Subacute ruminal acidosis induces pyroptosis via the mitophagy-mediated NLRP3 inflammasome activation in the livers of dairy cows fed a high-grain diet.

High-grain (HG) feeding can trigger subacute ruminal acidosis (SARA) and subsequent liver tissue injury. This study investigated pyroptosis and NLRP3 inflammasome activation in SARA-induced liver injury, and the role of mitophagy during this process. Twelve mid-lactating Holstein cows equipped with rumen fistulas were randomly divided into 2 groups: a low-grain (LG) diet group (grain:forage = 4:6) and a HG diet group (grain:forage = 6:4). Each group had 6 cows. The experiment lasted for 3 wk. The ruminal fluid was collected through the rumen fistula on experimental d 20 and 21, and the pH immediately measured. At the end of the experiment, all animals were slaughtered, and peripheral blood and liver tissue were collected. The ruminal pH was lower in the HG group than that in the LG group at all time points. In addition, the ruminal pH in the HG group was lower than 5.6 at 3 consecutive time points after feeding (4, 6, and 8 h on d 20; 2, 4, and 6 h on d 21), indicating that HG feeding induced SARA. The content of lipopolysaccharide, IL-1ß, and apoptosis-related cysteine protease 1 (caspase-1) and the activity of alanine aminotransferase and aspartate aminotransferase in the blood plasma of the HG group were all significantly increased. Hepatic caspase-1 activity was increased in the livers of the HG group. The increased expression levels of pyroptosis- and NLRP3 inflammasome-related genes IL1B, IL18, gasdermin D (GSDMD), apoptosis-associated speck-like protein containing a card (ASC), NLR family pyrin domain-containing 3 (NLRP3), and caspase-1 (CASP1) in liver tissue of the HG group were detected. Furthermore, western blot analysis showed that HG feeding led to increased expression of pyroptosis- and NLRP3 inflammasome-related proteins GSDMD N-terminal (GSDMD-NT), IL-1ß, IL-18, cleaved-caspase-1, ASC, NLRP3, and cleaved-caspase-11 and upregulated expression of mitophagy-related proteins microtubule-associated protein 1 light chain 3 II (MAP1LC3-II), beclin 1 (BECN1), Parkin, and PTEN-induced kinase 1 (PINK1) in liver tissue. Collectively, our results revealed that SARA caused increased mitophagy and activated the NLRP3 inflammasome, causing pyroptosis and subsequent liver injury in dairy cows fed a HG diet.

A high-concentrate diet induces mitochondrial dysfunction by activating the MAPK signaling pathway in the mammary gland of dairy cows.

