hepatocytes, but reduced the content of urea. In FFA-treated bovine hepatocytes, Nuc administration increased TFEB nuclear localization and the protein abundance of t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1 , decreased the contents of MDA and H 2 O 2 and the protein abundance of p-TFEB, and enhanced the activities of CAT and GSH-Px in a dose-dependent manner. Consistently, Nuc administration reduced the activities of ALT, AST, and LDH and increased the content of urea in the medium of FFA-treated hepatocytes. Importantly, knockdown of TFEB reduced the protein abundance of p-TFEB, t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1 , and impeded the beneficial effects of Nuc on FFA-induced oxidative damage in bovine hepatocytes. In addition, PPARGC1A silencing did not alter Nuc-induced nuclear translocation of TFEB, increase of the protein abundance of t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1 , or decrease of the protein abundance of p-TFEB, whereas it partially reduced the beneficial effects of Nuc on FFA-caused oxidative injury. Taken together, Nuc exerts protective effects against FFA-induced oxidative damage in bovine hepatocytes through activation of the TFEB/PGC-1α signaling pathway. [ABSTRACT FROM AUTHOR]

When ketosis occurs, supraphysiological concentrations of nonesterified fatty acids (NEFA) display lipotoxicity and are closely related to the occurrence of hepatic lipid accumulation, oxidative stress, and inflammation, resulting in hepatic damage and exacerbating the progression of ketosis. However, the mechanism of these lipotoxic effects caused by high concentrations of NEFA in ketosis is still unclear. Cluster antigen 36 (CD36), a fatty acid transporter, plays a vital role in the development of hepatic pathological injury in nonruminants. Thus, the aim of this study was to investigate whether CD36 plays a role in NEFA-induced hepatic lipotoxicity in dairy cows with clinical ketosis. Liver tissue and blood samples were collected from healthy (n = 10) and clinically ketotic (n = 10) cows at 3 to 15 d in milk. In addition, hepatocytes isolated from healthy calves were treated with 0, 0.6, 1.2, or 2.4 m M NEFA for 12 h; or infected with CD36 expressing adenovirus or CD36 silencing small interfering RNA for 48 h and then treated with 1.2 m M NEFA for 12 h. Compared with healthy cows, clinically ketotic cows had greater concentrations of serum NEFA and β-hydroxybutyrate and activities of aspartate aminotransferase and alanine aminotransferase but lower serum glucose. In addition, dairy cows with clinical ketosis displayed excessive hepatic lipid accumulation. More importantly, these alterations were accompanied by an increased abundance of hepatic CD36. In the cell culture model, exogenous NEFA (0, 0.6, 1.2, or 2.4 m M) treatment could dose-dependently increase the abundance of CD36. Meanwhile, NEFA (1.2 m M) increased the content of triacylglycerol, reactive oxygen species and malondialdehyde, and decreased the activities of glutathione peroxidase and superoxide dismutase. Moreover, NEFA upregulated phosphorylation levels of nuclear factor κB (NF-κB) and the inhibitor of NF-κB (IκB) α, along with the upregulation of protein abundance of NLR family pyrin domain containing 3 (NLRP3) and caspase-1, and mRNA abundance of IL1B , IL6 , and tumor necrosis factor α (TNFA). These alterations induced by NEFA in bovine hepatocytes were associated with increased lipid accumulation, oxidative stress and inflammation, which could be further aggravated by CD36 overexpression. Conversely, silencing CD36 attenuated these NEFA-induced detriments. Overall, these data suggest that CD36 may be a potential therapeutic target for NEFA-induced hepatic lipid accumulation, oxidative stress, and inflammation in dairy cows. [ABSTRACT FROM AUTHOR]

