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Subacute ruminal acidosis downregulates FOXA2, changes oxidative status, and induces autophagy in the livers of dairy cows fed a high-concentrate diet

  • Hongzhu Zhang
    Affiliations
    Ministry of Education Joint International Research Laboratory of Animal Health and Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu, P. R. China
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  • Yang Xue
    Affiliations
    Ministry of Education Joint International Research Laboratory of Animal Health and Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu, P. R. China
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  • Wan Xie
    Affiliations
    Ministry of Education Joint International Research Laboratory of Animal Health and Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu, P. R. China
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  • Yan Wang
    Affiliations
    Ministry of Education Joint International Research Laboratory of Animal Health and Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu, P. R. China
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  • Nana Ma
    Affiliations
    Ministry of Education Joint International Research Laboratory of Animal Health and Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu, P. R. China
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  • Guangjun Chang
    Affiliations
    Ministry of Education Joint International Research Laboratory of Animal Health and Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu, P. R. China
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  • Xiangzhen Shen
    Correspondence
    Corresponding author
    Affiliations
    Ministry of Education Joint International Research Laboratory of Animal Health and Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu, P. R. China
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Open AccessPublished:January 09, 2023DOI:https://doi.org/10.3168/jds.2022-22222

      ABSTRACT

      The purpose of this experiment was to investigate high-concentrate feeding-induced changed status of oxidative and autophagy in the livers of dairy cows. Hepatocyte nuclear factor 3β (FOXA2) was reported in cases of liver fibrosis, glucolipid metabolism, and hepatocyte differentiation, but not in cases liver damage in cows fed a high-concentrate diet. Therefore, we also aimed to explore the potential role of FOXA2 in SARA-induced liver damage. We divided 12 mid-lactating Holstein cows into 2 groups and fed them a high-concentrate (HC group, forage:concentrate = 4:6) and a low-concentrate (forage:concentrate = 6:4) diet. After a 2-wk adaptation period and a 3-wk experimental period, peripheral blood was collected for determination of antioxidant enzyme activity, and liver tissue was collected to examine genes and proteins. On d 20 and 21 of the experiment, rumen fluid was collected, and the pH was measured. A significant difference in rumen fluid pH was found between the 2 groups (low-concentrate: 6.10 ± 0.07 vs. HC: 5.59 ± 0.09). The rumen fluid pH in the HC group was less than 5.6 at 2 time points, indicating that SARA was successfully induced. Lipopolysaccharide (0.24 ± 0.10 vs. 0.42 ± 0.12) and malondialdehyde (1.46 ± 0.25 vs. 2.94 ± 0.65) increased, whereas superoxide dismutase (14.06 ± 0.63 vs. 11.71 ± 0.64), reduced glutathione (14.48 ± 2.25 vs. 6.82 ± 0.67), and the total antioxidant capacity (0.43 ± 0.03 vs. 0.30 ± 0.03) decreased in the peripheral blood of the HC group. Moreover, in liver tissue from the HC group, catalase (0.71 ± 0.03 vs. 0.49 ± 0.03) and superoxide dismutase (27.46 ± 1.90 vs. 20.32 ± 1.54) were decreased, whereas malondialdehyde (0.21 ± 0.03 vs. 0.28 ± 0.03) was elevated. Meanwhile, we observed lower gene expression of CAT (1.00 ± 0.15 vs. 0.64 ± 0.17), NAD(P)H quinone dehydrogenase 1 (NQO1; 1.00 ± 0.09 vs. 0.47 ± 0.14), glutathione peroxidase 1 (GPX1; 1.03 ± 0.27 vs. 0.55 ± 0.09), SOD1 (1.01 ± 0.17 vs. 0.76 ± 0.17), and SOD3 (1.02 ± 0.21 vs. 0.55 ± 0.16) in the liver tissue of the HC group. Furthermore, western blot analysis showed that high-concentrate feeding led to decreased sirtuin-1 (SIRT1) (1.00 ± 0.10 vs. 0.62 ± 0.15) and FOXA2 (1.02 ± 0.19 vs. 0.68 ± 0.18), elevated autophagy-related protein microtubule associated protein 1 light chain 3 II (MAP1LC3-II; 1.00 ± 0.32 vs. 1.98 ± 0.83) and autophagy related 5 (ATG5; 1.00 ± 0.30 vs. 1.80 ± 0.27), and suppressed antioxidant signaling pathway-related protein nuclear factor erythroid 2-like 2 (NFE2L2; 1.00 ± 0.18 vs. 0.61 ± 0.30) and heme oxygenase 1 (HMOX1; 1.00 ± 0.48 vs. 0.38 ± 0.25) in liver tissue. Overall, these data indicated that SARA elevated systematic oxidative status and enhanced autophagy in the liver, and suppressed SIRT1 and FOXA2 may mediate enhanced oxidative damage and autophagy in the livers of dairy cows fed a high-concentrate diet.

