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Forkhead box protein A2 alleviates toll-like receptor 4-mediated inflammation, endoplasmic reticulum stress, autophagy, and apoptosis induced by lipopolysaccharide in bovine hepatocytes

  • Wan Xie
    Affiliations
    Ministry of Education Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, P. R. China 210095
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  • Yang Xue
    Affiliations
    Ministry of Education Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, P. R. China 210095
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  • Xiaokun Song
    Affiliations
    Ministry of Education Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, P. R. China 210095
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  • Hongzhu Zhang
    Affiliations
    Ministry of Education Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, P. R. China 210095
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  • Guangjun Chang
    Affiliations
    Ministry of Education Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, P. R. China 210095
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  • Xiangzhen Shen
    Correspondence
    Corresponding author
    Affiliations
    Ministry of Education Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, P. R. China 210095
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Open AccessPublished:December 29, 2022DOI:https://doi.org/10.3168/jds.2022-22252

      ABSTRACT

      Lipopolysaccharide (LPS) is an important stimulus of inflammation via binding to toll-like receptor 4 (TLR4), but the role of TLR4 in LPS-induced cellular homeostasis disruption indicated by the increased level of endoplasmic reticulum (ER) stress, autophagy, and apoptosis is unknown in the liver of dairy cows. Previous studies show that forkhead box protein A2 (FOXA2) is an important transcriptional factor to maintain cellular metabolic homeostasis, but the mechanisms by which FOXA2 mediates cellular homeostasis disruption in response to LPS remains unclear. To achieve the aims, hepatocytes separated from dairy cows at ∼160 d in milk were pretreated with a specific TLR4 inhibitor TAK-242 for 12 h, followed by LPS treatment for another 12 h to investigate the role of TLR4 in LPS-induced disruption of cellular homeostasis. The results indicated that LPS-induced nuclear factor-κB (NF-κB)-mediated inflammatory cascades, ER stress, autophagy, and apoptosis via activating TLR4 and downregulating FOXA2 expression in bovine hepatocytes. The application of TLR4 inhibitor alleviated LPS-induced inflammation through inactivating NF-κB proinflammatory pathway, restored cell homeostasis by decreasing the level of ER stress, autophagy, and apoptosis, and upregulated FOXA2 expression. Furthermore, we also elevated FOXA2 expression with an overexpression plasmid to clarify its molecular role in response to LPS challenge. FOXA2 overexpression reduced LPS-caused inflammation by inhibiting NF-κB signaling pathway. Also, FOXA2 could alleviate ER stress to block unfolded protein response and suppress autophagic flux. In addition, FOXA2 enhanced mitochondrial membrane potential via reducing pro-apoptotic protein BAX, CASPASE3, and Cleaved CASPASE3 expression and elevating anti-apoptotic protein BCL-2 expression to mitigate LPS-induced apoptosis. Taken together, these findings suggested that FOXA2 is a mediator to alleviate TLR4-controlled inflammation, ER stress, autophagy, and apoptosis in LPS-treated bovine hepatocytes, it could serve as a potential target to intervene cell homeostasis disruption caused by LPS in the liver of dairy cows.

      Key words

      INTRODUCTION

      Overfeeding dairy cows with high-concentrate diets can meet the energy demand for milk yield in a short period, but for a long-term period, it can cause a decreased rumen pH and an increased concentration of bacterial endotoxin in the ruminal fluid (
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      Sodium butyrate mitigates iE-DAP induced inflammation caused by high-concentrate feeding in liver of dairy goats.
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      Sodium butyrate suppresses NOD1-mediated inflammatory molecules expressed in bovine hepatocytes during iE-DAP and LPS treatment.
      ). Deletion of FOXA2 disrupts hepatic bile acid homeostasis and results in ER stress in the mice fed a diet containing cholic acid (
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      Hepatocyte-specific ablation of Foxa2 alters bile acid homeostasis and results in endoplasmic reticulum stress.
      ). Recent publication demonstrates ER stress induced by tunicamycin reduces FOXA2 expression to facilitate cancer stem cell self-renewal via homeobox B9 (HOXB9)-miR-765 axis in human melanoma (
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      Regulation of cancer stem cell self-renewal by HOXB9 Antagonizes endoplasmic reticulum stress-induced melanoma cell apoptosis via the miR-765-FOXA2 axis.
      ). Moreover, γ-D-glutamyl-meso-diaminopimelic acid treatment activates inflammatory response and autophagy with inactive FOXA2 expression and in bovine hepatocytes (
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      γ-d-Glutamyl-meso-diaminopimelic acid induces autophagy in bovine hepatocytes during nucleotide-binding oligomerization domain 1-mediated inflammation.
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      Foxa2 may modulate hepatic apoptosis through the cIAP1 pathway.
      ). These studies demonstrate that FOXA2 monitors cell homeostasis including inflammation, ER stress, autophagy, and apoptosis, but the role of FOXA2 in regulating inflammatory response and cell stress remains unknown in bovine hepatocytes.
      We hypothesize that LPS treatment disrupts the cellular homeostasis indicated by active inflammatory response and the increased level of ER stress, autophagy, and apoptosis in a TLR4-dependent manner, and FOXA2 overexpression can alleviate these changes to restore cellular homeostasis in bovine hepatocytes. Our study will provide a novel sight to alleviate LPS-induced hepatic stress in dairy cows.

      MATERIALS AND METHODS

      Reagents and Chemicals

      Ultrapure LPS (Escherichia coli O111:B4, L2630, Sigma-Aldrich) and a specific-TLR4 inhibitor TAK-242 (243984–11–4, MedChem Express) were applied to treat the cells in current study. No animals were used in this study, and ethical approval for the use of animals was thus deemed unnecessary.

      Hepatocyte Isolation, Cell Culture, and Identification

      The primary bovine hepatocytes were kindly provided and shipped by Professor Juan J. Loor (Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana) for our current study. Hepatocytes were isolated from liver tissue obtained via puncture biopsy from mid-lactation Holstein multiparous cows (almost 160 d postpartum). The liver tissue was washed in 70% ethanol for 30 s to reduce superficial contaminants. After washing, the liver tissue was kept in 10 mL of sterile tubes containing 5 mL of HEPES with 0.05 mM EGTA, and transported to the laboratory within 30 min. After being finely minced with a scalpel blade, the tissue was washed with Ca2+- and Mg2+-free HBSS (Corning) by centrifugation at 50 × g for 3 min. About 50 mL of HBSS containing 1 mM calcium chloride and 150 U/mL type I collagenase (Sigma Chemical Co.) was used to resuspend the tissue. Subsequently, the minced tissue was incubated with collagenase I for 40 min at 37°C with gentle shaking. After incubation, medium (25 mL) with 10% fetal bovine serum (Thermo Fisher Scientific) was used to neutralize the collagenase. Subsequently, a 200-mesh cell strainer was used to filter the cell suspensions. After 3-time washing with PBS, the filtered cell suspensions were resuspending in growth medium (Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 1 nM glucagon, 10 nM dexamethasone, 10 ng/mL epidermal growth factor, 10 nM insulin, and 1% penicillin/streptomycin), and then cultured in T75 flasks (Corning). All medium supplements were from HyClone. The hepatocytes were incubated in an incubator (3111, Thermo Fisher Scientific) set at 37°C under a humidified atmosphere composed of 95% air and 5% CO2 to grow up to 60 to 70% confluency. At 60 to 70% confluency after 48 h incubation, cells were treated with LPS in serum-free media. In the present study, we used a confluent monolayer. Cells between 4 and 6 passage numbers were used in our present study. The hepatocyte marker genes albumin (ALB), betaine homocysteine s-methyltransferase (BHMT), and cytochrome P4501A1 (CYP1A1) were used to identify the bovine hepatocytes as reported in previous study (
      • Zhou Y.F.
      • Zhou Z.
      • Batistel F.
      • Martinez-Cortés I.
      • Pate R.T.
      • Luchini D.L.
      • Loor J.J.
      Methionine and choline supply alter transmethylation, transsulfuration, and cytidine 5′-diphosphocholine pathways to different extents in isolated primary liver cells from dairy cows.
      ).

      Experimental Design

      First of all, the mRNA expression and protein abundance of key genes related to inflammatory response, ER stress, autophagy, and apoptosis were tested to clarify the effect of LPS on inflammation and cell damage at different concentrations. Then, the optimum concentration (12 μg/mL) required for the induction of inflammation and cell damage was selected to treat bovine hepatocytes. To explore the role of TLR4 in the LPS-induced inflammation and cell damage, the mRNA expression of key genes related to inflammatory response, ER stress, autophagy, and apoptosis was measured at different doses of TAK-242 to find the optimum doses to alleviate the effect of LPS on bovine hepatocytes. We treated the cells with different doses of TAK-242 according to cell counting kit-8 assay, and the experiment was divided into the following 6 groups: the control group (without treatment; n = 3), the LPS-treated group (LPS; n = 3), the 0.1 μM TAK-242 with LPS-treated group (0.1 μM; n = 3), the 1.0 μM TAK-242 with LPS group (1.0 μM; n = 3), the 5.0 μM TAK-242 with LPS group (5.0 μM; n = 3), and the 10 μM TAK-242 with LPS group (10.0 μM; n = 3). For the LPS group, the hepatocytes were treated with 12 μg/mL LPS for 12 h. For the TAK-242 with LPS group, the cells were exposed to TAK-242 for 12 h at different doses followed by 12 μg/mL LPS treatment for another 12 h. Then, the optimum concentration (10 μM) was selected to alleviate the effect of LPS on inflammation and cell damage in bovine hepatocytes. The experiment was divided into the following 4 groups: the control group (without treatment; n = 3), the TAK-242-treated group (TAK-242; n = 3), the LPS-treated group (LPS; n = 3), and the TAK-242 with LPS group (TAK-242+LPS or TL, n = 3). For the TAK-242 and LPS group, the hepatocytes were treated with 10 μM TAK-242 and 12 μg/mL LPS for 12 h, respectively. For the TAK-242+LPS group, the cells were exposed to 10 μM TAK-242 for 12 h followed by 12 μg/mL LPS treatment for another 12 h. To explore the role of FOXA2 in mediating the LPS-induced inflammation and cell damage, the cells were divided into the following 4 groups: the control group (pcDNA; without treatment; n = 3), the overexpression group (pcFOXA2; n = 3), the LPS-treated group (pcDNA+LPS; n = 3), and the overexpression with LPS group (pcFOXA2+LPS; n = 3). For the pcDNA and pcFOXA2 group, the hepatocytes were transfected with pcDNA3.1 and pcFOXA2 (FOXA2 overexpression plasmid) for 48 h, respectively. For the pcDNA+LPS and pcFOXA2+LPS group, the hepatocytes were transfected with pcDNA3.1 and pcFOXA2 for 36 h, respectively, followed by 12 μg/mL LPS treatment for another 12 h.