Subacute rumen acidosis can lead to mastitis in dairy cows. Mitochondrial dysfunction is closely related to the inflammatory response. This experiment was conducted to investigate the effects of a high-concentrate diet on mammary gland inflammation and mitochondrial damage in dairy cows. Twelve Holstein dairy cows in mid-lactation were randomly divided into 2 groups and fed a 40% concentrate (low concentrate, LC) diet or a 60% concentrate (high concentrate, HC) diet. Cows were fed individually, and the experiment lasted for 3 wk. After the experiment, mammary gland tissue, blood, and rumen fluid were collected. Compared with the LC diet, the HC diet significantly decreased rumen pH; the pH was <5.6 for more than 3 h. The HC diet also increased the concentration of LPS in the blood (7.17 ± 1.25 µg/mL vs. 12.12 ± 1.26 µg/mL), which indicated that feeding the HC diet successfully induced subacute rumen acidosis. The HC diet also increased the concentration of Ca2+ (34.80 ± 4.23 µg/g vs. 46.87 ± 7.24 µg/g) in the mammary gland and upregulated the expression of inflammatory factors IL-6 (1,128.31 ± 147.53 pg/g vs. 1,538.42 ± 241.38 pg/g), IL-1ß (69.67 ± 5.86 pg/g vs. 90.13 ± 4.78 pg/g), and tumor necrosis factor-α (91.99 ± 10.43 pg/g vs. 131.75 ± 17.89 pg/g) in mammary venous blood. The HC diet also increased the activity of myeloperoxidase (0.41 ± 0.05 U/g vs. 0.71 ± 0.11 U/g) and decreased the content of ATP (0.47 ± 0.10 µg/mL vs. 0.32 ± 0.11 µg/mL) in the mammary gland. In addition, phosphorylation of JNK (1.00 ± 0.21 vs. 2.84 ± 0.75), ERK (1.00 ± 0.20 vs. 1.53 ± 0.31), and p38 (1.00 ± 0.13 vs. 1.47 ± 0.41) and protein expression of IL-6 (1.00 ± 0.22 vs. 2.21 ± 0.27) and IL-8 (1.00 ± 0.17 vs. 1.96 ± 0.26) were enhanced in cows of the HC group, indicating that the mitogen-activated protein kinase (MAPK) signaling pathway was activated. Compared with the LC diet, the HC diet reduced the protein expression of mitochondrial biogenesis-related proteins PGC-1α (1.00 ± 0.17 vs. 0.55 ± 0.12), NRF1 (1.00 ± 0.17 vs. 0.60 ± 0.10), TFAM (1.00 ± 0.10 vs. 0.73 ± 0.09), and SIRTI (1.00 ± 0.44 vs. 0.40 ± 0.10). The HC diet promoted mitochondrial fission and inhibited mitochondrial fusion by reducing protein expression of MFN1 (1.00 ± 0.31 vs. 0.49 ± 0.09), MFN2 (1.00 ± 0.19 vs. 0.69 ± 0.13), and OPA1 (1.00 ± 0.08 vs. 0.72 ± 0.07), and by increasing that of DRP1 (1.00 ± 0.09 vs. 1.39 ± 0.10), MFF (1.00 ± 0.15 vs. 1.89 ± 0.12), and TTC1/FIS1 (1.00 ± 0.08 vs. 1.76 ± 0.14), leading to mitochondrial dysfunction. The HC diet increased mitochondrial permeability by upregulating the protein expression of VDAC1 (1.00 ± 0.42 vs. 1.90 ± 0.44), ANT (1.00 ± 0.22 vs. 1.27 ± 0.17), and CYPD (1.00 ± 0.41 vs. 1.82 ± 0.43). Taken together, these results indicated that feeding the HC diet induced mitochondrial damage via the MAPK signaling pathway in the mammary gland of dairy cows.

AMPK-endoplasmic reticulum stress axis contributes to lipopolysaccharide-caused mitochondrial dysfunction by regulating mitochondria-associated membrane function in bovine hepatocytes.

Mitochondrial homeostasis is closely associated with cellular homeostasis process, whereas mitochondrial dysfunction contributes to apoptosis and mitophagy. Hence, analyzing the mechanism of lipopolysaccharide (LPS)-caused mitochondrial damage is necessary to understand how cellular homeostasis is maintained in bovine hepatocytes. Mitochondria-associated membranes (MAM), a connection between endoplasmic reticulum (ER) and mitochondria, is important to control mitochondrial function. To investigate the underlying mechanisms of the LPS-caused mitochondrial dysfunction, hepatocytes isolated from dairy cows at ∼160 d in milk (DIM) were pretreated with the specific inhibitors of adenosine 5'-monophosphate-activated protein kinase (AMPK), ER stress, RNA-activated protein kinase-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α), c-Jun N-terminal kinase, and autophagy followed by a 12 I1/4g/mL LPS treatment. The results showed that inhibiting ER stress with 4-phenylbutyric acid decreased the levels of autophagy and mitochondrial damage with AMPK inactivation in LPS-treated hepatocytes. The AMPK inhibitor compound C pretreatment alleviated LPS-induced ER stress, autophagy and mitochondrial dysfunction by regulating the expression of MAM-related genes, such as mitofusin 2 (MFN2), PERK, and IRE1α. Moreover, inhibiting PERK and IRE1α mitigated autophagy and mitochondrial dynamic disruption by regulating the MAM function. Additionally, blocking c-Jun N-terminal kinase, the downstream sensor of IRE1α, could reduce the levels of autophagy and apoptosis and restore the balance of mitochondrial fusion and fission by modulating the B cell leukemia 2 (BCL-2)/BCL-2 interacting protein 1 (BECLIN1) complex in the LPS-treated bovine hepatocytes. Furthermore, autophagy blockage with chloroquine could intervene in LPS-caused apoptosis to restore mitochondrial function. Collectively, these findings suggest that the AMPK-ER stress axis is involved in the LPS-caused mitochondrial dysfunction by mediating the MAM activity in bovine hepatocytes.