Ketosis is often accompanied by a reduction in milk production in dairy cows, but the molecular mechanism has not been fully elucidated. Ketotic cows possess systemic oxidative stress (OS), which may implicate apoptosis in mammary glands. Sirtuin 3 (SIRT3) is a vital regulator of cellular redox homeostasis and is under the control of AMP-activated protein kinase (AMPK) signaling in nonruminants. Thus, we aimed to investigate (1) the AMPK-SIRT3 and apoptosis status of mammary glands from ketotic cows, (2) the effect of SIRT3 on OS-induced apoptosis in bovine mammary epithelial cells (BMEC), and (3) the role of AMPK signaling on SIRT3-mediated effects on apoptosis. Mammary gland samples were reused from a previous study, which contained healthy and ketotic cows (both n = 15). BMEC were incubated with 0, 0.3, 0.6, or 0.9 m M H 2 O 2 for 6 h with/without a 30 min incubation of an antioxidant MitoQ (1 μ M). Then BMEC were incubated with SIRT3 overexpression adenovirus (Ad-SIRT3) for 6 h followed by a 6 h incubation with 0.6 m M H 2 O 2. Finally, BMEC were treated with the AMPK inhibitor Compound C (Cd C,10 μ M) for 30 min before the H 2 O 2 challenge, or cells were initially treated with the AMPK agonist MK8722 (10 μ M) for 30 min followed by a 30-h culture with/without si-SIRT3 and eventually the H 2 O 2 exposure. Ketotic cows displayed higher levels of Bax, Caspase-3 and Bax/Bcl-2 but lower levels of Bcl-2 in mammary glands. H 2 O 2 incubation displayed similar results, exhibiting a dose-dependent manner between the H 2 O 2 concentration and the apoptosis degree. Mito Q pretreatment reduced cellular reactive oxygen species and rescued cells from apoptosis. Ketotic cows had a lower mammary protein abundance of SIRT3. Similarly, H 2 O 2 incubation downregulated both mRNA and protein levels of SIRT3 in a dose- and time-dependent manner. Ad-SIRT3 infection lowered levels of cellular reactive oxygen species, Bax, Caspase-3 and Bax/Bcl-2 but increased levels of Bcl-2. TUNEL assays confirmed that Ad-SIRT3 infection mitigated H 2 O 2 -induced apoptosis. Both ketotic cows and H 2 O 2 -induced BMEC had lower levels of p-AMPK and p-AMPK/AMPK. Additionally, Cd C pretreatment decreased SIRT3 and Bcl-2 expression but increased levels of Bax and Caspase-3. Contrary to the inhibitor, MK8722 had opposite effects and reduced the percentage of apoptotic cells. However, these effects of MK8722 were reversed upon SIRT3 silencing. In conclusion, in vivo data confirmed that ketosis is associated with greater apoptosis and restricted AMPK-SIRT3 signaling in mammary glands; in vitro data indicated that SIRT3 mitigates OS-induced apoptosis via AMPK signaling. As such, there may be potential benefits for targeting the AMPK-SIRT3 axis to help counteract the negative effects of mammary glands during ketosis. [ABSTRACT FROM AUTHOR]

When ketosis occurs, supraphysiological levels of free fatty acids (FFA) can cause oxidative injury to the mammary gland and autophagy can regulate the cellular oxidative status. The aim of this study was to investigate the autophagy status of mammary tissue and its associations with oxidative stress in healthy and clinically ketotic dairy cows. Mammary tissue and blood samples were collected from healthy cows [n = 15, β-hydroxybutyrate (BHB) <0.6 m M ] and clinically ketotic cows (n = 15, BHB >3.0 m M) at 3 to 15 (average = 7) days in milk. For in vitro study, bovine mammary epithelial cells (BMEC) isolated from healthy cows were treated with 0, 0.3, 0.6, or 1.2 m M FFA for 24 h. Furthermore, BMEC were pretreated with 100 n M rapamycin, an autophagy activator, for 4 h or 50 m M 3-methyladenine (3-MA), an autophagy inhibitor, for 1 h, followed by treatment with or without FFA (1.2 m M) for another 24 h. Oxidation indicators and autophagy-related protein abundance were measured. Compared with healthy cows, serum concentrations of FFA, BHB, and malondialdehyde were greater in clinically ketotic cows, but milk production (kg/d), milk protein (kg/d), activities of superoxide dismutase, catalase, and glutathione peroxidase were lower. Abundances of mRNA and protein of autophagy-related gene 5 (ATG5) and 7 (ATG7) were lower, but sequestosome-1 (SQSTM1, also called p62) greater in mammary tissue of clinically ketotic cows. The mRNA abundance of microtubule-associated protein 1 light chain 3 (MAP1LC3 , also called LC3) and protein abundance of LC3-II were lower in mammary tissue of clinically ketotic cows. In vitro, exogenous FFA increased the content of malondialdehyde and reactive oxygen species, but decreased the activities of superoxide dismutase, catalase, and plasma glutathione peroxidase. Compared with the 0 m M FFA group, abundance of ATG5, ATG7, LC3-II was greater, but p62 was lower in the 0.6 m M FFA-treated cells. Similarly, abundance of ATG5, ATG7, and LC3-II was lower, but p62 greater in the 1.2 m M FFA-treated cells relative to 0 m M FFA group. Culture with rapamycin alleviated oxidative stress induced by 1.2 m M FFA, whereas 3-MA aggravated it. Overall, results indicated that a low concentration (0.6 m M) of FFA can induce oxidative stress and activate autophagy in BMEC. At higher concentrations of FFA (1.2 m M), autophagy is impaired and oxidative stress is aggravated. Autophagy is a mechanism for BMEC to counteract FFA-induced stress. As such, it could serve as a potential target for further development of novel strategies against oxidative stress. [ABSTRACT FROM AUTHOR]