      Key words

      INTRODUCTION

      High-concentrate feeding is necessary for the dairy industry because it leads to increased milk production. However, it is detrimental to the long-term health of cows. Long-term feeding of a high-concentrate diet that contains more grains and less forage, which is low in structure, high in energy, and easier to ferment, results in the accumulation of organic acids and a decrease in buffering capacity in the rumen (
      • Plaizier J.C.
      • Krause D.O.
      • Gozho G.N.
      • McBride B.W.
      Subacute ruminal acidosis in dairy cows: The physiological causes, incidence and consequences.
      ). Moreover, in the case of the shift from a dry period to an early-lactation diet, the ruminal papillae are too short to absorb much organic acid. The ruminal microbial population also cannot metabolize these organic acids in a timely manner (
      • Kleen J.L.
      • Hooijer G.A.
      • Rehage J.
      • Noordhuizen J.P.
      Subacute ruminal acidosis (SARA): A review.
      ). All these factors lead to decreased pH in the rumen, which eventually induces SARA. Subacute ruminal acidosis is characterized by persistent low postfeeding rumen pH below 5.6 for more than 3 h per day (
      • Gozho G.N.
      • Plaizier J.C.
      • Krause D.O.
      • Kennedy A.D.
      • Wittenberg K.M.
      Subacute ruminal acidosis induces ruminal lipopolysaccharide endotoxin release and triggers an inflammatory response.
      ). Cows with SARA may exhibit decreased DMI, loss of condition, milk fat depression, liver abscess, dehydration, diarrhea, and laminitis, which cause huge economic losses (
      • Nocek J.E.
      Bovine acidosis: Implications on laminitis.
      ;
      • Kleen J.L.
      • Hooijer G.A.
      • Rehage J.
      • Noordhuizen J.P.
      Subacute ruminal acidosis (SARA): A review.
      ). The condition is difficult to diagnose because some of these signs are not specific to SARA (
      • Nocek J.E.
      Bovine acidosis: Implications on laminitis.
      ). Therefore, farms often fail to provide the correct treatment. In the acidic rumen environment, gram-negative bacteria lyse and release amounts of lysates into rumen fluid, such as LPS, an important component of the cell wall of gram-negative bacteria. Previous studies identified that LPS in rumen fluid is implicated in high-concentrate diet-induced diseases such as ruminal acidosis and laminitis (
      • Dougherty R.W.
      • Coburn K.S.
      • Cook H.M.
      • Allison M.J.
      Preliminary study of appearance of endotoxin in circulatory system of sheep and cattle after induced grain engorgement.
      ;
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      • Fina L.R.
      • Anthony H.D.
      Relationship of rumen gram-negative bacteria and free endotoxin to lactic acidosis in cattle.
      ). Meanwhile, low rumen pH results in parakeratosis of the ruminal epithelium, enabling translocation of LPS into the bloodstream (
      • Dong G.
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      • Lei C.
      • Zhou J.
      • Zhang S.
      Diet-induced bacterial immunogens in the gastrointestinal tract of dairy cows: Impacts on immunity and metabolism.
      ), which reaches the liver and many other organs through the portal vein, thus provoking systemic inflammation (
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      • Amasheh S.
      • Penner G.B.
      Symposium review: The importance of the ruminal epithelial barrier for a healthy and productive cow.
      ).
      Oxidative stress is a result of an imbalance between oxidants and antioxidants. Reactive oxygen species and reactive nitrogen species are normal by-products of metabolism and lead to oxidative stress when produced in excess (
      • Ma Q.
      Transcriptional responses to oxidative stress: Pathological and toxicological implications.
      ;
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      Signal transduction by reactive oxygen species.
      ). A previous study revealed that unbalanced nutrient feeding leads to oxidative stress (
      • Perfeito R.
      • Cunha-Oliveira T.
      • Rego A.C.
      Revisiting oxidative stress and mitochondrial dysfunction in the pathogenesis of Parkinson disease—Resemblance to the effect of amphetamine drugs of abuse.
      ) and disorders in physiology, metabolism, and even pathology in cows (
      • Miller J.K.
      • Brzezinska-Slebodzinska E.
      • Madsen F.C.
      Oxidative stress, antioxidants, and animal function.
      ). Autophagy, a process of cellular self-phagocytosis, is important in maintaining physical function. Staphylococcus aureus-treated bovine mammary epithelial cells showed elevated autophagy (
      • Geng N.
      • Liu K.
      • Lu J.
      • Xu Y.
      • Wang X.
      • Wang R.
      • Liu J.
      • Liu Y.
      • Han B.
      Autophagy of bovine mammary epithelial cell induced by intracellular Staphylococcus aureus.
      ). Meanwhile, it has been reported that autophagy is closely associated with oxidative stress (
      • Ichimura Y.
      • Waguri S.
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      • Ishimura R.
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      • Mizushima T.
      • Motohashi H.
      • Lee M.S.
      • Yoshimori T.
      • Tanaka K.
      • Yamamoto M.
      • Komatsu M.
      Phosphorylation of p62 activates the Keap1-Nrf2 pathway during selective autophagy.
      ;
      • Yun H.R.
      • Jo Y.H.
      • Kim J.
      • Shin Y.
      • Kim S.S.
      • Choi T.G.
      Roles of autophagy in oxidative stress.
      ). Autophagy has been studied in dairy cows with ketosis, fatty liver, and many other diseases (
      • Chen M.
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      • Zhai Q.
      • Liang Y.
      • Yu H.
      • Du X.
      • Shen T.
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      • Zhu Y.
      • Song Y.
      • Liu G.
      • Li X.
      • Wang Z.
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      Short communication: Enhanced autophagy activity in liver tissue of dairy cows with mild fatty liver.
      ;
      • Shen T.
      • Xu F.
      • Fang Z.
      • Loor J.J.
      • Ouyang H.
      • Chen M.
      • Jin B.
      • Wang X.
      • Shi Z.
      • Zhu Y.
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      • Wang Z.
      • Li X.
      • Du X.
      • Liu G.
      Hepatic autophagy and mitophagy status in dairy cows with subclinical and clinical ketosis.
      ), but studies on autophagy in cows with SARA have not been reported.
      Sirtuin-1 (SIRT1) is an NAD+-dependent deacetylase and is vital in regulating many biological processes (
      • Lee I.H.
      • Cao L.
      • Mostoslavsky R.
      • Lombard D.B.
      • Liu J.
      • Bruns N.E.
      • Tsokos M.
      • Alt F.W.
      • Finkel T.
      A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy.
      ;
      • Xu C.
      • Wang L.
      • Fozouni P.
      • Evjen G.
      • Chandra V.
      • Jiang J.
      • Lu C.
      • Nicastri M.
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      • Garcia B.A.
      • Adams P.D.
      • Ott M.
      • Tong W.
      • Johansen T.
      • Dou Z.
      • Berger S.L.
      SIRT1 is downregulated by autophagy in senescence and ageing.
      ). As an upstream regulator of multiple signaling pathways, SIRT1 has been reported to regulate oxidative stress and autophagy in vivo and in vitro. A previous study showed that SIRT1 can regulate autophagy-related proteins via deacetylation (
      • Lee I.H.
      • Cao L.
      • Mostoslavsky R.
      • Lombard D.B.
      • Liu J.
      • Bruns N.E.
      • Tsokos M.
      • Alt F.W.
      • Finkel T.
      A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy.
      ). In addition, many studies have reported that SIRT1 exerts antioxidative damage effects by regulating nuclear factor erythroid 2-like 2 (NFE2L2), which activates the expression of downstream antioxidant genes (
      • Shah S.A.
      • Khan M.
      • Jo M.H.
      • Jo M.G.
      • Amin F.U.
      • Kim M.O.
      Melatonin stimulates the SIRT1/Nrf2 signaling pathway counteracting lipopolysaccharide (LPS)-induced oxidative stress to rescue postnatal rat brain.
      ;
      • Wang G.
      • Xie X.
      • Yuan L.
      • Qiu J.
      • Duan W.
      • Xu B.
      • Chen X.
      Resveratrol ameliorates rheumatoid arthritis via activation of SIRT1-Nrf2 signaling pathway.
      ). Hepatocyte nuclear factor 3β, also named transcription factor forkhead box A2 (FOXA2), regulates embryonic differentiation, bile acid, glucose, and lipid metabolism (
      • Lee K.
      • Cho H.
      • Rickert R.W.
      • Li Q.V.
      • Pulecio J.
      • Leslie C.S.
      • Huangfu D.
      FOXA2 is required for enhancer priming during pancreatic differentiation.
      ;
      • Liu M.
      • Lee D.
      • Chen C.
      • Yen C.
      • Li L.
      • Lee H.
      • Chang C.
      • Chang W.
      • Hsu J.
      • Kuo H.
      • Xia W.
      • Wei Y.
      • Chiu P.
      • Chou C.
      • Du Y.
      • Dhar D.
      • Karin M.
      • Chen C.
      • Hung M.
      IKKα activation of NOTCH links tumorigenesis via FOXA2 suppression.
      ), and plays a vital role in preventing the development of liver disease in humans, but has not been reported in the livers of dairy cows. In addition, FOXA2 is also reported to be related to autophagy (
      • Peng Q.
      • Qin J.
      • Zhang Y.
      • Cheng X.
      • Wang X.
      • Lu W.
      • Xie X.
      • Zhang S.
      Autophagy maintains the stemness of ovarian cancer stem cells by FOXA2.
      ). Our previous study revealed that high-concentrate feeding induced liver damage and increased the secretion of inflammatory factors, but did not focus on the role of FOXA2 in SARA-induced liver damage (
      • Chang G.
      • Zhang K.
      • Xu T.
      • Jin D.
      • Seyfert H.M.
      • Shen X.
      • Zhuang S.
      Feeding a high-grain diet reduces the percentage of LPS clearance and enhances immune gene expression in goat liver.
      ,
      • Chang G.
      • Liu X.
      • Ma N.
      • Yan J.
      • Dai H.
      • Roy A.C.
      • Shen X.
      Dietary addition of sodium butyrate contributes to attenuated feeding-induced hepatocyte apoptosis in dairy goats.
      ). Thus, based on our previous research, we hypothesize that high-concentrate feeding may induce alternation of oxidative status and autophagy in the livers of dairy cows, and that SIRT1 and FOXA2 participate in this process.