      Cell Viability

      One × 104 bovine hepatocytes were placed in each 96-well microplate well and repeated for 6 times for each treatment followed by 24-h incubation in an incubator (3111, Thermo Fisher Scientific) set at 37°C under a humidified atmosphere composed of 95% air and 5% CO2. Hepatocytes were treated with LPS for 12 h or TAK-242 for 12 h followed by cell counting kit-8 (CCK-8, CA1210) analysis for measuring the live cell percentage. Ten microliters of cell counting kit-8 solution was added into each well and the microplates were incubated in an incubator (3111, Thermo Fisher Scientific) set at 37°C under a humidified atmosphere composed of 95% air and 5% CO2.

      Measurement of IL-1β and IL-8 Concentration by ELISA

      The concentration of IL-1β and IL-8 in the cell culture supernatants was measured by a commercially available ELISA assay kit (MEIMIAN, Jiangsu Meimian Industrial Co., Ltd.) as directed by the manufacturers.

      Extraction of RNA and Performance of Quantitative Reverse-Transcription PCR

      The total RNA was extracted from liver tissue (n = 3 cows;
      • Xie W.
      • Xue Y.
      • Zhang H.
      • Wang Y.
      • Meng M.
      • Chang G.
      • Shen X.
      A high-concentrate diet provokes inflammatory responses by downregulating Forkhead box protein A2 (FOXA2) through epigenetic modifications in the liver of dairy cows.
      ), bovine hepatocytes, and mammary tissue (stored at −80°C, n = 6 cows;
      • 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.
      ) using RNA isoPlus reagent (9109, Takara) according to manufacturer's instructions. HiScript III RT SuperMix for qPCR (+gDNA wiper, R323–01, Vazyme) was mixed with 500 ng/μL of RNA to synthesize cDNA by reverse transcription. All information on primers used in this study are listed in Supplemental Table S1 (https://data.mendeley.com/datasets/k6jzcmbg4g/draft?a=55f9b312-1aa9-4975-9839-4538d88eda83;
      • Xie W.
      Supplementary figures and tables. Mendeley Data, v2.
      ). The quantitative reverse-transcription PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme) on 7300 real-time PCR system (Applied Biosystems). Glyceraldehyde phosphate dehydrogenase served as the housekeeping gene for the normalization of gene expression, and our previous studies have reported its validity as a reference for normalizing gene expression in the liver of dairy cows (
      • Xu T.
      • Tao H.
      • Chang G.
      • Zhang K.
      • Xu L.
      • Shen X.
      Lipopolysaccharide derived from the rumen down-regulates stearoyl-CoA desaturase 1 expression and alters fatty acid composition in the liver of dairy cows fed a high-concentrate diet.
      ;
      • Xie W.
      • Xue Y.
      • Zhang H.
      • Wang Y.
      • Meng M.
      • Chang G.
      • Shen X.
      A high-concentrate diet provokes inflammatory responses by downregulating Forkhead box protein A2 (FOXA2) through epigenetic modifications in the liver of dairy cows.
      ) and in bovine hepatocytes (
      • Chandra Roy A.
      • Wang Y.
      • Zhang H.
      • Roy S.
      • Dai H.
      • Chang G.
      • Shen X.
      Sodium butyrate mitigates iE-DAP induced inflammation caused by high-concentrate feeding in liver of dairy goats.
      ;
      • Roy A.C.
      • Chang G.
      • Ma N.
      • Wang Y.
      • Roy S.
      • Liu J.
      • Aabdin Z.U.
      • Shen X.
      Sodium butyrate suppresses NOD1-mediated inflammatory molecules expressed in bovine hepatocytes during iE-DAP and LPS treatment.
      ). The 2−△△Ct method was used to for relative quantification. The results were presented as the fold change relative to the control.

      Western Blot Analysis

      Radio immunoprecipitation assay lysis buffer with 1 mM phenylmethylsulfonyl fluoride (PMSF, R0010, Solarbio) was used to extract the total protein of bovine hepatocytes. The protein concentration was measured using a bicinchoninic acid protein assay kit (23225, Thermo Fisher Scientific) as directed by the manufacturer. Samples were diluted to the same concentration and denatured with 5 × SDS loading buffer (BL502A, Biosharp life Sciences). The same quantity of protein was subjected to SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. Membranes were blocked using 7% skim milk for 2 h at room temperature followed by incubated with the primary antibodies for 16 h at 4°C. Subsequently, the membranes were washed with 1 × Tris-buffered saline containing Tween 20 and then incubated with horseradish peroxidase-conjugated secondary antibodies. The primary and secondary antibodies are listed in Supplemental Table S2 (https://data.mendeley.com/datasets/k6jzcmbg4g/draft?a=55f9b312-1aa9-4975-9839-4538d88eda83;
      • Xie W.
      Supplementary figures and tables. Mendeley Data, v2.
      ). An ECL Plus Kit (E411–05, Vazyme) was used to visualize the bands on the membranes, and BIO-RAD Molecular Imager ChemiDoc XRS + Imaging System (Bio-rad, Berkeley) was used to capture the band signals. The intensity of each band was analyzed with Image Lab software (Bio-rad, Berkeley). Glyceraldehyde phosphate dehydrogenase or β-actin (ACTB) was chosen to normalize the protein expression. The final result of each target protein was presented as relative abundance to GAPDH or ACTB.

      Plasmid Construction and Transfection

      The FOXA2 overexpression plasmid pcDNA3.1-FOXA2 (named pcFOXA2) was designed and synthesized by Shanghai Sangon Biotechnology Co. Ltd. Bovine hepatocytes were cultured in microplate wells and were transfected with pcDNA3.1 plasmid as the control group and pcFOXA2 plasmid as the treatment group using Lipofectamine 3000 (L3000015, Thermo Fisher Scientific) when the cell density reached 70% confluency according to the manufacturer's protocol. Thirty-six hours posttransfection, the hepatocytes were exposed to LPS for another 12 h.

      Measurement of Autophagic Flux

      In 24-well microplates, 5 × 104 bovine hepatocytes were cultured on coverslips in each well at 37°C under a humidified atmosphere composed of 95% air and 5% CO2, and transfected with the tandem fluorescent monomeric red fluorescent protein (mRFP)-enhanced green fluorescent protein (eGFP)-LC3 (mRFP-eGFP-LC3) plasmid (Beyotime) to detect autophagic flux using Lipofectamine 3000 when cells reached 70% confluency. Thirty-six hours posttransfection, the hepatocytes were exposed to LPS for another 12 h. The fluorescence of mRFP-eGFP-LC3 was observed under a LSM 710 confocal laser microscope system (Zeiss). Experiments were performed in triplicate.

      Immunofluorescence Analysis

      In each 24-well, 5 × 104 bovine hepatocytes were seeded on circular coverslips for 24-h incubation in an incubator (3111, Thermo Fisher Scientific) set at 37°C under a humidified atmosphere composed of 95% air and 5% CO2. After indicated treatment, the cells were fixed with 4% paraformaldehyde (P1110, Solarbio) for 20 min and then incubated with 0.25% Triton X-100 (T8200, Solarbio) for 15 min at room temperature to increase permeability. We used 5% BSA in PBS to block hepatocytes at room temperature for 1 h. After blocked, the hepatocytes were incubated with the primary antibodies at 4°C for 16 h. The cells were then incubated with FITC- or Cy3-labeled secondary antibodies at 37°C for 1 h in the dark. Subsequently, the cell nuclei were counter-stained with 4',6-diamidino-2-phenylindole staining solution (C0060, Solarbio) at room temperature for 15 min in the dark. Antifade mounting medium (P0126, Beyotime) was used to mount the coverslips on glass slides. The fluorescence was observed under a confocal laser microscope (Zeiss). The primary antibodies and secondary antibodies are listed in Supplemental Table S2.

      Measurement of Apoptotic Cell by Flow Cytometry

      In each 24-well, 5 × 104 bovine hepatocytes were seeded for 24 h incubation in an incubator (3111, Thermo Fisher Scientific) set at 37°C under a humidified atmosphere composed of 95% air and 5% CO2. After indicated treatment, the hepatocytes were collected through digestion using EDTA-free trypsin (T1350, Solarbio). The apoptotic cells were stained using an Annexin V-FITC Apoptosis Detection Kit (with propidium iodide, 88–8005–74, Thermo Fisher Scientific) according to manufacturer's instructions and detected by BD FACS-Verse Flow Cytometer (BD Biosciences). The data were analyzed by Flowjo software.

      Acridine Orange, Lyso-Tracker Red, Lyso-Sensors Green, and Tetraethylbenzimidazolyl Carbocyanine Iodide Probe Staining

      In each 24-well, 5 × 104 bovine hepatocytes were seeded on circular coverslips for 24 h incubation in an incubator (3111, Thermo Fisher Scientific) set at 37°C under a humidified atmosphere composed of 95% air and 5% CO2. After indicated treatment, the hepatocytes stained with 5 μM acridine orange (AO) solution (A3568, Thermo Fisher Scientific) in cell culture medium for 15 min at 37°C were analyzed for autolysosome formation. To analyze lysosome mass, the cells were stained with 0.5 μM Lyso-Tracker red (C1046, Beyotime) in cell culture medium for 30 min at 37°C. The cells were stained with Lyso-Sensors green (40767ES50, Yeasen) in cell culture medium for 20 min at 37°C to analyze the lysosome pH. The mitochondrial membrane potential (MMP, ΔΨm) was detected by tetraethylbenzimidazolyl carbocyanine iodide (JC-1) probe. The cells were stained with 10 μg/mL JC-1 solution (40705ES08, Yeasen) in cell culture medium for 15 min at 37°C. After washing with PBS, the processed cells were detected by laser confocal microscope (Zeiss) to determine fluorescence.

      Statistical Analysis

      The 2-tailed Student's t-test was used to analyze the results between 2 groups. The data from different concentrations of LPS and TAK-242 treatment was analyzed using 1-way ANOVA with Dunnett's post hoc test, the rest of data was analyzed using ANOVA with least significant difference post hoc test by IBM SPSS 20.0 statistics for Windows (IBM Inc.). The results are expressed as the mean and standard error of the mean (mean ± SEM). The P < 0.05 is considered statistically significant and P < 0.01 is considered highly significant. All the graphs were created using GraphPad Prism 8 (GraphPad Software).

      RESULTS

      Identification of Bovine Hepatocytes

      To identify the bovine hepatocytes, the expression of ALB, BHMT, and CYP1A1 was detected. As shown in Supplemental Figure S1 (https://data.mendeley.com/datasets/k6jzcmbg4g/draft?a=55f9b312-1aa9-4975-9839-4538d88eda83;
      • Xie W.
      Supplementary figures and tables. Mendeley Data, v2.
      ), the bands of ALB, BHMT, and CYP1A1 showed the strong identity in the hepatocytes and the liver of dairy cows, whereas ALB, BHMT, and CYP1A1 expression could not be detected in bovine mammary gland tissues, which confirmed the phenotype of the cells was the same as bovine liver and the cells could be used for studying the bovine liver function in vitro.