High-concentrate diet elevates histone lactylation mediated by p300/CBP through the upregulation of lactic acid and induces an inflammatory response in mammary gland of dairy cows.

High-concentrate diet can cause metabolic diseases, such as subacute ruminal acidosis (SARA), and secondary mastitis. To investigate the effect of SARA induced by high-concentrate diet on the lysine lactylation (Kla) and inflammatory responses in the mammary gland of dairy cows and the mechanism between them, we selected twelve mid-lactation Holstein cows with similar body conditions for modelling. They were randomly divided into two groups, fed a low-concentrate diet (LC) and a high-concentrate diet (HC) for 21 days. Our results showed that high-concentrate diet feeding significantly reduced ruminal pH, and the pH was below 5.6 for more than 3 h per day, indicating successful induction of the SARA model. Lactic acid concentrations in mammary gland and plasma were higher in the HC group than that in the LC group. HC diet feeding significantly up-regulated the expression levels of the Pan Kla, H3K18la, p300/CBP and monocarboxylate transporter 1 (MCT1) in the mammary gland. In addition, the mRNA expression levels of inflammatory factors were significantly regulated, including IL-1ß, IL-1α, IL-6, IL-8, SAA3, and TNF-α, while the anti-inflammatory factor IL-10 was down-regulated. The mammary gland of HC group was structurally disorganized with incomplete glandular vesicles, with a large number of detached mammary epithelial cells and inflammatory cells infiltration. The up-regulation of TLR4, TNF-α, p-p65, and p-IκBα indicated that the TLR4/NF-κB signaling pathway was activated. In conclusion, this study found that HC diet feeding can induce SARA and increase the concentration of lactic acid in mammary gland and plasma. Then, lactic acid could be transported into cells by MCT1 and up-regulate the expression level of histone lactylation mediated by p300/CBP, and subsequently promote the activation of TLR4/NF-κB signaling pathway, ultimately causing inflammatory responses in the mammary gland.

iE-DAP Induced Inflammatory Response and Tight Junction Disruption in Bovine Mammary Epithelial Cells via NOD1-Dependent NF-κB and MLCK Signaling Pathway.

γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP), a bacterial cell wall component, can trigger an inflammatory response. A mammary inflammatory response causes tight junction (TJ) dysfunction. This study aimed to explore the effects and involved mechanisms of iE-DAP-induced inflammatory response on the TJ integrity in bovine mammary epithelial cells (BMECs). The results showed that iE-DAP-induced inflammatory response and TJ disruption was associated with increased expression levels of inflammatory cytokines and decreased gene expression of ZO-1 and Occludin, as well as a reduction in transepithelial electrical resistance and elevation in paracellular dextran passage. While MLCK inhibitor ML-7 reversed the TJ disruption induced by iE-DAP. NF-κB inhibitor BAY 11-7085 hindered the activation of NF-κB and MLCK signaling pathways, the inflammatory response and TJ disruption induced by iE-DAP. NOD1-specific shRNA also inhibited the activation of the NOD1/NF-κB signaling pathway and reversed the inflammatory response and TJ injury in iE-DAP-treated BMECs. Above results suggest that iE-DAP activated the NF-κB and MLCK signaling pathway in NOD1-dependent manner, which promoted the transcription of inflammatory cytokines and altered the expression and distribution of tight junction proteins, finally caused inflammatory response and TJ disruption. This study might provide theoretical basis and scientific support for the prevention and treatment of mastitis.

The protective role of caffeic acid on bovine mammary epithelial cells and the inhibition of growth and biofilm formation of Gram-negative bacteria isolated from clinical mastitis milk.

As a first-line barrier against bacterial infection of mammary tissues, bovine mammary epithelial cells (bMECs) are generally believed to be involved in the immune response due to exogenous stress. Due to the escalating crisis of antibiotic resistance, there is an urgent need for new strategies to combat pathogenic bacteria-infected bovine mastitis. In this study, isolated bMECs and Institute of Cancer Research (ICR) mice were used for Escherichia coli infection and caffeic acid (CA) pretreatment experiments in vitro and in vivo. The inhibitory effect of CA on bacterial growth and biofilm formation was also demonstrated with bacteria strains isolated from mastitis-infected milk. It was demonstrated that CA supplementation prohibits the growth of the predominant strains of bacteria isolated from clinical bovine mastitis milk samples. CA was found to disrupt the biofilm formation of E. coli B1 in a sub-minimum inhibitory concentration (sub-MIC) and inhibited the adherence property of E. coli on bMECs by decreasing the staining of bacteria on cell surfaces in vitro. In addition, CA was found to attenuate proinflammatory and oxidative responses in cells infected with E. coli. The pretreatment of bMECs with CA also restored altered lipid homeostasis caused by E. coli stimulation. The protective role of CA was further confirmed via the administration of CA in mice followed by representative Gram-negative bacterial infection. Collectively, these findings highlight the potential of CA to mediate Gram-negative infections and indicate that it has the potential to be developed as a novel antibacterial drug.