      MATERIALS AND METHODS

      Ethics Statement

      The animal experiments were examined and approved by the Animal Care and Use Committee of Nanjing Agricultural University (No. SYXK-2017–0027), and all animal operations strictly complied with the experimental protocols under the Ministry of Science and Technology's Law on Experimental Animals (2006, Beijing, China).

      Animals, Diet, and Experimental Design

      Sample size determination was described in our previously published study (
      • Ma N.
      • Abaker J.A.
      • Wei G.
      • Chen H.
      • Shen X.
      • Chang G.
      A high-concentrate diet induces an inflammatory response and oxidative stress and depresses milk fat synthesis in the mammary gland of dairy cows.
      ). Briefly, sample size determination was based on assumed LPS concentrations of the lacteal vein [the low-concentrate (LC) group is 0.12 endotoxin units/mL and the high-concentrate (HC) group is almost 0.24 endotoxin units/mL] of dairy cows with a standard error of less than 0.05 (P = 0.05, 95% power). We used an online tool (https://www.bu.edu/researchsupport/compliance/animal-care/working-with-animals/research/sample-size-calculations-iacuc/) to calculate an estimate of the sample size. Five cows of each group are needed for a difference (P < 0.05) in the mean of LPS concentration. Twelve healthy mid-lactating Holstein dairy cows (parities 2–3) were installed with rumen fistulas and randomly divided into HC group (forage:concentrate = 4:6) and LC group (forage:concentrate = 6:4). Their BW and lactation days are 651 ± 54 kg and 233 ± 16 d, respectively. Each group was fed at 0400, 1200, and 2000 h per day. The specific ingredients in the diet are listed in Table 1. Both groups were fed adaptive diets for 2 wk followed by HC and LC diets for 3 wk. During the experimental period, cows were housed in individual tiestalls and were free to access water. The DMI was recorded daily and is listed in Table 2. All cows were healthy, and their body condition was monitored daily by evaluating rectal temperature, respiratory rate, and feed intake.
      Table 1Nutrient composition and ingredients of high-concentrate (HC) and low-concentrate (LC) diets
      ItemDiet
      LCHC
      Ingredient (% of DM)
       Ground corn19.4024.92
       Soybean meal13.5013.48
       Ground barley012.00
       Distillers dried grains with solubles3.805.91
       Limestone0.801.48
       Calcium hydrophosphate1.100.92
       Salt0.400.37
       Premix
      Premix ingredients: vitamin A, 22.5 kIU/kg; vitamin D3, 5.0 kIU/kg; vitamin E, 37.5 kIU/kg; vitamin K3, 5.0 mg/kg; Mn, 63.5 mg/kg; Zn, 111.9 mg/kg; Cu, 25.6 mg/kg; Fe, 159.3 mg/kg.
      1.00.92
       Silage corn12.006.00
       Alfalfa hay24.0017.00
       Tall oat grass24.0017.00
       Forage:concentrate (F:C)6:44:6
      Nutritional composition (% of DM)
       CP16.1616.21
       Ca1.141.18
       P0.520.51
       NDF36.1429.92
       NFC35.3942.34
       Net energy (MJ/kg)1.571.64
       NFC/NDF0.990.71
       Ash5.974.87
       Fat3.053.05
       Starch17.9627.82
      1 Premix ingredients: vitamin A, 22.5 kIU/kg; vitamin D3, 5.0 kIU/kg; vitamin E, 37.5 kIU/kg; vitamin K3, 5.0 mg/kg; Mn, 63.5 mg/kg; Zn, 111.9 mg/kg; Cu, 25.6 mg/kg; Fe, 159.3 mg/kg.
      Table 2The effect of low-concentrate (LC) and high-concentrate (HC) diet feedings on milk composition in dairy cows
      ItemDietSEMP-value
      LCHC
      Milk fat (%)4.493.920.100.001
      Milk protein (%)3.864.130.090.049
      Milk lactose (%)4.964.940.050.747
      TS (%)13.6913.340.160.135
      SCC (× 103 cells/mL)189.50212.5852.240.758
      Urea nitrogen (mg/dL)17.9518.820.390.134
      Milk yield (kg/d)17.0517.711.830.722
      DMI (kg/d)22.1923.693.150.436