      Effects of LPS on Inflammatory Response, ER Stress, Autophagy, and Apoptosis in Bovine Hepatocytes

      To study the effects of LPS on the inflammatory response, ER stress, autophagy, and apoptosis in bovine hepatocytes, we treated the cells with different concentrations of LPS for 12 h, and analyzed the cell viability. We found no change in cell viability percentage at all indicated doses of LPS (Figure 1a). As shown in Figure 1b, compare with control group, LPS increased the mRNA expression of TLR4 at the doses of 8, 12, and 16 μg/mL (P < 0.05 for all). The mRNA expression of IL-8 and SAA3 was increased after LPS treatment at all doses (P < 0.05 for all), whereas the FOXA2 mRNA expression was decreased at the doses of 2, 4, and 12 μg/mL (P < 0.01 for all). As showed in Figure 1c, LPS promoted the protein expression of TLR4 at the 16 μg/mL dose (P = 0.005), phosphorylated inhibitor of nuclear factor kappa-B (IκBα) at all indicated doses (P < 0.01 for all), IκBα at the doses of 2 and 12 μg/mL (P = 0.006 and P = 0.022, respectively), and phosphorylated NF-κB P65 at the doses of 4, 8, and 12 μg/mL (P < 0.01 for all), and diminished FOXA2 protein expression at the doses of 8, 12, and 16 μg/mL (P < 0.05 for all). As shown in Figure 1d, compared with the control group (0 μg/mL), the production of IL-1β significantly and sharply increased in the 2 and 16 μg/mL (P = 0.033 and P = 0.03, respectively) groups and the concentration of IL-8 highly increased in the 8 and 12 μg/mL groups (P = 0.025 and P = 0.011, respectively). These results indicated that LPS activated TLR4/NF-κB proinflammatory signaling pathway and triggered inflammatory response in bovine hepatocytes.
      Figure thumbnail gr1a
      Figure 1Effects of different concentrations of LPS on inflammatory response, endoplasmic reticulum (ER) stress, autophagy, and apoptosis in bovine hepatocytes. (a) The percentage of cell viability during treatment of the hepatocytes with LPS at different concentrations for 12 h. Cell viability was measured using cell counting kit-8. (b) The relative mRNA expression of inflammatory genes. (c) The relative expression of FOXA2 and inflammatory proteins in the TLR4/NF-κB signaling pathway (left), and the small bar graphs (right) represent the relative intensity of proteins. (d) The secretion of IL-1β and IL-8 in cell supernatant measured by ELISA kit. (e) The relative mRNA expression of ER stress-related genes. (f) The relative expression of ER stress-related proteins (left), and the small bar graphs (right) represent the relative intensity of proteins. (g) The relative mRNA expression of autophagy-related genes. (h) The relative expression of apoptosis-related proteins. (i) The relative protein expression of autophagy-related genes. (j) The relative expression of apoptosis-related proteins. The relative mRNA expression and relative protein intensity are determined by quantitative reverse-transcription PCR and western blot, respectively. Glyceraldehyde 3-phosphate dehydrogenase and β-actin (ACTB) serve as the loading controls for normalization. The data are expressed as the mean ± SEM and analyzed by ANOVA with Dunnett's post hoc test. The P ≤ 0.05 is considered statistically significant. These data are representative of 3 independent experiments (n = 3).
      Figure thumbnail gr1b
      Figure 1Effects of different concentrations of LPS on inflammatory response, endoplasmic reticulum (ER) stress, autophagy, and apoptosis in bovine hepatocytes. (a) The percentage of cell viability during treatment of the hepatocytes with LPS at different concentrations for 12 h. Cell viability was measured using cell counting kit-8. (b) The relative mRNA expression of inflammatory genes. (c) The relative expression of FOXA2 and inflammatory proteins in the TLR4/NF-κB signaling pathway (left), and the small bar graphs (right) represent the relative intensity of proteins. (d) The secretion of IL-1β and IL-8 in cell supernatant measured by ELISA kit. (e) The relative mRNA expression of ER stress-related genes. (f) The relative expression of ER stress-related proteins (left), and the small bar graphs (right) represent the relative intensity of proteins. (g) The relative mRNA expression of autophagy-related genes. (h) The relative expression of apoptosis-related proteins. (i) The relative protein expression of autophagy-related genes. (j) The relative expression of apoptosis-related proteins. The relative mRNA expression and relative protein intensity are determined by quantitative reverse-transcription PCR and western blot, respectively. Glyceraldehyde 3-phosphate dehydrogenase and β-actin (ACTB) serve as the loading controls for normalization. The data are expressed as the mean ± SEM and analyzed by ANOVA with Dunnett's post hoc test. The P ≤ 0.05 is considered statistically significant. These data are representative of 3 independent experiments (n = 3).
      Figure 1e shows that the mRNA abundance of 78 kDa glucose-regulated protein (GRP78) was higher in the 12 μg/mL group (P = 0.011) than the control group, and C/EBP homologous protein (CHOP) transcriptionally increased in the 8 and 12 μg/mL groups (P = 0.038 and P = 0.002, respectively) compared with the control group. In Figure 1f, LPS could elevate the protein expression of GRP78 at the doses of 12 and 16 μg/mL (P < 0.01 for all), double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) at the doses of 12 and 16 μg/mL (P < 0.05 for all), inositol-requiring enzyme 1α (IRE1α) at the doses of 4, 12, and 16 μg/mL (P < 0.05 for all), and CHOP at the doses of 12 and 16 μg/mL (P < 0.05 for all). These results suggested LPS treatment induced ER stress and UPR in bovine hepatocyte. ER stress is associated with autophagy and apoptosis. Hence, we investigated the effects of LPS on autophagy and apoptosis. As shown in Figures 1g and h, LPS elevated the mRNA abundance of unc-51-like kinase 1 (ULK1) at 2 and 8 μg/mL dose points (P = 0.011 and P = 0.007, respectively), BECLIN1 at 12 and 16 μg/mL dose points (P = 0.001 and P = 0.019, respectively), CASPASE3 at 2 and 8 μg/mL dose points (P < 0.01, for all), and diminished that of sequestosome 1 (SQSTM1) at 4, 12, and 16 μg/mL dose points (P < 0.05 for all), and B cell leukemia-2 (BCL-2) at 16 μg/mL dose point (P = 0.045). As shown in Figures 1i and j, LPS treatment increased the protein abundance of ULK1 at the doses of 8, 12, and 16 μg/mL (P < 0.05 for all), autophagy-related gene 5 (ATG5) at the doses of 12 and 16 μg/mL (P = 0.037 and P = 0.006, respectively), BECLIN1 at the doses of 8, 12, and 16 μg/mL (P < 0.05 for all), lysosomal-associated membrane protein 2A (LAMP2A) at the dose of 8 μg/mL (P = 0.049), BCL2-associated X protein (BAX) at the doses of 8, 12, and 16 μg/mL (P < 0.05 for all), Cleaved CASPASE3 at the doses of 12 and 16 μg/mL (P = 0.05 and P = 0.004, respectively), and CASPASE3 at the dose of 16 μg/mL (P = 0.049). As shown in Figure 1i, the protein expression of mechanistic target of rapamycin kinase (mTOR) in the 16 μg/mL group (P = 0.015), and SQSTM1 in the 4 and 12 μg/mL groups (P = 0.019 and P = 0.009, respectively).
      To further explore the effects of LPS on inflammatory response, ER stress, autophagy, and apoptosis in bovine hepatocytes, we treated the cells with 12 μg/mL LPS for 12 h, and the immunofluorescence data showed that compared with the control group, FOXA2 showed poor staining (Supplemental Figure S2a; https://data.mendeley.com/datasets/k6jzcmbg4g/draft?a=55f9b312-1aa9-4975-9839-4538d88eda83;
      • Xie W.
      Supplementary figures and tables. Mendeley Data, v2.
      ), whereas LPS treatment increased the activation of NF-κB P65 (Supplemental Figure S2b). As shown in Supplemental Figure S2c, compared with the control group, there was strong staining for GRP78 and CHOP in LPS-treated cells. Moreover, LPS treatment increased the fluorescence intensity of autophagy-related protein autophagy-related gene16-like 1 (ATG16L1), microtubule-associated protein 1 light chain 3 (LC3), and lysosomal-associated membrane protein (LAMP1), and decreased the fluorescence intensity of SQSTM1 in LPS-treated cells (Supplemental Figure S2d). Additionally, LPS treatment increased the fluorescence intensity of mRFP-eGFP-LC3 (Supplemental Figure S2e) indicating that LPS enhanced autophagic flux. The result of AO staining declared that LPS treatment increased the formation of autolysosomes (Supplemental Figure S2f). Flow cytometry results showed that LPS treatment significantly increased the apoptotic cells (P = 0.012; Supplemental Figure S2g).

      Inhibition of TLR4 Inactivated NF-κB Signaling Pathway in LPS-stimulated Bovine Hepatocytes

      In the current study, a specific TLR4 inhibitor TAK-242 was used to inhibit the activity of TLR4. The cell viability during 0.1 to 20 μM TAK-242 treatment for 12 h was not influenced by TAK-242 within 10 μM dose, and significantly decreased at 20 μM dose (P = 0.001; Supplemental Figure S3a; https://data.mendeley.com/datasets/k6jzcmbg4g/draft?a=55f9b312-1aa9-4975-9839-4538d88eda83;
      • Xie W.
      Supplementary figures and tables. Mendeley Data, v2.
      ). Pretreated with TAK-242 for 12 h could decrease the mRNA level of MYD88 at the doses of 1, 5, and 10 μM (P < 0.01 for all), TRAF6 at the doses of 0.1 and 10 μM (P < 0.01 for all), transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) at the doses of 0.1 and 1 μM (P < 0.05 for all), IκBα at all the doses (P < 0.05 for all), TNFα at the dose of 10 μM (P = 0.044), IL-1β, IL-8,IL-6, and SAA3 at all indicated doses (P < 0.05 for all), and increase the mRNA abundance of FOXA2 at the doses of 0.1,1, and 10 μM (P < 0.01 for all) in LPS-treated hepatocytes (Supplemental Figure S3b and S3c). In addition, inhibiting TLR4 with 10 μM TAK-242 could upregulate FOXA2 protein expression (P = 0.013) and downregulate the protein abundance of phosphorylated IκBα (P = 0.006), phosphorylated NF-κB P65 (P = 0.001), and TNFα (P = 0.009) in LPS-exposed cells (Figure 2a). Immunofluorescence analysis showed that inactivating TLR4 increased the protein expression of FOXA2 and reduced the activation of NF-κB P65 in LPS-stimulated hepatocytes (Figure 2b). These results indicated that inhibiting TLR4 could inactivate NF-κB signaling pathway with upregulating FOXA2 expression during LPS treatment.
      Figure thumbnail gr2a
      Figure 2Inhibition of TLR4 mitigates LPS-induced inflammatory response and endoplasmic reticulum (ER) stress in bovine hepatocytes. The hepatocytes were treated with 10 μM TAK-242 for 12 h and incubated with LPS (12 μg/mL) for another 12 h. (a) Representative image of western blot for the relative expression of inflammatory proteins in NF-κB signaling pathway. (b) The protein expression of FOXA2 and P65 determined by immunofluorescence staining. The immunofluorescence analysis was performed with anti-FOXA2 antibody (green), anti-P65 (green) antibody, and 4ʹ,6-diamidino-2-phenylindole (DAPI; blue; nuclei); bar = 10 μm. (c) Representative image of western blot for the relative expression of ER stress-related proteins. (d) The protein expression of JNK, GRP78, and CHOP determined by immunofluorescence staining. The immunofluorescence analysis was performed with anti-GRP78 antibody (green), anti-JNK antibody (green), anti-CHOP (green) antibody, and DAPI (blue; nuclei); bar = 10 μm. The relative protein expression is determined by western blot, and GAPDH and β-actin (ACTB) serve as the loading controls for normalization. The data are expressed as the mean ± SEM and analyzed by 1-way ANOVA with LSD post hoc test. The P ≤ 0.05 is considered statistically significant. These data are representative of 3 independent experiments (n = 3).
      Figure thumbnail gr2b
      Figure 2Inhibition of TLR4 mitigates LPS-induced inflammatory response and endoplasmic reticulum (ER) stress in bovine hepatocytes. The hepatocytes were treated with 10 μM TAK-242 for 12 h and incubated with LPS (12 μg/mL) for another 12 h. (a) Representative image of western blot for the relative expression of inflammatory proteins in NF-κB signaling pathway. (b) The protein expression of FOXA2 and P65 determined by immunofluorescence staining. The immunofluorescence analysis was performed with anti-FOXA2 antibody (green), anti-P65 (green) antibody, and 4ʹ,6-diamidino-2-phenylindole (DAPI; blue; nuclei); bar = 10 μm. (c) Representative image of western blot for the relative expression of ER stress-related proteins. (d) The protein expression of JNK, GRP78, and CHOP determined by immunofluorescence staining. The immunofluorescence analysis was performed with anti-GRP78 antibody (green), anti-JNK antibody (green), anti-CHOP (green) antibody, and DAPI (blue; nuclei); bar = 10 μm. The relative protein expression is determined by western blot, and GAPDH and β-actin (ACTB) serve as the loading controls for normalization. The data are expressed as the mean ± SEM and analyzed by 1-way ANOVA with LSD post hoc test. The P ≤ 0.05 is considered statistically significant. These data are representative of 3 independent experiments (n = 3).