ß-carotene alleviates LPS-induced inflammation through regulating STIM1/ORAI1 expression in bovine mammary epithelial cells.

ß-carotene has anti-inflammatory properties. STIM1(Stromol interaction molecule 1)/ORAI1 (Orai calcium release-activated calcium modulator 1) is an important inflammatory receptor, participating in the regulation of intracellular calcium signals by inflammation. The aim of this study was to clarify the correlation between STIM1/ORAI1-mediated Ca2+ signaling and inflammation and the anti-inflammatory effect of ß-carotene on lipopolysaccharide (LPS)-induced bovine mammary epithelial cells (BMECs). The results showed that LPS activated SOCE channels and induced Ca2+ influx via up-regulating the expression of STIM1 and ORAI1, leading to cell injury. STO-609, BTP2, STIM1 or ORAI1 sclienced attenuated LPS-induced inflammation by inhibiting NF-κB signaling. However, overexpression of STIM1 or ORAI1 induced inflammatory response by activating NF-κB signaling pathway, and which had synergistic effect with LPS. ß-carotene inhibited NF-κB activation by decreasing STIM1/ORAI1 expression, and thus alleviated LPS-induced inflammation in BMECs. Therefore, SOCE-targeting inhibitors are promising as new anti-inflammatory agents, and ß-carotene may be considered for the prevention of mastitis in dairy cows.

Lentinan inhibits oxidative stress and alleviates LPS-induced inflammation and apoptosis of BMECs by activating the Nrf2 signaling pathway.

Lentinan (LNT) has been reported to have a wide range of functions, including anti-inflammatory, antioxidant and anticancer properties. LNT may provide a protective effect in dairy cow mastitis. In this study, we investigated the effect of LNT on lipopolysaccharide (LPS)-induced injury of bovine mammary epithelial cells (BMECs) and the possible mechanism. First, we treated BMECs with different concentrations of LPS to study the effects of LPS on oxidative stress and inflammation in BMECs. Then, we examined the effects of LNT by dividing the cells into seven groups: the control group (CON), LPS treatment group (LPS), Acetyl-l-cysteine (NAC) pretreatment group (NAC + LPS), LNT pretreatment group (LNT + LPS), ML385 and LNT pretreatment group (ML385 + LNT + LPS), LNT treatment group (LNT) and NAC treatment group (NAC). The results showed that LPS-triggered intracellular ROS production and the downregulation of Nrf-2 and HO-1 in BMECs were blocked by LNT pretreatment. LNT inhibited the expression of inflammatory genes and proteins by inhibiting of NF-κB and MAPK. In addition, LNT attenuated LPS induced-apoptosis in BMECs. However, ML385 reversed the protective effect of LNT. Taken together, LNT can be used as a natural protective agent against LPS-triggered BMECs damage through its anti-inflammatory, antioxidant and antiapoptotic effects through modulation of the Nrf2 pathway.

A high-concentrate diet provokes inflammatory responses by downregulating Forkhead box protein A2 (FOXA2) through epigenetic modifications in the liver of dairy cows.