      Sample Collection

      Rumen fluid was collected through the rumen fistula every 2 h for a total of 6 times on d 20 and 21, and the first collection was conducted before feeding in the morning. The samples were filtered by 4 layers of gauze, and filtrates were assessed immediately with a pH meter (HI 9125, Hanna Instruments). On d 20 and 21 of the experiment, milk composition analysis was conducted immediately after milk collection (Milko Scan TM FT1, Foss). On d 21, peripheral blood was collected with a heparin sodium vacuum tube, and plasma was collected after centrifugation (1,000 × g, 15 min) and stored in a −80°C refrigerator. Then, cows were slaughtered, and liver tissue was divided into small pieces, which were stored in liquid nitrogen.

      RNA Extraction, cDNA Synthesis, and Real-Time PCR

      Tissue fragments were ground into powder with liquid nitrogen. We used RNAiso Plus (Cat # 9108, TaKaRa) to obtain total RNA. Briefly, 100 mg of powder and 1 mL of RNAiso Plus were added to a 1.5-mL Eppendorf tube and mixed for 10 min. After adding chloroform and isopropanol and centrifuging, the RNA precipitate was collected and dissolved in 30 μL of nuclease-free water. The RNA concentration was measured with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher). To obtain cDNA, 500 ng of RNA, 2 μL of 4 × gDNA wiper, and 2 μL of 5 × PrimeScript RT Master Mix (Cat # R323, Vazyme) were mixed in nuclease-free water, and reactions were carried out in Mastercycler Nexus (Eppendorf).
      The cDNA was diluted with nuclease-free water. Then, 5 μL of 2 × ChamQ Universal SYBR qPCR Master Mix (Cat # Q711, Vazyme), 2 μL of cDNA, 0.4 μL each of 10 μM forward and reserve primers, and 2.6 μL of nuclease-free water were added to a 200-μL tube. The primers used in this experiment (Supplemental Table S1; https://data.mendeley.com/datasets/vs89s37y2x;
      • Zhang H.
      Supplemental Table S1. Mendeley Data, V1.
      ) were designed by Oligo 7 software (Molecular Biology Insights Inc.). Reactions were conducted in an ABI 7300 Real Time PCR System (Thermo Fisher). Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as an internal reference, and the 2−ΔΔCt method was used for analysis.

      Protein Isolation and Western Blot Analysis

      Tissue powder (100 mg), 10 μL of protease inhibitor, 10 μL of protein phosphatase inhibitor, and 1 mL of protein lysates (Cat # 0100, Cat # P1260, Cat # R0010, Solarbio) were added to a 1.5-mL Eppendorf tube and mixed for 10 min. After centrifugation (15,984 × g, 4°C, 15 min), the protein supernatant was collected, and the concentration was measured with a BCA Protein Assay Kit (Cat # 23225, Thermo Fisher). All samples were diluted to the same concentration.
      Different concentrations of PAGE gels (Cat # PG111, Cat # PG113, Epizyme, Shanghai, China) were made for different proteins. We used 12.5% of gels for LC3, and 7.5% of gels were used for the others. Five × SDS protein loading buffer (Cat # BL502A, Hefei, China) was added to the protein samples. After heating at 99°C for 5 min, the samples were added to gels and separated. Then, the separated proteins were transferred to polyvinylidene fluoride membranes (Cat # p2938, Millipore). Membranes were incubated in 5% BSA (Cat # A8010, Solarbio) or skim milk for 2 h and then incubated in primary antibody at 4°C for approximately 12 h followed by incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. We obtained the following: MAP1LC3 (14600–1-AP) and GAPDH (60004–1-Ig) from Proteintech; FOXA2 (ab256493) from Abcam; ATG5 (AF2269), BECN1 (AF5123), SQSTM1 (AF5312), SIRT1 (AF1267), HMOX1 (AF1333), and HRP-conjugated secondary antibodies (A0208, A0216) from Beyotime; p-NFE2L2 (DF7519) and NFE2L2 (AF0639) from Affinity Biosciences. All antibodies were diluted in Tris-buffered saline/Tween at a ratio of 1:1,000. After washing with Tris-buffered saline/Tween 6 times, the membranes were incubated with chemiluminescence reagent (Cat # E412, Vayzme), and blots were detected by a ChemDoc XRS+ Imaging System (Bio-Rad).

      Measurement of Plasma LPS

      We added 100 μL of plasma and 900 μL of sample treatment solution to a 1.5-mL Eppendorf tube to obtain a 10× diluted sample and heated in a metal bath at 70°C for 10 min. After cooling to room temperature, the LPS concentration was measured with a Chromogenic End-point Tachypleus Amebocyte Lysate Assay Kit (Chinese Horseshoe Crab Reagent Manufactory Co. Ltd.).