      Inhibition of TLR4 Alleviated ER Stress to Block UPR in LPS-Stimulated Bovine Hepatocytes

      As shown in Supplemental Figure S3d, inhibiting TLR4 with TAK-242 for 12 h could decrease the mRNA level of ATF6 at the doses of 1, 5, and 10 μM (P < 0.05 for all), TNF receptor-associated factor 2 (TRAF2) at the doses of 5 and 10 μM (P < 0.05 for all), X-box binding protein 1 (XBP1), and activating transcription factor 4 (ATF4) and CHOP at all doses (P < 0.05 for all) in LPS-treated hepatocytes. The western blot analysis results showed that 10 μM TAK-242 pretreatment significantly decreased the abundance of ER stress-related protein, including GRP78 (P = 0.025), PERK (P = 0.026), phosphorylated PERK (P = 0.002), ATF6 (P = 0.003), IRE1α (P = 0.001), phosphorylated c-Jun N-terminal kinase (JNK, P = 0.009), and CHOP (P = 0.001) in LPS-exposed cells (Figure 2c). Immunofluorescence analysis showed that compared with the control group, LPS stimulation increased the immunofluorescence intensity of GRP78, JNK, and CHOP, whereas inactivating TLR4 under LPS could alleviate this phenomenon (Figure 2d). These results implied that TLR4 modulated LPS-induced ER stress to promote UPR in bovine hepatocytes.

      Inhibition of TLR4 Attenuated Autophagy Induced by LPS in Bovine Hepatocytes

      As shown in Supplemental Figure S3e, inhibiting TLR4 with TAK-242 for 12 h could transcriptionally downregulate ATG5 at the doses of 1, 5, and 10 μM (P < 0.01 for all), autophagy-related gene 12 (ATG12) at all doses (P < 0.05 for all), and autophagy-related gene 4A (ATG4A) at the doses of 1, 5, and 10 μM (P < 0.05 for all), and upregulate SQSTM1 at all doses (P < 0.01 for all). The western blot analysis results showed that 10 μM TAK-242 pretreatment decreased the abundance of autophagy-related protein, including ULK1 (P = 0.001), BECLIN1 (P = 0.002), autophagy-related gene 14 (ATG14, P = 0.006), ATG16L1 (P = 0.03), ATG5 (P = 0.003), LC3-II (P = 0.001), LAMP2A (P = 0.003), synaptosomal-associated protein 29 (SNAP29, P = 0.001), vesicle-associated membrane protein 8 (VAMP8, P = 0.009), and syntaxin 17 (STX17, P = 0.032), and significantly increased SQSTM1 protein abundance (P = 0.005) in LPS-challenged hepatocytes (Figure 3a). The immunofluorescence results showed that compared with the control group, LPS treatment increased the immunofluorescence intensity of ATG16L1, LC3, and LAMP1, and decreased that of SQSTM1, whereas inhibition of TLR4 under LPS could alleviate this phenomenon (Figure 3b). Moreover, the results of Lyso-Tracker red and AO staining showed that LPS increased the formation of lysosomes and autolysosomes, respectively, and Lyso-Sensors green assay revealed that LPS caused a lower pH of acidic organelles in hepatocytes. These results suggested that LPS enhanced the function of lysosome to promote autophagy in bovine hepatocytes, and inhibiting TLR4 activity reduced these changes (Figures 3c and d). Our data showed that inhibitingTLR4 could inhibit autophagy induced by LPS in bovine hepatocytes.
      Figure thumbnail gr3a
      Figure 3Inhibition of TLR4 attenuates LPS-induced autophagy in bovine hepatocytes. The hepatocytes were treated with 10 μM TAK-242 for 12 h, and incubated with LPS (12 μg/mL) for another 12 h. (a) Western blot analysis was performed to determine the relative expression of autophagy-related proteins (left). The bar graphs (right) represent the relative intensity of autophagy-related proteins. (b) The expression of autophagy-related proteins determined by immunofluorescence staining. The immunofluorescence analysis was performed with anti-SQSTM1 antibody (green), anti-ATL16L1 (green) antibody, anti-LC3 antibody (green), anti-LAMP1 antibody (red), and 4ʹ,6-diamidino-2-phenylindole (DAPI) (blue; nuclei); bar = 10 μm. (c) The hepatocytes were stained by acridine orange (AO) and the AO fluorescence was determined by the laser confocal microscope. The arrows indicated the autolysosomes. (d) The hepatocytes were stained by Lyso-Tracker red and Lyso-Sensors green, respectively, and the fluorescence were determined by laser confocal microscope. β-actin (ACTB) serves as the loading control for normalization. The data are expressed as the mean ± SEM and analyzed by 1-way ANOVA with LSD post hoc test. The P ≤ 0.05 is considered statistically significant. These data are representative of 3 independent experiments (n = 3).
      Figure thumbnail gr3b
      Figure 3Inhibition of TLR4 attenuates LPS-induced autophagy in bovine hepatocytes. The hepatocytes were treated with 10 μM TAK-242 for 12 h, and incubated with LPS (12 μg/mL) for another 12 h. (a) Western blot analysis was performed to determine the relative expression of autophagy-related proteins (left). The bar graphs (right) represent the relative intensity of autophagy-related proteins. (b) The expression of autophagy-related proteins determined by immunofluorescence staining. The immunofluorescence analysis was performed with anti-SQSTM1 antibody (green), anti-ATL16L1 (green) antibody, anti-LC3 antibody (green), anti-LAMP1 antibody (red), and 4ʹ,6-diamidino-2-phenylindole (DAPI) (blue; nuclei); bar = 10 μm. (c) The hepatocytes were stained by acridine orange (AO) and the AO fluorescence was determined by the laser confocal microscope. The arrows indicated the autolysosomes. (d) The hepatocytes were stained by Lyso-Tracker red and Lyso-Sensors green, respectively, and the fluorescence were determined by laser confocal microscope. β-actin (ACTB) serves as the loading control for normalization. The data are expressed as the mean ± SEM and analyzed by 1-way ANOVA with LSD post hoc test. The P ≤ 0.05 is considered statistically significant. These data are representative of 3 independent experiments (n = 3).

      Inhibition of TLR4 Alleviates Apoptosis Induced by LPS in Bovine Hepatocytes

      Supplemental Figure S3f showed that LPS challenge significantly upregulated the mRNA expression of CASPASE3 (P = 0.02) and BAX (P = 0.01), and significantly downregulated that of BCL-2 (P = 0.048) in bovine hepatocytes, whereas pretreatment with TAK-242 under LPS could alleviate the effect of LPS on the mRNA expression of CASPASE3 at the doses of 1, 5, and 10 μM (P < 0.05 for all), BAX at the dose of 10 μM (P = 0.036), and BCL-2 at the doses of 0.1, 1, and 10 μM (P < 0.05 for all). Figure 4a shows that pretreatment with 10 μM TAK-242 followed by LPS stimulation could significantly decrease the protein abundance of CASPASE3 (P = 0.015), Cleaved CASPASE3 (P = 0.006), and BAX (P = 0.005), and increase that of BCL-2 (P = 0.005) compared with the LPS group. Mitochondrial membrane potential (ΔΨm) is one of the indicators of mitochondrial function. Generally, the reduced MMP is indicated that mitochondrial dysfunction and early apoptosis. In the current study, JC-1 fluorescent probe was used to determine the changes of MMP. When the physiological conditions are normal, JC-1 usually aggregates in the matrix of mitochondrial, forming a polymer emitting red fluorescence. When MMP is reduced, we detected green fluorescence, suggesting the polymer disintegrated. As shown in Figure 4b, the red fluorescence decreased and the green fluorescence increased in the LPS-stimulated group. In contrast, TAK-242-pretreatment improved the above changes, and the red fluorescence increased compared with the LPS group. Compared with the control group, the percentage of bovine hepatocytes apoptosis significantly increased in the LPS-exposed group (P = 0.001), and TAK-242 pretreatment could sharply reduce the apoptotic hepatocytes caused by LPS (P = 0.001; Figure 4c). These results suggested that TAK-242 alleviated the apoptotic level of bovine hepatocytes induced by LPS.
      Figure thumbnail gr4
      Figure 4Inhibiting TLR4 alleviates LPS-induced apoptosis in bovine hepatocytes. The hepatocytes were treated with 10 μM TAK-242 for 12 h, and incubated with LPS (12 μg/mL) for another 12 h. (a) The relative expression of apoptosis-related proteins determined by western blot analysis (left). The bar graphs (right) represent the relative intensity of apoptosis-related proteins. (b) Mitochondrial membrane potential (△Ψ) was detected by tetraethylbenzimidazolyl carbocyanine iodide (JC-1) fluorescence probe. (c) Apoptotic cell was evaluated using Annexin V and propidium iodide (PI) staining assay followed by flow cytometry analysis (left). The bar graph (right) represents the percentage of apoptotic cells. β-actin (ACTB) serves as the loading control for normalization. The data are expressed as the mean ± SEM and analyzed by 1-way ANOVA with LSD post hoc test. The P ≤ 0.05 is considered statistically significant. These data are representative of 3 independent experiments (n = 3).