A high-concentrate diet has been reported to promote an inflammatory response in dairy cows. The purpose of this study was to clarify the effect of the high-concentrate (HC) diet on hepatic Forkhead box protein A2 (FOXA2) expression and uncover the molecular mechanisms in inflammatory responses in the liver. The results showed that the HC diet reduced the ruminal fluid pH and elevated the secretion of SAA3, IL-1α, and IL-8 and reduced that of IL-10 in peripheral blood plasma. Compared with the low-concentrate (LC) group, the concentration of myeloperoxidase (MPO) was higher in the liver of dairy cows in the HC group. In addition, the relative mRNA expression of acute phase proteins (HP, SAA3, and LBP), proinflammatory cytokines (TNFα, IL-1α, IL-1ß, IL-8), TLR4, MyD88, TRAF6, TRIF, IκBα, p65, p38 and JNK1 was upregulated and that of IL-10 was downregulated in the liver of the HC group. Consistently, the protein abundance of TLR4, TNFα and phosphorylation of proteins involved in NF-κB (IκBα and p65) and MAPK (p38 and JNK) pathways were significantly increased in the HC group compared with the LC group. And both the mRNA and protein abundance of FOXA2 were downregulated in the HC group. Further epigenetic analysis results demonstrated that chromatin compaction and DNA hypermethylation contributed to inhibiting FOXA2 expression, in which the demethylase ten-eleven translocation 1 (TET1) and histone deacetylase 3 (HDAC3) might participate. Overall, these findings demonstrated that the high-concentrate diet triggered inflammatory cascades and downregulated FOXA2 by epigenetic modifications in the liver of dairy cows.

Protective Effects of N-Acetylcysteine on Lipopolysaccharide-Induced Respiratory Inflammation and Oxidative Stress.

As the leading cause of bovine respiratory disease (BRD), bacterial pneumonia can result in tremendous losses in the herd farming industry worldwide. N-acetylcysteine (NAC), an acetylated precursor of the amino acid L-cysteine, has been reported to have anti-inflammatory and antioxidant properties. To explore the protective effect and underlying mechanisms of NAC in ALI, we investigated its role in lipopolysaccharide (LPS)-induced bovine embryo tracheal cells (EBTr) and mouse lung injury models. We found that NAC pretreatment attenuated LPS-induced inflammation in EBTr and mouse models. Moreover, LPS suppressed the expression of oxidative-related factors in EBTr and promoted gene expression and the secretion of inflammatory cytokines. Conversely, the pretreatment of NAC alleviated the secretion of inflammatory cytokines and decreased their mRNA levels, maintaining stable levels of antioxidative gene expression. In vivo, NAC helped LPS-induced inflammatory responses and lung injury in ALI mice. The relative protein concentration, total cells, and percentage of neutrophils in BALF; the level of secretion of IL-6, IL-8, TNF-α, and IL-1ß; MPO activity; lung injury score; and the expression level of inflammatory-related genes were decreased significantly in the NAC group compared with the LPS group. NAC also ameliorated LPS-induced mRNA level changes in antioxidative genes. In conclusion, our findings suggest that NAC affects the inflammatory and oxidative response, alleviating LPS-induced EBTr inflammation and mouse lung injury, which offers a natural therapeutic strategy for BRD.

Long-term high-concentrate diet feeding induces apoptosis of rumen epithelial cells and inflammation of rumen epithelium in dairy cows.

The aim of the present study was to evaluate how long-term high-concentrate diet feeding affected rumen epithelium (RE) of dairy cows. So, 12 mid-lactating multiparous cows were divided into two groups randomly fed either with high-concentrate diet (HC, concentrate: forage = 6: 4) or low-concentrate diet (LC, concentrate: forage = 4:6) for 20 weeks. Remarkable upregulation of lipopolysaccharide (LPS) level and depress of pH in rumen fluid were induced by HC compared with LC group. mRNA abundance of interleukin-6 (IL-6), interleukin-8 (IL-8), C-C motif chemokine ligand 5 (CCL5), caspase-3, caspase-8, and caspase-9 were elevated in RE of HC group compared with LC group. Greater protein abundance of phosphorylated NF-κB p65, IL-6, and tumor necrosis factor α (TNF-α) was observed in RE of cows fed HC than that fed LC. Abundance of protein related to proapoptotic response (cytochrome c, BAX and caspase-3) in HC group was greater than that in LC group, while the abundance of anti-apoptotic factor protein (Bcl-2) was lower in HC group than LC group. Therefore, the present study demonstrated that long-term high-concentrate diet feeding upregulated LPS level in rumen fluid and induced the proinflammatory response in the rumen epithelium and apoptosis of rumen epithelial cells.

Sodium butyrate pretreatment mitigates lipopolysaccharide-induced inflammation through the TLR4/NF-κB signaling pathway in bovine embryo trachea cells.