      Determination of Antioxidant-Related Enzymes

      For plasma, samples/standard substance were mixed with reaction reagent in a 96-well plate and incubated for 5 to 10 min at room temperature. Then, the optical density values were measured by a microplate reader (Thermo Fisher). The concentrations of the samples were calculated according to a standard curve.
      For liver tissue, the sample was ground with liquid nitrogen and diluted with saline. Then, the diluted sample was mined with reaction reagent. The remaining steps are consistent with the above procedure. Catalase (CAT; EC 1.11.1.6; A007–1-1), superoxide dismutase (SOD; A001–3), reduced glutathione (EC 1.6.4.2; A006–2-1), total antioxidant capacity (A015–2-1), and malondialdehyde (MDA; A003–1) detection kits were obtained from Nanjing Jiancheng Bioengineering Institute.

      Statistical Analysis

      All statistical analyses were performed using SPSS 26.0 (IBM Inc.). The rumen fluid pH value was analyzed using 2-way ANOVA with a univariate general linear model. Other data were analyzed using an independent-samples t-test. The primary endpoints were defined as the expression of autophagy-related genes and proteins, oxidative stress-related genes and proteins, and antioxidant enzymes activities in the liver tissue. The secondary endpoints were gene and protein expression of FOXA2 and SIRT1 in the liver tissue. The rumen fluid pH value and duration of rumen fluid pH ≤5.6 were used to determine the occurrence of SARA. The results are expressed as the mean and standard error of the mean (mean ± SEM). The residuals for each variable were used to assess normality. The data were considered statistically significant at P < 0.05.

      RESULTS

      Rumen Fluid pH and LPS Concentration in Plasma

      As shown in Figure 1, the rumen fluid pH in the HC group was always lower than that in the LC group (P < 0.001). In addition, with high-concentrate diet feeding, the rumen fluid pH in the HC group was lower than 5.6 at 4 and 6 h and was close to 5.6 at 2 and 8 h. Meanwhile, in the HC group, the LPS concentration in peripheral blood was significantly higher than that in the LC group (P = 0.016). The results above indicated that feeding a high-concentrate diet successfully induced SARA.
      Figure thumbnail gr1
      Figure 1pH values in the rumen and LPS concentrations in plasma. (A) The pH value was measured at different times after feeding on the 20th and 21st days of the experiment. (B) The mean rumen fluid pH values of the low-concentrate (LC) and high-concentrate (HC) groups. (C) Peripheral blood was collected to measure the LPS concentration. We had 6 cows per group, and the values are the mean ± SEM. EU = endotoxin units. *P < 0.05 and **P < 0.01.

      Antioxidant Enzyme Content in Peripheral Blood and Liver

      Then, we detected antioxidant-related enzymes in the plasma and liver. As shown in Figure 2, compared with those in the LC group, the reduced glutathione content (P < 0.001), SOD activity (P < 0.001), and total antioxidant capacity (P < 0.001) were significantly decreased in the HC group, whereas the MDA content (P < 0.001) was significantly increased and no significant difference in CAT activity (P = 0.118) was found between the 2 groups. Consistent with the plasma results, the liver tissue results revealed a significant decrease in CAT (P < 0.001) and SOD activity (P < 0.001) and a significant increase in the MDA content (P < 0.001) in the HC group compared with the LC group. Moreover, the serum levels of alanine aminotransferase (P = 0.012) and aspartate aminotransferase (P < 0.001) were also increased in the HC group (Figure 3).
      Figure thumbnail gr2
      Figure 2Levels of antioxidant-related enzymes in plasma. (A) Catalase (CAT) activity; (B) reduced glutathione (GSH) content; (C) total antioxidant capacity (T-AOC); (D) total superoxide dismutase (SOD) activity; (E) malondialdehyde (MDA) content. We had 6 cows per group, and the values are the mean ± SEM. LC = low concentrate; HC = high concentrate. **P < 0.01.
      Figure thumbnail gr3
      Figure 3Levels of antioxidant-related enzymes in liver tissue. (A) Catalase (CAT) activity; (B) total superoxide dismutase (SOD) activity; (C) malondialdehyde (MDA) content; (D) serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). We had 6 cows per group, and values are the mean ± SEM. LC = low concentrate; HC = high concentrate. *P < 0.05 and **P < 0.01.

      Expression of Antioxidant-Related Genes and Proteins in the Liver

      Quantitative PCR and western blot analyses were used to detect the expression of antioxidant-related genes and proteins in liver tissue. The results showed that, compared with those in the LC group, the mRNA expression levels of CAT (P = 0.002), NAD(P)H quinone dehydrogenase 1 (NQO1; P < 0.001), glutathione peroxidase 1 (GPX1; P = 0.002), superoxide dismutase 1 (SOD1; P = 0.031), SOD3 (P = 0.002), heme oxygenase 1 (HMOX1; P = 0.011), heme oxygenase 2 (HMOX2; P = 0.003), and glutathione S-transferase pi 1 (GSTP1; P = 0.009) were significantly decreased in the HC group (Figure 4A–C). In addition, we detected protein expression in the NFE2L2/HMOX1 pathway, which participates in the transcription of antioxidant-related genes. As shown in Figure 4D, E, the protein expression of HMOX1 (P = 0.019) and the ratio of p-NFE2L2 to NFE2L2 (P = 0.022) were significantly decreased.
      Figure thumbnail gr4
      Figure 4The expression of antioxidant-related genes and proteins in liver tissue. (A–C) Antioxidant-related gene expression in liver tissue was measured by quantitative PCR, and GAPDH was selected as the reference gene. (D, E) The relative protein expression of p-NFE2L2, NFE2L2, and HMOX1 against the reference GAPDH protein. We had 6 cows per group, and the values are the mean ± SEM. LC = low concentrate; HC = high concentrate. *P < 0.05; **P < 0.01.