      FOXA2 Mitigates LPS-triggered Inflammatory Response Via NF-κB Signaling Pathway in Bovine Hepatocytes

      FOXA2 has been recently identified as a crucial modulator of cellular stress response (
      • Bochkis I.M.
      • Rubins N.E.
      • White P.
      • Furth E.E.
      • Friedman J.R.
      • Kaestner K.H.
      Hepatocyte-specific ablation of Foxa2 alters bile acid homeostasis and results in endoplasmic reticulum stress.
      ), but no studies have addressed the effect of FOXA2 on bovine hepatocytes. In current study, FOXA2 was downregulated with active inflammatory response, ER stress, autophagy, and apoptosis induced by LPS, and inhibiting TLR4 could upregulate FOXA2 expression and mitigate the effects of LPS on inflammation, ER stress, autophagy, and apoptosis. Based on these results, we hypothesized that FOXA2 could mediate LPS-induced cellular stress response. To investigate the possible involvement of FOXA2 in LPS-induced inflammatory response, we upregulated FOXA2 expression in bovine hepatocytes using a FOXA2 overexpression plasmid, and the results showed that transfecting FOXA2 overexpression plasmid significantly upregulated the expression of FOXA2 at mRNA and protein levels in the Figures 5a and b (P = 0.001 and P = 0.003, respectively). Overexpressing FOXA2 significantly reduced the mRNA level of NF-κB P65 (P = 0.007), IκBα (P = 0.001), and IL-8 (P = 0.001) in LPS-challenged cells (Figure 5a). The western blot analysis showed that compared with the LPS group, FOXA2 overexpression significantly downregulated the protein expression of phosphorylated IκBα (P = 0.01), phosphorylated NF-κB P65 (P = 0.021), and TNFα (P = 0.018) in LPS-stimulated hepatocytes (Figure 5c). Additionally, overexpressing FOXA2 could suppress the activation of NF-κB P65 (Figure 5d) and significantly reduce the secretion of the inflammatory factors IL-1β (P = 0.004) and IL-8 (P = 0.027) induced by LPS (Figure 5e). These results demonstrated that FOXA2 overexpression suppressed the activation of NF-κB signaling pathway induced by LPS treatment, suggesting that FOXA2 could modulate inflammatory response by inhibiting NF-κB in LPS-exposed bovine hepatocytes.
      Figure thumbnail gr5
      Figure 5FOXA2 protects bovine hepatocytes against LPS-induced inflammatory damage. (a) The relative mRNA expression of FOXA2 and inflammatory genes in NF-κB signaling pathway analyzed by quantitative reverse-transcription PCR. (b) The relative expression of FOXA2 protein in bovine hepatocytes during FOXA2 overexpressing. (c) The relative expression of inflammatory proteins in NF-κB signaling pathway determined by western blot (left). The small bar graphs (right) represent the relative intensity of inflammatory proteins. (d) The P65 protein expression in the hepatocytes determined by immunofluorescence staining (green: P65; blue: nuclei; bar = 10 μm); DAPI = 4ʹ,6-diamidino-2-phenylindole. (e) The secretion of cytokines (IL-1β and IL-8) in cell supernatant measured by ELISA kit. The GAPDH and β-actin (ACTB) serve as the loading controls for normalization. The data are expressed as the mean ± SEM and analyzed by 1-way ANOVA with LSD post hoc test. The P ≤ 0.05 is considered statistically significant. These data are representative of 3 independent experiments (n = 3). pcDNA = control group; pcFOXA2 = overexpression group; pcDNA+LPS = the LPS-treated group; pcFOXA2+LPS = overexpression with LPS group.

      FOXA2 Relieves LPS-induced ER Stress to Block UPR in Bovine Hepatocytes.

      As shown in Figure 6a, compared with the pcDNA group, the mRNA expression of ATF6, ATF4, GRP78, PERK, CHOP, and JNK1 was increased in the pcDNA+LPS group (P = 0.011, P = 0.001, P = 0.022, P = 0.032, P = 0.003, and P = 0.015, respectively), and transfecting FOXA2 overexpression plasmid could suppress these changes. Consistent with mRNA results, compared with the pcDNA+LPS group, the protein intensity of GRP78 (P = 0.002), PERK (P = 0.005), phosphorylated PERK (P = 0.001), ATF6 (P = 0.001), IRE1α (P = 0.006), JNK (P = 0.005), and CHOP (P = 0.002) was downregulated in the pcFOXA2+LPS group (Figure 6b). The immunofluorescence results showed that the immunofluorescence intensity of GRP78, JNK, and CHOP was higher in the pcDNA+LPS group. In contrast, overexpression FOXA2 could reduce the immunofluorescence intensity of JNK, GRP78, and CHOP under LPS treatment (Figure 6c). These results suggested that FOXA2 could alleviate LPS-induced ER stress to inactivate UPR signaling pathway in bovine hepatocytes.
      Figure thumbnail gr6
      Figure 6FOXA2 alleviates LPS-induced endoplasmic reticulum (ER) stress in bovine hepatocytes. The hepatocytes were transfected with pcDNA3.1 or pcFOXA2 overexpression plasmid by lipofectamine 3000 (Thermo Fisher Scientific) for 36 h followed by LPS for another 12 h. (a) The relative mRNA expression of ER stress-related genes determined by quantitative reverse-transcription PCR. (b) The relative expression of ER stress-related proteins determined by western blot analysis. (c) The protein expression of GRP78, JNK, and CHOP determined by immunofluorescence staining. The immunofluorescence analysis was performed with anti-GRP78 (green) antibody, anti-JNK antibody (green), anti-CHOP antibody (green), and 4',6-diamidino-2-phenylindole (DAPI; blue; nuclei); bar = 10 μm. The GAPDH and β-actin (ACTB) serve as the loading controls for normalization. The data are expressed as the mean ± SEM and analyzed by 1-way ANOVA with LSD post hoc test. The P ≤ 0.05 is considered statistically significant. These data are representative of 3 independent experiments (n = 3). pcDNA = control group; pcFOXA2 = overexpression group; pcDNA+LPS = the LPS-treated group; pcFOXA2+LPS = overexpression with LPS group.

      FOXA2 Inhibits LPS-induced Autophagy in Bovine Hepatocytes

      We demonstrated that FOXA2 could suppress LPS-induced ER stress and UPR, but whether FOXA2 is involved in the LPS-induced autophagy in bovine hepatocytes remains unknown. Hence, we transfected bovine hepatocytes with FOXA2 overexpression vector to determine the molecular mechanisms of FOXA2 in regulating the LPS-induced autophagy. As shown in Figure 7a, compared with the pcDNA+LPS group, the mRNA expression of ATG5 (P = 0.001), BECLIN1 (P = 0.001), ULK1 (P = 0.001), and microtubule-associated protein 1 light chain 3 α (MAP1LC3A; P = 0.001) was lower, and that of SQSTM1 was higher (P = 0.018) in the pcFOXA2+LPS group. The western blot analysis showed that FOXA2 overexpression could significantly reduce the protein abundance of BECLIN1 (P = 0.001), ATG16L1 (P = 0.011), ATG5 (P = 0.001), LC3-II (P = 0.001), LAMP2A (P = 0.014), SNAP29 (P = 0.029), VAMP8 (P = 0.001), and STX17 (P = 0.001), and significantly upregulate SQSTM1 protein expression (P = 0.045) in LPS-stimulated hepatocytes (Figure 7b). Additionally, FOXA2 overexpression could upregulate the immunofluorescence intensity of SQSTM1 and downregulate that of ATG16L1, LC3, and LAMP1 in LPS-challenged cells (Figure 7c). The autophagic processes was evaluated by mRFP-eGFP-LC3 fluorescence microscopy. As shown in Figure 7d, compared with the pcDNA group, mRFP-eGFP-LC3 fluorescence was increased in the pcDNA +LPS group, whereas FOXA2 overexpression could reduce the fluorescence intensity of mRFP-eGFP-LC3 in LPS-treated hepatocytes. Additionally, compared with the pcDNA group, AO staining, Lyso-Tracker red, and Lyso-Sensor green showed the strong staining in the pcDNA+LPS group, whereas FOXA2 overexpression reduced these changes in LPS-treated cells (Figures 7e and f). These results suggested that FOXA2 could alleviate the effect of LPS on autophagy via inhibiting autophagic flux.
      Figure thumbnail gr7a
      Figure 7FOXA2 inhibits LPS-induced autophagy in bovine hepatocytes. The hepatocytes were transfected with pcDNA3.1 as control or pcFOXA2 overexpression plasmid by lipofectamine 3000 (Thermo Fisher Scientific) for 36 h followed by LPS for another 12 h. (a) The mRNA relative expression of autophagy-related genes determined by quantitative reverse-transcription PCR. (b) The protein relative expression of autophagy-related genes determined by western blot. (c) The expression of autophagy-related proteins determined by immunofluorescence staining. The immunofluorescence analysis was performed with anti-SQSTM1 antibody (green), anti-ATG16L1 (green) antibody, anti-LC3 antibody (green), anti-LAMP1 antibody (red), and 4ʹ,6-diamidino-2-phenylindole (DAPI; blue; nuclei); bar = 10 μm. (d) The hepatocytes were co-transfected with mRFP-eGFP-LC3 plasmid and pcFOXA2 plasmid. Thirty-six post-transfection, the cells were exposed to LPS for 12 h and the fluorescence was observed by laser confocal microscope. (e) The hepatocytes were stained by acridine orange (AO) and the AO fluorescence was determined by laser confocal microscope. The arrows indicated the autolysosomes. (f) The hepatocytes were stained by Lyso-Tracker red and Lyso-Sensors green, and the fluorescence were determined by laser confocal microscope. The GAPDH and β-actin (ACTB) serve as the loading controls for normalization. The data are expressed as the mean ± SEM and analyzed by 1-way ANOVA with LSD post hoc test. The P ≤ 0.05 is considered statistically significant. These data are representative of 3 independent experiments (n = 3). pcDNA = control group; pcFOXA2 = overexpression group; pcDNA+LPS = the LPS-treated group; pcFOXA2+LPS = overexpression with LPS group.
      Figure thumbnail gr7b
      Figure 7FOXA2 inhibits LPS-induced autophagy in bovine hepatocytes. The hepatocytes were transfected with pcDNA3.1 as control or pcFOXA2 overexpression plasmid by lipofectamine 3000 (Thermo Fisher Scientific) for 36 h followed by LPS for another 12 h. (a) The mRNA relative expression of autophagy-related genes determined by quantitative reverse-transcription PCR. (b) The protein relative expression of autophagy-related genes determined by western blot. (c) The expression of autophagy-related proteins determined by immunofluorescence staining. The immunofluorescence analysis was performed with anti-SQSTM1 antibody (green), anti-ATG16L1 (green) antibody, anti-LC3 antibody (green), anti-LAMP1 antibody (red), and 4ʹ,6-diamidino-2-phenylindole (DAPI; blue; nuclei); bar = 10 μm. (d) The hepatocytes were co-transfected with mRFP-eGFP-LC3 plasmid and pcFOXA2 plasmid. Thirty-six post-transfection, the cells were exposed to LPS for 12 h and the fluorescence was observed by laser confocal microscope. (e) The hepatocytes were stained by acridine orange (AO) and the AO fluorescence was determined by laser confocal microscope. The arrows indicated the autolysosomes. (f) The hepatocytes were stained by Lyso-Tracker red and Lyso-Sensors green, and the fluorescence were determined by laser confocal microscope. The GAPDH and β-actin (ACTB) serve as the loading controls for normalization. The data are expressed as the mean ± SEM and analyzed by 1-way ANOVA with LSD post hoc test. The P ≤ 0.05 is considered statistically significant. These data are representative of 3 independent experiments (n = 3). pcDNA = control group; pcFOXA2 = overexpression group; pcDNA+LPS = the LPS-treated group; pcFOXA2+LPS = overexpression with LPS group.