Reduced feed intake during the transition period renders cows unable to meet their energy needs for maintenance and lactation, leading to a state of negative energy balance. Severe negative energy balance initiates fat mobilization and increases circulating levels of free fatty acids (FFA), which could induce hepatic mitochondrial dysfunction, oxidative stress, and apoptosis. Enhancing the hepatic supply of propionate (major gluconeogenic substrate) is a feasible preventive and therapeutic strategy to alleviate hepatic metabolic disorders during the transition period. Whether propionate supply affects pathways beyond gluconeogenesis during high FFA loads is not well known. Thus, the objective of this study was to investigate whether propionate supply could protect calf hepatocytes from FFA-induced mitochondrial dysfunction, oxidative stress, and apoptosis. Hepatocytes were isolated from 5 healthy calves (1 d old, female, 30–40 kg, fasting) and treated with various concentrations of propionate (0, 1, 2, and 4 m M propionate for 12 h) or for different times (2 m M propionate for 0, 3, 6, 12 and 24 h). Furthermore, hepatocytes were treated with propionate (2 m M), fatty acids (1.2 m M), or both for 12 h with or without 50 n M PGC-1α (peroxisome proliferator-activated receptor-gamma coactivator-1 alpha) small interfering RNA. Compared with the control group, protein abundance of PGC-1α was greater with 2 and 4 m M propionate treatment groups. Furthermore, protein abundance of TFAM (mitochondrial function marker mitochondrial transcription factor A) and VDAC1 (voltage-dependent anion channel 1) was greater with 1, 2, and 4 m M propionate, and COX4 (cyclooxygenase 4) was greater with 2 and 4 m M propionate groups. In addition, propionate supply led to an increase in protein abundance of PGC-1α, TFAM, VDAC1, and COX4 over time. Flow cytometry revealed that propionate treatment increased the number of mitochondria in hepatocytes compared with control group, but inhibition of PGC-1α abolished these beneficial effects. The lower protein abundance of PGC-1α, TFAM, COX4, and VDAC1 and activities of superoxide dismutase and glutathione peroxidase, along with greater production of reactive oxygen species, malondialdehyde, and apoptosis rate in response to treatment with high concentrations of FFA suggested an impairment of mitochondrial function and induction of oxidative stress and apoptosis. In contrast, propionate treatment hastened these negative effects. Knockdown of PGC-1α by small interfering RNA impeded the beneficial role of propionate on FFA-induced mitochondrial dysfunction, oxidative stress, and apoptosis. Overall, results demonstrated that propionate supply alleviates mitochondrial dysfunction, oxidative stress, and apoptosis in FFA-treated calf hepatocytes by upregulating PGC-1α. Together, the data suggest that PGC-1α may be a promising target for preventing or improving hepatic function during periods such as the transition into lactation where the FFA load on the liver increases. [ABSTRACT FROM AUTHOR]

Ketosis is a common metabolic disorder in high-producing dairy cows during the peripartal period. Negative energy balance leads to increased circulating levels of nonesterified fatty acids (NEFA) and β-hydroxybutyrate (BHB), consequently increasing the risk of ketosis. It is well-known that NEFA and BHB can induce lipotoxicity and oxidative stress in bovine tissues/organs including the liver and adipose tissue. Although the mammary gland is one important site for NEFA and BHB metabolism, whether an overload in their concentrations within mammary cells causes oxidative stress during ketosis remains unclear. Thus, the present study compared oxidative stress status and mitochondrial function in mammary tissues harvested by biopsy from healthy (n = 15) and clinically ketotic (n = 15) dairy cows within 2 to 3 wk postpartum. Compared with healthy cows, ketotic cows had depressed daily