      Expression of Autophagy-Related Genes and Proteins in the Liver

      To investigate autophagy in the livers of dairy cows fed a high-concentration diet, we detected the expression of autophagy-related genes and proteins. The results showed that the mRNA expression of microtubule associated protein 1 light chain 3 β (MAP1LC3B) (P = 0.002) and autophagy related 5 (ATG5; P = 0.003) was increased in the HC group compared with the LC group, whereas Beclin1 (BECN1; P = 0.059) and sequestosome 1 (SQSTM1; P = 0.319) showed no significant difference (Figure 5A). Compared with that in the LC group, the protein expression of microtubule associated protein 1 light chain 3 II (MAP1LC3-II; P = 0.022), ATG5 (P = 0.033), and BECN1 (P = 0.092) was elevated, whereas SQSTM1 (P = 0.239) expression was decreased in the HC group.
      Figure thumbnail gr5
      Figure 5The expression of autophagy-related genes and proteins in liver tissue. (A) Gene expression of MAP1LC3B, BECN1, ATG5, and SQSTM1 was measured by quantitative PCR, and GAPDH was selected as the reference gene. (B, C) The related protein expression of MAP1LC3-II, BECN1, ATG5, and SQSTM1 against the reference GAPDH protein. We had 6 cows per group, and the values are the mean ± SEM. LC = low concentrate; HC = high concentrate. *P < 0.05 and **P < 0.01.

      Gene and Protein Expression of FOXA2 and SIRT1 in the Liver

      As shown in Figure 6, the mRNA expression of FOXA2 (P = 0.01) and SIRT1 (P < 0.001) was decreased in the HC group compared with the LC group (Figure 6A). Consistent with mRNA expression, the protein expression of FOXA2 (P = 0.007) and SIRT1 (P = 0.305) was also decreased (Figure 6B–C).
      Figure thumbnail gr6
      Figure 6The gene and protein expression of SIRT1 and FOXA2 in liver tissue. (A) Gene and (B, C) protein expression of SIRT1 and FOXA2 was measured by quantitative PCR and western blot, and GAPDH was selected as the reference gene and protein. We had 6 cows per group, and the values are the mean ± SEM. LC = low concentrate; HC = high concentrate. **P < 0.01.