      FOXA2 Protects Bovine Hepatocytes from LPS-induced Apoptosis

      Endoplasmic reticulum stress and autophagy are connected with apoptosis, and we confirmed that FOXA2 plays a crucial role in modulating LPS-triggered ER stress and autophagy, we hypothesized that FOXA2 could regulate LPS-induced apoptosis. As expected, overexpressing FOXA2 significantly downregulated the mRNA expression of CASPASE3 (P = 0.001) and BAX (P = 0.003), and significantly upregulated that of BCL-2 (P = 0.001) in LPS-exposed hepatocytes (Figure 8a). Consistently, compared with the pcDNA+LPS group, the protein intensity of BAX (P = 0.001), CASPASE3 (P = 0.001), and Cleaved CASPASE3 (P = 0.015) was downregulated and that of BCL-2 was upregulated (P = 0.033) in the pcFOXA2+LPS group (Figure 8b). As shown in Figure 8c, FOXA2 overexpression increased the JC-1 red fluorescence and decreased the JC-1 green fluorescence in the LPS-stimulated cells, which indicates that FOXA2 could suppress LPS-induced apoptosis by restoring mitochondrial function. The percentage of apoptotic cells was significantly lower in the pcDNA+LPS group than the pcFOXA2+LPS group (P = 0.001; Figure 8d). These results demonstrated that FOXA2 could repress the LPS-induced apoptosis in bovine hepatocytes.
      Figure thumbnail gr8
      Figure 8FOXA2 alleviates LPS-induced apoptosis in bovine hepatocytes. The hepatocytes were transfected with pcDNA3.1 as control and pcFOXA2 overexpression plasmid by lipofectamine 3000 (Thermo Fisher Scientific) for 36 h followed by LPS for another 12 h. (a) The relative mRNA expression of apoptosis-related genes determined by quantitative reverse-transcription PCR. (b) Mitochondrial membrane potential (△Ψ) was detected by tetraethylbenzimidazolyl carbocyanine iodide (JC-1) fluorescence probe. (c) The relative expression of apoptosis-related proteins determined by western blot analysis (left). The bar graphs (right) represent the relative intensity of apoptosis-related proteins. (d) Apoptotic cell was evaluated using Annexin V and propidium iodide (PI) staining assay followed by flow cytometry analysis (left). The bar graph (right) represents the percentage of apoptotic cells. The GAPDH and β-actin (ACTB) serve as loading controls for normalization. The data are expressed as the mean ± SEM and analyzed by 1-way ANOVA with LSD post hoc test. The P ≤ 0.05 is considered statistically significant. These data are representative of 3 independent experiments (n = 3). pcDNA = control group; pcFOXA2 = overexpression group; pcDNA+LPS = the LPS-treated group; pcFOXA2+LPS = overexpression with LPS group.