milk yield (median: 28.92 vs. 21.56 kg) and dry matter intake (median: 22.36 vs. 19.92 kg/d), accompanied by elevated plasma NEFA (median: 0.32 vs. 1.26 mM), BHB (median: 0.52 vs. 3.69 m M), and lower plasma glucose (median: 4.55 vs. 2.13 m M). As detected by a commercial kit, a greater level of reactive oxygen species in mammary epithelial cells of ketotic cows, and greater oxidant indices including hydrogen peroxide and malondialdehyde coupled with lower antioxidant indices including glutathione peroxidase, catalase, and superoxide dismutase activities as detected by the respective biochemical kits in the homogenate of mammary tissue of ketotic cows indicated increased oxidative stress status. Lower citrate synthase activity and ATP production as detected by the respective commercial kits coupled with lower mRNA and protein abundance of mitochondrial respiratory chain oxidative phosphorylation complexes I–V (CO I–V) in ketotic cows suggested an impairment of mitochondrial function. This was supported by lower mRNA and protein abundance of nucleus-derived mitochondrial function regulators including peroxisome proliferator activated receptor gamma coactivator 1 α, mitofusin 2, nuclear respiratory factor 1, and mitochondrial transcription factor A. Lower mitochondrial membrane potential evaluated via the tetraethylbenzimidazolylcarbocyanine iodide (JC-1) labeling method and swollen mitochondria in mammary epithelial cells of ketotic cows suggested the existence of mitochondrial damage. Overall, the present study revealed extensive mitochondrial dysfunction and oxidative stress in the mammary gland of clinically ketotic cows. As such, data suggest that reduced milk yield in cows with ketosis is partly due to enhanced oxidative stress along with mitochondrial dysregulation in the mammary gland. [ABSTRACT FROM AUTHOR]

Severe negative energy balance around parturition is an important contributor to ketosis, a metabolic disorder that occurs most frequently in the peripartal period. Autophagy and mitophagy are important processes responsible for breaking down useless or toxic cellular material, and in particular damaged mitochondria. However, the role of autophagy and mitophagy during the occurrence and development of ketosis is unclear. The objective of this study was to investigate autophagy and mitophagy in the livers of cows with subclinical ketosis (SCK) and clinical ketosis (CK). We assessed autophagy by measuring the protein abundance of microtubule-associated protein 1 light chain 3-II (LC3-II; encoded by MAP1LC3) and sequestosome-1 (p62, encoded by SQSTM1), as well as the mRNA abundance of autophagy-related genes 5 (ATG5), 7 (ATG7), and 12 (ATG12), beclin1 (BECN1), and phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3). Mitophagy was evaluated by measuring the protein abundance of the mitophagy upstream regulators PTEN-induced putative kinase 1 (PINK1) and Parkin. Liver and blood samples were collected from healthy cows [n = 15; blood β-hydroxybutyrate (BHB) concentration <1.2 m M , cows with SCK (n = 15; blood BHB concentration 1.2 to 3.0 m M) and cows with CK (n = 15; blood BHB concentration >3.0 m M with clinical signs) with similar lactation numbers (median = 3, range = 2 to 4) and days in milk (median = 6, range = 3 to 9). The serum activity of aspartate aminotransferase and alanine aminotransferase was greater in cows with CK than in healthy cows. Levels of oxidative stress biomarkers malondialdehyde and hydrogen peroxide were also higher in liver tissue from ketotic cows (SCK and CK) than from healthy cows. Compared with cows with CK and healthy cows, the hepatic mRNA abundance of MAP1LC3 , SQSTM1 , ATG5 , ATG7 , ATG12 , and PIK3C3 was upregulated in cows with SCK. Compared with healthy cows, cows with SCK had a lower abundance of p62 and a greater abundance of LC3-II, but levels of both were higher in cows with CK. The mRNA abundance of ATG12 was lower in cows with CK than in healthy cows. Furthermore, the hepatic protein abundance of PINK1 and Parkin was greater in cows with SCK and slightly lower in cows with CK than in healthy cows. These data demonstrated differences in the hepatic activities of autophagy and mitophagy in cows with SCK compared with cows with CK. Although the precise mechanisms for these differences could not be discerned, autophagy and mitophagy seem to be involved in ketosis. [ABSTRACT FROM AUTHOR]