      DISCUSSION

      Subacute ruminal acidosis has always been a challenge in dairy herds due to the economic loss it causes and diagnostic difficulty. In most cases, the best method to diagnose SARA is measuring the rumen pH. Previous study provided several standards for SARA definition.
      • Gozho G.N.
      • Plaizier J.C.
      • Krause D.O.
      • Kennedy A.D.
      • Wittenberg K.M.
      Subacute ruminal acidosis induces ruminal lipopolysaccharide endotoxin release and triggers an inflammatory response.
      defined SARA as a rumen fluid pH value <5.6 for more than 3 h per day.
      • Duffield T.
      • Plaizier J.C.
      • Fairfield A.
      • Bagg R.
      • Vessie G.
      • Dick P.
      • Wilson J.
      • Aramini J.
      • McBride B.
      Comparison of techniques for measurement of rumen pH in lactating dairy cows.
      proposed that thresholds for abnormal pH indicating SARA should be 5.5, 5.8, and 5.9 when rumen fluid samples are collected by rumenocentesis, cannula, and oral probe, respectively. Considering the feasibility and convenience of the experiment, we applied the first standard in our experiment. Although the rumen fluid pH in our experiment was detected at different time points rather than using an intraruminal pH meter, which yielded consecutive rumen fluid pH values, the changing trend of rumen fluid pH was obvious. The pH values at 2 and 8 h were very close to 5.6, and the pH values at 4 and 6 h were lower than 5.6. The rumen fluid pH first decreased and then increased after feeding. We believe that feeding a high-concentrate diet induced SARA in our experiment.
      Lipopolysaccharide is the major cause of liver damage in SARA (
      • Plaizier J.C.
      • Krause D.O.
      • Gozho G.N.
      • McBride B.W.
      Subacute ruminal acidosis in dairy cows: The physiological causes, incidence and consequences.
      ). Long-term feeding with a high-concentrate diet increases the content of VFA, such as lactic acid, in the rumen and decreases the buffering capacity of the rumen, resulting in decreased rumen pH. In this condition, ruminal gram-negative bacteria lyse and release a large amount of free LPS. Low rumen pH also disrupts the integrity of the rumen epithelium, leading to the translocation of LPS from the rumen into the circulatory system. Increased LPS in blood can cause systemic inflammation and organ damage. Our previous study reported that feeding a high-concentrate diet increased the content of LPS in both the lacteal vein and portal vein (
      • Abaker J.A.
      • Xu T.L.
      • Jin D.
      • Chang G.J.
      • Zhang K.
      • Shen X.Z.
      Lipopolysaccharide derived from the digestive tract provokes oxidative stress in the liver of dairy cows fed a high-grain diet.
      ;
      • Memon M.A.
      • Wang Y.
      • Xu T.
      • Ma N.
      • Zhang H.
      • Roy A.
      • Aabdin Z.U.
      • Shen X.
      Lipopolysaccharide induces oxidative stress by triggering MAPK and Nrf2 signalling pathways in mammary glands of dairy cows fed a high-concentrate diet.
      ). Moreover, LPS-induced cellular oxidative damage and autophagy have been widely reported (
      • Chen Y.
      • Jin S.
      • Teng X.
      • Hu Z.
      • Zhang Z.
      • Qiu X.
      • Tian D.
      • Wu Y.
      Hydrogen sulfide attenuates LPS-induced acute kidney injury by inhibiting inflammation and oxidative stress.
      ;
      • Zhang J.
      • Dong X.
      • Yan Q.
      • Ren W.
      • Zhang R.
      • Jiang X.
      • Geng Z.
      • Xu X.
      • Liu C.
      • Zhang S.
      • Liu D.
      • Liu Y.
      Galectin-1 inhibited LPS-induced autophagy and apoptosis of human periodontal ligament stem cells.
      ). In this study, consistent with our previous research, we observed an increased concentration of LPS in peripheral blood, indicating the potential role of LPS in inducing hepatic oxidative damage and autophagy.
      Reactive oxygen species such as O2, OH, and H2O2 are always produced in vivo and neutralized by the antioxidant system. However, under some conditions, such as disease, metabolism disorder, and aging, antioxidant function may decrease, and oxidative stress occurs. Antioxidant enzymes constitute an important form of antioxidative damage (
      • Elias R.J.
      • Kellerby S.S.
      • Decker E.A.
      Antioxidant activity of proteins and peptides.
      ). Measuring antioxidant enzymes in plasma and tissue has been a necessary method in research on oxidative stress. In our study, we detected several antioxidants in plasma and liver tissue to determine physical antioxidant levels under SARA conditions. As the first discovered antioxidant enzyme, CAT is a general term for a class of antioxidants with different functions and plays an important role in the metabolism of H2O2 (
      • Glorieux C.
      • Calderon P.B.
      Catalase, a remarkable enzyme: targeting the oldest antioxidant enzyme to find a new cancer treatment approach.
      ). It participates in many biological activities, including proliferation and apoptosis (
      • Sies H.
      Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress.
      ). Previous studies have reported that CAT-deficient mice were more susceptible to oxidative damage, indicating that it plays an important role in resistance to oxidation (
      • Izawa S.
      • Inoue Y.
      • Kimura A.
      Importance of catalase in the adaptive response to hydrogen peroxide: Analysis of acatalasaemic Saccharomyces cerevisiae.
      ). Another important role of CAT is to assist GPX, which is also an important antioxidant enzyme. SOD can scavenge excessive free radicals derived from extracellular stimulants. According to its combined metal ions, it can be classified into the following 3 distinct isoforms: copper-zinc SOD (SOD1), manganese SOD (SOD2), and extracellular SOD (SOD3;
      • Miao L.
      • St. Clair D.K.
      Regulation of superoxide dismutase genes: Implications in disease.
      ). Researchers have reported that loss of SOD is linked with Parkinson's disease (
      • Trist B.G.
      • Davies K.M.
      • Cottam V.
      • Genoud S.
      • Ortega R.
      • Roudeau S.
      • Carmona A.
      • De Silva K.
      • Wasinger V.
      • Lewis S.
      • Sachdev P.
      • Smith B.
      • Troakes C.
      • Vance C.
      • Shaw C.
      • Al-Sarraj S.
      • Ball H.J.
      • Halliday G.M.
      • Hare D.J.
      • Double K.L.
      Amyotrophic lateral sclerosis-like superoxide dismutase 1 proteinopathy is associated with neuronal loss in Parkinson's disease brain.
      ), Alzheimer's disease (
      • Choi J.
      • Rees H.D.
      • Weintraub S.T.
      • Levey A.I.
      • Chin L.S.
      • Li L.
      Oxidative modifications and aggregation of Cu,Zn-superoxide dismutase associated with Alzheimer and Parkinson diseases.
      ), neuron degradation (
      • Trist B.G.
      • Hilton J.B.
      • Hare D.J.
      • Crouch P.J.
      • Double K.L.
      Superoxide dismutase 1 in health and disease: How a frontline antioxidant becomes neurotoxic.
      ), and lifespan reduction (
      • Landis G.N.
      • Tower J.
      Superoxide dismutase evolution and life span regulation.
      ). Malondialdehyde is the best biomarker of lipid peroxidation (
      • Tsikas D.
      Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples: Analytical and biological challenges.
      ) and is generated when lipid peroxidation occurs. Therefore, MDA is positively correlated with the oxidation level, and many researchers have reported that MDA is increased with oxidative stress in vivo/in vitro. In this study, we collected peripheral blood to detect these antioxidation enzymes, and the results showed that CAT and SOD were decreased, whereas while MDA was elevated in the HC group compared with the LC group. In addition, reduced glutathione, a ubiquitous intracellular antioxidant enzyme, and total antioxidant capacity were also decreased. These results indicated that high-concentrate feeding leads to decreased antioxidant capacity in the circulatory system.
      • Kleen J.L.
      • Hooijer G.A.
      • Rehage J.
      • Noordhuizen J.P.
      Subacute ruminal acidosis (SARA): A review.