      DISCUSSION

      Immune and inflammatory systems can be activated by LPS. The binding of LPS and TLR4 complex signals via the adaptor protein MYD88 recruits IL-1 receptor-associated kinase-1 (IRAK-1), IRAK-4, and TRAF-6, which subsequently links with TAK1 and activates its activity (
      • Yamamoto M.
      • Takeda K.
      • Akira S.
      TIR domain-containing adaptors define the specificity of TLR signaling.
      ). Activated TAK1 phosphorylation activates the IκB kinase (IKK) complex and causes the phosphorylation of IκBα. Phosphorylated IκBα dissociates from the NF-κB complex, initiating NF-κB activity and inducing nuclear translocation of phosphorylated NF-κB P65 to trigger inflammatory cascades (
      • Kanayama A.
      • Seth R.B.
      • Sun L.
      • Ea C.K.
      • Hong M.
      • Shaito A.
      • Chiu Y.H.
      • Deng L.
      • Chen Z.J.
      TAB2 and TAB3 activate the NF-kB pathway through binding to polyubiquitin chains.
      ). In our study, LPS treatment increased the expression of MYD88, TRAF6, and TAK1, promoted the activation of IκBα and NF-κB P65, and elevated the expression of the proinflammatory factors (TNFα, IL-1β, IL-6, IL-8, and SAA3), which indicated that inflammatory cascades were activated by LPS via NF-κB signaling pathway in bovine hepatocytes. A specific inhibitor of TLR4 was applied to investigate the outcome of TLR4 inhibition in inflammation and cell damage. The results showed that inhibiting TLR4 reduced the LPS-induced inflammatory cascades by suppressing the expression of inflammatory genes in NF-κB signaling pathway in bovine hepatocytes, which were consistent with the publication demonstrating that TAK-242, the specific inhibitor of TLR4, can completely prevent the LPS-induced activation of NF-κB and the increase of proinflammatory cytokines production (
      • Hussey S.E.
      • Liang H.
      • Costford S.R.
      • Klip A.
      • DeFronzo R.A.
      • Sanchez-Avila A.
      • Ely B.
      • Musi N.
      TAK-242, a small-molecule inhibitor of Toll-like receptor 4 signalling, unveils similarities and differences in lipopolysaccharide- and lipid-induced inflammation and insulin resistance in muscle cells.
      ).
      • Walters K.A.
      • Olsufka R.
      • Kuestner R.E.
      • Wu X.
      • Wang K.
      • Skerrett S.J.
      • Ozinsky A.
      Prior infection with Type A Francisella tularensis antagonizes the pulmonary transcriptional response to an aerosolized Toll-like receptor 4 agonist.
      demonstrated that TLR4 mediates LPS-triggered inflammation with reducing FOXA2 expression in Francisella tularensis infection. Treating mice with LPS contributes to the downregulation of FOXA2 protein expression in the liver (
      • Zollner G.
      • Wagner M.
      • Fickert P.
      • Geier A.
      • Fuchsbichler A.
      • Silbert D.
      • Gumhold J.
      • Zatloukal K.
      • Kaser A.
      • Tilg H.
      • Denk H.
      • Trauner M.
      Role of nuclear receptors and hepatocyte-enriched transcription factors for Ntcp repression in biliary obstruction in mouse liver.
      ) and FOXA2 silencing abolishes the inhibition of monocyte chemotactic protein-1 (MCP-1) expression by propofol in LPS-administered HepG2 cells (
      • Ma X.
      • Zhao J.Y.
      • Zhao Z.L.
      • Ye J.
      • Li S.F.
      • Fang H.H.
      • Gu M.N.
      • Hu Y.W.
      • Qin Z.S.
      Propofol attenuates lipopolysaccharide-induced monocyte chemoattractant protein-1 production through enhancing apoM and foxa2 expression in HepG2 cells.
      ). Studies show that FOXA2 regulates the promoter activity of β-defensin1 to control its expression in Th2-dominated airway inflammation (
      • Tang X.
      • Liu X.J.
      • Tian C.
      • Su Q.
      • Lei Y.
      • Wu Q.
      • He Y.
      • Whitsett J.A.
      • Luo F.
      Foxa2 regulates leukotrienes to inhibit Th2-mediated pulmonary inflammation.
      ;
      • Wei C.
      • Tang X.
      • Wang F.
      • Li Y.
      • Sun L.
      • Luo F.
      Molecular characterization of pulmonary defenses against bacterial invasion in allergic asthma: The role of Foxa2 in regulation of β-defensin 1.
      ). Consistently, we found that LPS could inactivate FOXA2 in TLR4-mediated inflammatory response in bovine hepatocytes and FOXA2 might play a protective role against LPS-induced inflammation. To further explore the anti-inflammation role of FOXA2, we elevated FOXA2 expression with an overexpression plasmid in the LPS-treated hepatocytes, and found that FOXA2 overexpression attenuated the effects of LPS on the bovine hepatocytes by suppressing the activation of NF-κB P65 and reducing the expression and secretion of proinflammatory factors. Deletion of FOXA2 causes the accumulation of bile acid and increases the expression of inflammatory genes signal transducer and activator of transcription 3 (STAT3), interferon regulatory factor 3 (IRF3), and NF-κB in the liver of mice (
      • Bochkis I.M.
      • Shin S.
      • Kaestner K.H.
      Bile acid-induced inflammatory signaling in mice lacking Foxa2 in the liver leads to activation of mTOR and age-onset obesity.
      ). Our previous studies demonstrate that FOXA2 is downregulated with the activation of NF-κB signaling pathway and the increase of proinflammatory factors in vivo and in vitro (
      • Chandra Roy A.
      • Wang Y.
      • Zhang H.
      • Roy S.
      • Dai H.
      • Chang G.
      • Shen X.
      Sodium butyrate mitigates iE-DAP induced inflammation caused by high-concentrate feeding in liver of dairy goats.
      ;
      • Roy A.C.
      • Chang G.
      • Ma N.
      • Wang Y.
      • Roy S.
      • Liu J.
      • Aabdin Z.U.
      • Shen X.
      Sodium butyrate suppresses NOD1-mediated inflammatory molecules expressed in bovine hepatocytes during iE-DAP and LPS treatment.
      ). Therefore, FOXA2 mediated the inflammatory response by suppressing NF-κB activation in bovine hepatocytes.
      Under physiological conditions, GRP78 binds to and inhibits the 3 sensors (PERK, IRE1α, and ATF6). When ER stress occurs, the accumulated unfolded proteins bind to and recruit GRP78 away from PERK, IRE1α, and ATF6, leading to initiating the downstream cascades, including autophagy and apoptosis (
      • Song S.
      • Tan J.
      • Miao Y.
      • Li M.
      • Zhang Q.
      Crosstalk of autophagy and apoptosis: Involvement of the dual role of autophagy under ER stress.
      ). Evidence has shown that ER stress is blunted by TLR4 deletion, suggesting TLR4 is essential for the inhibition of ER stress pathway (
      • de Vicente L.G.
      • Pinto A.P.
      • Muñoz V.R.
      • Rovina R.L.
      • da Rocha A.L.
      • Gaspar R.C.
      • da Silva L.
      • Simabuco F.M.
      • Frantz F.G.
      • Pauli J.R.
      • de Moura L.P.
      • Cintra D.E.
      • Ropelle E.R.
      • da Silva A.S.R.
      Tlr4 participates in the responses of markers of apoptosis, inflammation, and ER stress to different acute exercise intensities in mice hearts.
      ,
      • de Vicente L.G.
      • Pinto A.P.
      • Muñoz V.R.
      • Rovina R.L.
      • da Rocha A.L.
      • Gaspar R.C.
      • da Silva L.E.C.M.
      • Simabuco F.M.
      • Frantz F.G.
      • Pauli J.R.
      • de Moura L.P.
      • Cintra D.E.
      • Ropelle E.R.
      • da Silva A.S.R.
      Tlr4 participates in the responses of markers of apoptosis, inflammation, and ER stress to different acute exercise intensities in mice hearts.
      ) and TLR4 knockout abolishes a high-fat diet-induced ER stress via reducing the expression of GRP78, XBP1, and CHOP in mice (
      • Pierre N.
      • Deldicque L.
      • Barbé C.
      • Naslain D.
      • Cani P.D.
      • Francaux M.
      Toll-like receptor 4 knockout mice are protected against endoplasmic reticulum stress induced by a high-fat diet.
      ). Consistently, we demonstrated that LPS increased the expression of GRP78, PERK, IRE1α, ATF6, and CHOP, indicating the activation of ER stress, and TLR4 inhibition mitigated the LPS-induced ER activation. It has been demonstrated that the TLR4/TRAF6 pathway is involved in regulating ER stress in ventilation-induced lung injury, employment of TLR4 and TRAF6 inhibitor ameliorates NF-κB-mediated inflammation and prevents ER stress by reducing GRP78 and CHOP expression in murine model (
      • Zeng Q.
      • Ye L.
      • Ling M.
      • Ma R.
      • Li J.
      • Chen H.
      • Pan L.
      TLR4/TRAF6/NOX2 signaling pathway is involved in ventilation-induced lung injury via endoplasmic reticulum stress in murine model.
      ). Publications have declared that TLR4 promotes the activation of IRE1α through TRAF6-IRE1α interaction and activates IRE1α-XBP1 signaling pathway to regulate proinflammatory cytokine IL-6 production (
      • Martinon F.
      • Chen X.
      • Lee A.H.
      • Glimcher L.H.
      TLR activation of the transcription factor XBP1 regulates innate immune responses in macrophages.
      ;
      • Qiu Q.
      • Zheng Z.
      • Chang L.
      • Zhao Y.S.
      • Tan C.
      • Dandekar A.
      • Zhang Z.
      • Lin Z.
      • Gui M.
      • Li X.
      • Zhang T.
      • Kong Q.
      • Li H.
      • Chen S.
      • Chen A.
      • Kaufman R.J.
      • Yang W.L.
      • Lin H.K.
      • Zhang D.
      • Perlman H.
      • Thorp E.
      • Zhang K.
      • Fang D.
      Toll-like receptor-mediated IRE1α activation as a therapeutic target for inflammatory arthritis.
      ). Additionally, during ER stress, IRE1α forms a complex with TRAF2 and apoptosis signal-regulating kinase 1 (ASK1) to activate JNK and the activation of JNK promotes BECLIN1 releasing from BECLIN1/BCL-2 complex to trigger autophagy (
      • Pattingre S.
      • Tassa A.
      • Qu X.
      • Garuti R.
      • Liang X.H.
      • Mizushima N.
      • Packer M.
      • Schneider M.D.
      • Levine B.
      Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy.
      ;
      • Ron D.
      • Hubbard S.R.
      How IRE1 reacts to ER stress.
      ). Our data verified that LPS-induced ER stress through TLR4-TRAF6 axis and TLR4 inhibitor attenuated these changes in bovine hepatocytes. As a consequence of FOXA2 deletion, the hepatic bile acid homeostasis disruption and ER stress activation are caused by a diet containing cholic acid in mice (
      • Bochkis I.M.
      • Rubins N.E.
      • White P.
      • Furth E.E.
      • Friedman J.R.
      • Kaestner K.H.
      Hepatocyte-specific ablation of Foxa2 alters bile acid homeostasis and results in endoplasmic reticulum stress.
      ). Hepatocytes-specific knockout of FOXA2 enhances ER stress, whereas FOXA2 overexpression can suppress ER stress in mouse fibrotic liver, hence hepatic FOXA2 has an inhibitory effect on ER stress activation (
      • Wang W.
      • Yao L.J.
      • Shen W.
      • Ding K.
      • Shi P.M.
      • Chen F.
      • He J.
      • Ding J.
      • Zhang X.
      • Xie W.F.
      FOXA2 alleviates CCl4-induced liver fibrosis by protecting hepatocytes in mice.
      ). Additionally, tunicamycin-induced ER stress causes FOXA2 downregulation to facilitate cancer stem cell self-renewal via HOXB9-miR-765 axis in human melanoma (
      • Lin J.
      • Zhang D.
      • Fan Y.
      • Chao Y.
      • Chang J.
      • Li N.
      • Han L.
      • Han C.
      Regulation of cancer stem cell self-renewal by HOXB9 Antagonizes endoplasmic reticulum stress-induced melanoma cell apoptosis via the miR-765-FOXA2 axis.
      ). Our study here revealed that FOXA2 overexpression could block the increase of GRP78, PERK, IRE1α, ATF6, ATF4, and CHOP induced by LPS to alleviate ER stress; hence, hepatic FOXA2 played an inhibitory role in LPS-induced ER stress in bovine hepatocytes.
      Autophagy is a cellular degradation process initiated in response to stress, which attempts to restore metabolic homeostasis through the catabolic lysis of aggregated proteins, unfolded/misfolded proteins or damaged subcellular organelles (
      • Mizushima N.
      Autophagy: Process and function.
      ). Autophagy initiation is regulated by ULK1/2 complex consisting of ULK1, autophagy-related gene 13 (ATG13), autophagy-related gene 101 (ATG101), and family interacting protein 200kD (FIP200), which is inhibited by mTOR (
      • Mercer C.A.
      • Kaliappan A.
      • Dennis P.B.
      A novel, human Atg13 binding protein, Atg101, interacts with ULK1 and is essential for macroautophagy.
      ;
      • Mizushima N.
      The role of the Atg1/ULK1 complex in autophagy regulation.
      ). Evidence implicates the regulation of mTOR in response to UPR and its role in ER stress-induced autophagy. Tunicamycin-induced ER stress inhibits the activity of mTOR via ATF4 and CHOP in Hela cells (
      • Jin H.O.
      • Seo S.K.
      • Woo S.H.
      • Kim E.S.
      • Lee H.C.
      • Yoo D.H.
      • An S.
      • Choe T.B.
      • Lee S.J.
      • Hong S.I.
      • Rhee C.H.
      • Kim J.I.
      • Park I.C.
      Activating transcription factor 4 and CCAAT/enhancer-binding protein-beta negatively regulate the mammalian target of rapamycin via Redd1 expression in response to oxidative and endoplasmic reticulum stress.
      ) and ER stress contributes to the inactivation of mTOR in an AMP-activated protein kinase (AMPK)-independent fashion to enhance autophagy (
      • Qin L.
      • Wang Z.
      • Tao L.
      • Wang Y.
      ER stress negatively regulates AKT/TSC/mTOR pathway to enhance autophagy.
      ). Research on hepatitis C virus shows that ER stress activation inhibits mTOR and activates ULK1 to promote autophagy induction in Huh7 cells (
      • Huang H.
      • Kang R.
      • Wang J.
      • Luo G.
      • Yang W.
      • Zhao Z.
      Hepatitis C virus inhibits AKT-tuberous sclerosis complex (TSC), the mechanistic target of rapamycin (MTOR) pathway, through endoplasmic reticulum stress to induce autophagy.
      ). Herein, LPS administration activated ER stress to enhance autophagy by downregulating mTOR expression and upregulating ULK1 expression. In contrast, inhibition of TLR4 could reverse the changes.
      Evidence has demonstrated that TLR4-mediated TRAF6 is responsible for JNK phosphorylation and the activation of JNK promotes BECLIN1 releasing from BECLIN1/BCL-2 complex to trigger autophagy (
      • Pattingre S.
      • Tassa A.
      • Qu X.
      • Garuti R.
      • Liang X.H.
      • Mizushima N.
      • Packer M.
      • Schneider M.D.
      • Levine B.
      Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy.
      ;
      • Wu L.
      • Wang C.
      • Li J.
      • Li S.
      • Feng J.
      • Liu T.
      • Xu S.
      • Wang W.
      • Lu X.
      • Chen K.
      • Xia Y.
      • Fan X.
      • Guo C.
      Hepatoprotective effect of quercetin via TRAF6/JNK pathway in acute hepatitis.
      ). Another publication shows that TLR4 can promote the K63-linked ubiquitination of BECLIN1, a key component of a class III phosphatidylinositol 3-kinase complex (PI3KC3) that initiates autophagosome formation, through TRAF6 to induce autophagy (
      • Shi C.S.
      • Kehrl J.H.
      Traf6 and A20 differentially regulate TLR4-induced autophagy by affecting the ubiquitination of Beclin 1.
      ,
      • Shi C.-S.
      • Kehrl J.H.
      TRAF6 and A20 regulate lysine 63-linked ubiquitination of Beclin-1 to control TLR4-induced autophagy.
      ), and the active ULK1/2 complex facilitates the phagophore formation, in which PtdIns3K, BECLIN1, phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3), phosphoinositide 3-kinase regulatory subunit 4 (PIK3R4), autophagy-related gene 14 (ATG14), and UVRAG involve (
      • Martelli A.M.
      • Chiarini F.
      • Evangelisti C.
      • Cappellini A.
      • Buontempo F.
      • Bressanin D.
      • Fini M.
      • McCubrey J.A.
      Two hits are better than one: Targeting both phosphatidylinositol 3-kinase and mammalian target of rapamycin as a therapeutic strategy for acute leukemia treatment.
      ). Currently, we found that LPS administration increased the expression of BECLIN1 and ATG14 to phagophore formation and the inhibition of TLR4 activity suppressed these changes under LPS challenge. Hence, TLR4 might mediate autophagy via TRAF6-BECLIN1 axis and ER stress-regulated ULK1 in LPS-challenged bovine hepatocytes.
      At phagophore expansion stage, ATG12-ATG5-ATG16L1 complex takes part in phagophore elongation and stimulates the recruitment and conversion of proteolytically processed cytosolic MAP1LC3/LC3, LC3-I, to the membrane-bound, lipidated form, LC3-II, which serves as the marker protein for the autophagy activation (
      • Hanada T.
      • Noda N.N.
      • Satomi Y.
      • Ichimura Y.
      • Fujioka Y.
      • Takao T.
      • Inagaki F.
      • Ohsumi Y.
      The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy.
      ;
      • Matsushita M.
      • Suzuki N.N.
      • Obara K.
      • Fujioka Y.
      • Ohsumi Y.
      • Inagaki F.
      Structure of Atg5∙Atg16, a complex essential for autophagy.
      ) and SQSTM1, a cargo receptor, recruits cargo destined for autophagic degradation to LC3-II on forming autophagosomes (
      • Lamark T.
      • Kirkin V.
      • Dikic I.
      • Johansen T.
      NBR1 and p62 as cargo receptors for selective autophagy of ubiquitinated targets.
      ). Currently, LPS treatment enhanced phagophore elongation and autophagosome formation by increasing the expression of ATG12, ATG5, ATG16L1, and LC3-II and diminishing that of SQSTM1, and TLR4 inhibitor could suppress the effects of LPS on phagophore elongation and autophagosome formation. The fusion of autophagosomes with lysosomes is a critical step during the autophagic pathway, and this step is mediated among other factors—by autophagosomal and lysosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins VAMP8, STX17, and SNAP29 (
      • Itakura E.
      • Kishi-Itakura C.
      • Mizushima N.
      The hairpin-type tail-anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes.
      ). Also, lysosomal membrane proteins LAMP1/2 accelerate the autophagosome-lysosome fusion (
      • Eskelinen E.L.
      Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy.
      ). Herein, TLR4 controlled the LPS-induced autophagic flux via accelerating SNARE-mediated autophagosome-lysosome fusion. Previous studies have revealed that FOXA2 overexpression abolishes the autophagy inhibition by BafA1 to maintain the self-renewal ability of ovarian cancer stem cells (
      • 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.
      ) and FOXA2 expression is upregulated by autophagy inducer rapamycin and is downregulated during autophagy inhibition in bovine hepatocytes (
      • Roy A.C.
      • Chang G.
      • Roy S.
      • Ma N.
      • Gao Q.
      • Shen X.
      γ-d-Glutamyl-meso-diaminopimelic acid induces autophagy in bovine hepatocytes during nucleotide-binding oligomerization domain 1-mediated inflammation.
      ). However, we found that overexpression of FOXA2 suppressed autophagy through reducing the expression of autophagy-related genes, which was confirmed by reduced mRFP-eGFP-LC3 expression. Evidence shows that ER stress augments the transcription of genes involved in the formation, elongation and function of the autophagosome, including BECLIN1, MAP1LC3B, multiple autophagy-related genes (ATGs), and SQSTM1 via ATF4 and CHOP induced by ER stress sensors PERK, IRE1α, and ATF6 (
      • Jin H.O.
      • Seo S.K.
      • Woo S.H.
      • Kim E.S.
      • Lee H.C.
      • Yoo D.H.
      • An S.
      • Choe T.B.
      • Lee S.J.
      • Hong S.I.
      • Rhee C.H.
      • Kim J.I.
      • Park I.C.
      Activating transcription factor 4 and CCAAT/enhancer-binding protein-beta negatively regulate the mammalian target of rapamycin via Redd1 expression in response to oxidative and endoplasmic reticulum stress.
      ;
      • B'chir W.
      • Maurin A.-C.
      • Carraro V.
      • Averous J.
      • Jousse C.
      • Muranishi Y.
      • Parry L.
      • Stepien G.
      • Fafournoux P.
      • Bruhat A.
      The eIF2α/ATF4 pathway is essential for stress-induced autophagy gene expression.
      ;
      • Rashid H.O.
      • Yadav R.K.
      • Kim H.R.
      • Chae H.J.
      ER stress: Autophagy induction, inhibition and selection.
      ). Currently, we found that FOXA2 could inhibit LPS-induced ER stress. Hence, FOXA2 might prevent LPS-induced autophagy by inhibiting ER stress in bovine hepatocytes.
      Growing evidence has emphasized the molecular function of TLR4 in mediating apoptosis. Deletion of TLR4 abolishes the pro-apoptosis effect of LPS on mice pulmonary endothelial by downregulating the expression of BAX and Cleaved CASPASE3, and upregulating BCL-2 (
      • Wu Y.
      • Wang Y.
      • Gong S.
      • Tang J.
      • Zhang J.
      • Li F.
      • Yu B.
      • Zhang Y.
      • Kou J.
      Ruscogenin alleviates LPS-induced pulmonary endothelial cell apoptosis by suppressing TLR4 signaling.
      ). In TLR4-knockout mice, TLR4 deficiency causes the reduction in the CASPASE3 activity, the number of TdT-mediated dUTP nick-end labeling-positive cells and the level of BAX, whereas elevates the expression of BCL-2 under LPS administration in liver or in hepatic macrophages (
      • Chen S.N.
      • Tan Y.
      • Xiao X.C.
      • Li Q.
      • Wu Q.
      • Peng Y.Y.
      • Ren J.
      • Dong M.L.
      Deletion of TLR4 attenuates lipopolysaccharide-induced acute liver injury by inhibiting inflammation and apoptosis.
      ). Also, TLR4 activation can induce loss of mitochondrial transmembrane potential, cytochrome c release, and caspase activation to activate the apoptotic pathway in a TRAF6-mediated JNK fashion in human microvascular endothelial cells-1 (
      • Hull C.
      • McLean G.
      • Wong F.
      • Duriez P.J.
      • Karsan A.
      Lipopolysaccharide signals an endothelial apoptosis pathway through TNF receptor-associated factor 6-mediated activation of c-Jun NH2-terminal kinase.
      ). Additionally, TRAF6 is responsible for JNK phosphorylation and the JNK activation induces BCL-2 dissociating from BECLIN1/BCL-2 complex to trigger apoptosis (
      • Pattingre S.
      • Tassa A.
      • Qu X.
      • Garuti R.
      • Liang X.H.
      • Mizushima N.
      • Packer M.
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      Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy.
      ;
      • Wu L.
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      Hepatoprotective effect of quercetin via TRAF6/JNK pathway in acute hepatitis.
      ).
      • Wei Y.
      • Sinha S.
      • Levine B.
      Dual role of JNK1-mediated phosphorylation of Bcl-2 in autophagy and apoptosis regulation.
      demonstrate that nutrient starvation promotes BCL-2 free from BECLIN1 and BAX interaction, and increases the activity of CASPASE3 to apoptosis initiation via the JNK/BCL-2 signaling pathway. In our study, LPS treatment enhanced the percentage of apoptotic cells and the expression of BAX, Cleaved CASPASE3, and CASPASE3, and reduced the level of BCL-2. In contrast, TLR4 inhibitor pretreatment could alleviate these changes in bovine hepatocytes. It is known that the involvement of mitochondria is mediated by BAX and Bak, which act by altering the MMP (ΔΨm) to facilitate the release of apoptotic proteins such as cytochrome c, which activates the cascade of Caspase 9 and Caspase 3 during apoptosis (
      • Hanawa N.
      • Shinohara M.
      • Saberi B.
      • Gaarde W.A.
      • Han D.
      • Kaplowitz N.
      Role of JNK translocation to mitochondria leading to inhibition of mitochondria bioenergetics in acetaminophen-induced liver injury.
      ). In our study, JC-1 fluorescent probe was used to detect the MMP, and the results showed that LPS reduced the MMP as indicted by the increase of JC-1 green fluorescence and the decrease of JC-1 red fluorescence, whereas TAK-242 pretreatment inhibited the LPS-induced reduction of MMP. Also, during ER stress, CHOP servers as the vital pro-apoptotic mediator to initiate apoptotic cascades which are regulated by PERK, IRE1α, and ATF6 (
      • Gorman A.M.
      • Healy S.J.
      • Jäger R.
      • Samali A.
      Stress management at the ER: Regulators of ER stress-induced apoptosis.
      ). Once there is an increase in the ER stress-induced CHOP expression, it may induce BAX translocation from cytosol to mitochondria (
      • Gotoh T.
      • Terada K.
      • Oyadomari S.
      • Mori M.
      hsp70-DnaJ chaperone pair prevents nitric oxide- and CHOP-induced apoptosis by inhibiting translocation of Bax to mitochondria.
      ). Hence, TLR4 might intervene the LPS-induced mitochondrial dysfunction for apoptosis initiation via TRAF6-JNK axis and UPR signaling pathway in bovine hepatocytes. Hepatocyte apoptosis induced by glycochenodeoxycholate or LPS causes a downregulation of FOXA2 expression. Overexpressing FOXA2 inhibits apoptosis in LPS-treated HepG2 cells, whereas silencing FOXA2 by siRNA increases HepG2 apoptosis. Further, FOXA2 can enhance anti-apoptotic gene cIAP1 expression by binding to its promoter to alleviate hepatocyte apoptosis in HepG2 cells (
      • Wang K.
      • Brems J.J.
      • Gamelli R.L.
      • Holterman A.-X.
      Foxa2 may modulate hepatic apoptosis through the cIAP1 pathway.
      ). Currently, we found that LPS increased the apoptosis level via upregulating pro-apoptotic proteins (BAX, CASPASE3, and Cleaved CASPASE3), downregulating anti-apoptotic protein BCL-2 and reducing MMP indicated by JC-1 probe, whereas FOXA2 overexpression could reverse these changes, indicating that FOXA2 has an anti-apoptotic effect on LPS-treated bovine hepatocytes. Another study shows that hepatocyte-specific knockout of FOXA2 enhances ER stress and apoptosis, which can be mitigated by FOXA2 overexpression in mouse liver (
      • Wang W.
      • Yao L.J.
      • Shen W.
      • Ding K.
      • Shi P.M.
      • Chen F.
      • He J.
      • Ding J.
      • Zhang X.
      • Xie W.F.
      FOXA2 alleviates CCl4-induced liver fibrosis by protecting hepatocytes in mice.
      ). Considering apoptosis can be initiated by ER stress-mediated CHOP, and we demonstrated that FOXA2 overexpression alleviated the effects of LPS on ER stress in bovine hepatocytes, hence, FOXA2 might mitigate the pro-apoptotic effect of LPS on bovine hepatocytes through suppressing ER stress.