      reported that ruminal bacteria could translocate into the portal vein via damaged rumen epithelium and then flow into the liver, leading to liver damage. Our previous study also revealed that SARA induces elevated LPS concentrations in the portal vein and that the liver plays a role in LPS clearance by measuring the LPS concentrations in the portal vein and hepatic vein (
      • Chang G.
      • Zhang K.
      • Xu T.
      • Jin D.
      • Seyfert H.M.
      • Shen X.
      • Zhuang S.
      Feeding a high-grain diet reduces the percentage of LPS clearance and enhances immune gene expression in goat liver.
      ). Moreover, in our experiment, the HC group showed increased serum levels of alanine aminotransferase and aspartate aminotransferase. Therefore, we hypothesized that oxidative damage may have occurred in the liver. Then, we detected the same antioxidant enzymes in liver tissue. The results in liver tissue showed consistency with plasma, which verified our assumption.
      Generally, excessive oxidants lead to a decrease in antioxidant enzymes by inhibiting signaling pathways rather than antioxidant enzymes themselves. Many studies have reported that NFE2L2, a transcription factor, is necessary for antioxidant production. Usually, NFE2L2 has limited functionality but is activated by phosphorylation in response to stimuli, after which it is translocated to the nucleus and acts as a transcription factor to initiate the production of antioxidant enzymes. The region to which this transcription factor binds is collectively known as the antioxidant response element. Then, we investigated whether high-concentrate feeding affected NFE2L2 activity and the transcriptional activity of antioxidant genes. The results showed that compared with those in the LC group, many antioxidants, such as CAT, NQO1, and HMOX1, were decreased in the HC group, indicating that high-concentrate feeding inhibited the transcription of antioxidant genes. In addition, the protein expression of NFE2L2 and HMOX1 showed a trend similar to that of antioxidant genes. We believe that SARA decreases NFE2L2 activity, which contributes to the low transcriptional activity of antioxidant genes further leading to oxidative stress.
      Autophagy, a process of maintaining the stability of intracellular material and metabolism, has been widely studied in vitro and in other animal models, but in dairy cows, few studies have reported it. Recently, researchers have shown that autophagy was enhanced in the livers of dairy cows with clinical ketosis and fatty liver (
      • Chen M.
      • Loor J.J.
      • Zhai Q.
      • Liang Y.
      • Yu H.
      • Du X.
      • Shen T.
      • Fang Z.
      • Shi Z.
      • Wang X.
      • Zhu Y.
      • Song Y.
      • Liu G.
      • Li X.
      • Wang Z.
      • Li X.
      Short communication: Enhanced autophagy activity in liver tissue of dairy cows with mild fatty liver.
      ;
      • Shen T.
      • Xu F.
      • Fang Z.
      • Loor J.J.
      • Ouyang H.
      • Chen M.
      • Jin B.
      • Wang X.
      • Shi Z.
      • Zhu Y.
      • Liang Y.
      • Ju L.
      • Song Y.
      • Wang Z.
      • Li X.
      • Du X.
      • Liu G.
      Hepatic autophagy and mitophagy status in dairy cows with subclinical and clinical ketosis.
      ), indicating that autophagy plays an important role in maintaining metabolic stability in the cow liver. Under normal physiological conditions, autophagy is expressed only at low levels to maintain cell homeostasis (
      • Rabinowitz J.D.
      • White E.
      Autophagy and metabolism.
      ). However, when cells are subjected to exogenous stimuli such as bacteria and toxins, autophagy is adaptively enhanced to engulf bacteria or intracellular harmful metabolites such as reactive oxygen species, which maintains cell homeostasis (
      • Levine B.
      • Kroemer G.
      Autophagy in the pathogenesis of disease.
      ). Our study showed that SARA induced oxidative stress and elevated autophagy in the livers of dairy cows fed a high-concentrate diet. Meanwhile, we think that autophagy in hepatocytes is adaptively elevated to cope with SARA-induced liver oxidative damage. However, no powerful evidence demonstrated the protective effect of autophagy in this experiment. Further studies need to be conducted to investigate the role of autophagy in SARA-induced oxidative damage in the livers of dairy cows.
      In addition, we focused on 2 proteins that may mediate alternation of oxidative status and enhanced autophagy in the livers of dairy cows with SARA. SIRT1, an NAD+-dependent deacetylase, is essential for many biological processes. It regulates many signaling pathways, including TLR4/NF-κB and NFE2L2/HMOX1, and is correlated with many diseases. SIRT1 has been reported to regulate autophagy activity by changing the acetylation level of autophagy-related proteins (
      • Lee I.H.
      • Cao L.
      • Mostoslavsky R.
      • Lombard D.B.
      • Liu J.
      • Bruns N.E.
      • Tsokos M.
      • Alt F.W.
      • Finkel T.
      A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy.
      ). Moreover, a study revealed that SIRT1 exerts its antioxidative damage function by mediating NFE2L2 translocation from the cytoplasm to the nucleus (
      • Shah S.A.
      • Khan M.
      • Jo M.H.
      • Jo M.G.
      • Amin F.U.
      • Kim M.O.
      Melatonin stimulates the SIRT1/Nrf2 signaling pathway counteracting lipopolysaccharide (LPS)-induced oxidative stress to rescue postnatal rat brain.
      ). SIRT1 also has a deacetylation regulatory effect on FOXA2, another protein on which we focused (
      • van Gent R.
      • Di Sanza C.
      • van den Broek N.J.
      • Fleskens V.
      • Veenstra A.
      • Stout G.J.
      • Brenkman A.B.
      SIRT1 mediates FOXA2 breakdown by deacetylation in a nutrient-dependent manner.
      ). It has been widely demonstrated that FOXA2 is widely demonstrated to be crucial for embryonic development and is highly expressed in endoderm-derived organs such as the lung, pancreas, thyroid gland, and liver (
      • Friedman J.R.
      • Kaestner K.H.
      The Foxa family of transcription factors in development and metabolism.
      ). Decreased expression of FOXA2 has been noted in many diseases, including cancer, liver fibrosis, cholestatic liver injury, and inflammation (
      • Wang W.
      • Yao L.
      • Shen W.
      • Ding K.
      • Shi P.
      • Chen F.
      • He J.
      • Ding J.
      • Zhang X.
      • Xie W.
      FOXA2 alleviates CCl4-induced liver fibrosis by protecting hepatocytes in mice.
      ;
      • Ploton M.
      • Mazuy C.
      • Gheeraert C.
      • Dubois V.
      • Berthier A.
      • Dubois-Chevalier J.
      • Maréchal X.
      • Bantubungi K.
      • Diemer H.
      • Cianférani S.
      • Strub J.
      • Helleboid-Chapman A.
      • Eeckhoute J.
      • Staels B.
      • Lefebvre P.
      The nuclear bile acid receptor FXR is a PKA- and FOXA2-sensitive activator of fasting hepatic gluconeogenesis.
      ). FOXA2 also plays a vital role in glucose and lipid metabolism. In addition, the NF-κB signaling pathway has been shown to regulate FOXA2. IKKα, a vital protein in activating NF-κB, can regulate the phosphorylation of FOXA2, indicating the potential effect of FOXA2 in inflammation (
      • Liu M.
      • Lee D.
      • Chen C.
      • Yen C.
      • Li L.
      • Lee H.
      • Chang C.
      • Chang W.
      • Hsu J.
      • Kuo H.
      • Xia W.
      • Wei Y.
      • Chiu P.
      • Chou C.
      • Du Y.
      • Dhar D.
      • Karin M.
      • Chen C.
      • Hung M.
      IKKα activation of NOTCH links tumorigenesis via FOXA2 suppression.
      ). In high-concentrate diet-fed cows, rumen-derived bacterial lysates stimulate the liver by inhibiting SIRT1 and FOXA2, leading to a damaged antioxidant system caused by attenuated NFE2L2 activity and disordered metabolism, which eventually induce oxidative stress and autophagy in the liver.
      Our study revealed that SARA could induce alternation oxidative status and enhance autophagy in the livers of dairy cows. SIRT1 and FOXA2 were downregulated in SARA and may regulate oxidative status and autophagy in this condition.

      ACKNOWLEDGMENTS

      This study was financially supported by grants from the National Natural Science Foundation of China (Grant 31872528). The authors have not stated any conflicts of interest.

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