      CONCLUSIONS

      In conclusion, LPS administration activates inflammatory response via NF-κB signaling pathway, induces ER stress to promote UPR, increases the level of autophagy and apoptosis, and diminishes FOXA2 expression in a TLR4-dependent manner (Figure 9). Overexpressing FOXA2 can mitigate LPS-induced inflammation, ER stress, autophagy, and apoptosis to maintain cell homeostasis in bovine hepatocytes which indicates that FOXA2 could serve as a new potential target to restore liver function under LPS challenge. We will focus on the interaction of FOXA2 with genes or proteins in inflammation, ER stress, autophagy, and apoptosis to further clarify the regulatory role of FOXA2 in liver homeostasis.
      Figure thumbnail gr9
      Figure 9Schematic diagram of the potential mechanism of FOXA2-mediated inflammation and cellular homeostasis in LPS-treated bovine hepatocytes. Lipopolysaccharide is recognized by TLR4 and triggers inflammatory response via activating NF-κB proinflammatory pathway, induces endoplasmic reticulum (ER) stress to promote unfolded protein response, and causes autophagy and apoptosis in bovine hepatocytes. FOXA2 is negatively regulated in TLR4-modulated inflammation and cell damage. FOXA2 attenuates LPS-induced inflammatory response via activating NF-κB pathway and protects bovine hepatocytes against ER stress, autophagy, and apoptosis.

      ACKNOWLEDGMENTS

      This work was supported by National Natural Science Foundation of China (No. 31872528 and No. 32172933), the Key R&D Program of Ningxia Hui Autonomous Region of China (21BEF02019), and Natural Science Foundation of Ningxia Hui Autonomous Region (2022AAC02072). The authors have not stated any conflicts of interest.

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