The present study investigated the anti-inflammatory effects and potential mechanisms of sodium butyrate (SB) in bovine embryo tracheal cells (EBTr) stimulated with lipopolysaccharide (LPS). EBTr were exposed to either 1 mmol/L SB for 18 h for the SB group (SB) or to 0.4 µg/mL LPS for 6 h for the LPS group (LPS). PBS was added to EBTr for a control group (CON). EBTr were pretreated with SB for 18 h followed by 6 h of LPS stimulation for the LSB group (LSB). Results showed that with LPS stimulation, the gene expression of TLR4, NF-κB, IL6, and IL8, as well as cytokine production of IL6 and TNF-α, were significantly increased compared with the CON group. In contrast, protein expression of IL10 was decreased. However, these inflammatory effects induced by LPS were reversed in the LSB group. Compared with the CON group, protein expression of TLR4, phospho-NF-κB p65, phospho-IκBα, and IL1α were increased in the LPS group and these were decreased in the LSB group. Similarly, increased nuclear translocation of phospho-NF-κB p65 in the LPS group was suppressed with SB pretreatment. In conclusion, SB can reduce inflammation induced by LPS in EBTr, and this positive effect is mediated through the TLR4 and NF-κB signaling pathway.

Sodium butyrate promotes lipopolysaccharide-induced innate immune responses by enhancing mitogen-activated protein kinase activation and histone acetylation in bovine mammary epithelial cells.

The innate immune response plays a crucial role in recovery from infectious diseases by promoting the clearance of pathogens. Sodium butyrate (NaB) is an energy source for cellular processes with the potential to regulate the innate immune response. The present study aimed to evaluate the effect of NaB on the innate immune response in a bovine mammary alveolar cell line (MAC-T) initiated by lipopolysaccharides (LPS). Thus, treatments were conducted as follows: treated with 1× PBS for 24 h (control), pretreated with 1 mM NaB (optimized by cell viability assays and dose-dependent experiment) for 18 h followed by treatment of 1× PBS for 6 h (NaB), pretreated with 1× PBS for 18 h followed by stimulation with LPS (1 µg/mL) for 6 h (LPS), and pretreated with 1 mM NaB for 18 h followed by stimulation with LPS (1 µg/mL) for 6 h (NaB + LPS). Different inhibitors were also used to elucidate the underlying mechanism. Furthermore, cells were treated with NaB and heat-inactivated Escherichia coli to test the effect of NaB on transcription of genes related to the innate immune response triggered by the major causative pathogen of mastitis. Each treatment had 3 replicates and was repeated 3 times. Proinflammatory cytokines, chemokines, and ß-defensins are crucial secretion factors in innate immunity, and transcription of these factors was increased by NaB during challenge with LPS or heat-inactivated E. coli in MAC-T cells. Acetylation of histone H3 protein, which promotes gene expression by affecting the structure of chromatin, was also upregulated by NaB in response to LPS stimulation. P38 mitogen-activated protein kinases (MAPK), JNK, and Erk 1 and 2 are key upstream regulators of the expression of proinflammatory cytokines, chemokines, and ß-defensins, and their activity was enhanced by NaB during LPS stimulation. Furthermore, inhibitors were used to assess the role of MAPK signaling in the effects of NaB. The results showed that inhibitors of p38 MAPK, Erk, and JNK attenuated the NaB-induced upregulation of TNF and ß-defensin 5 (DEFB5) transcription, and that the inhibitor of Erk attenuated the NaB-induced upregulation of IL1B transcription during LPS challenge. Enhanced transcription of CXCL8 by NaB was blocked by the inhibitor of Erk and p38 MAPK during LPS stimulation. Overall, NaB boosted the LPS-induced innate immune response by promoting the expression of proinflammatory cytokines, chemokines, and ß-defensins, which was associated with enhanced MAPK signaling activation and histone H3 acetylation.

Sodium valproate attenuates the iE-DAP induced inflammatory response by inhibiting the NOD1-NF-κB pathway and histone modifications in bovine mammary epithelial cells.

The anti-inflammatory effects of sodium valproate (VPA) in vivo and in vitro have been demonstrated in recent studies. The aim of this study was to evaluate whether VPA can suppress inflammation in bovine mammary epithelial cells (BMECs) stimulated by γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP). First, the concentration and treatment points of iE-DAP and VPA were optimized. Then, BMECs were cultured in complete media and separated into four groups: untreated control cells (CON group), cells stimulated by 10 µg/mL iE-DAP for 6 h (DAP group), cells stimulated by 0.5 mmol/L VPA for 6 h (VPA group), and cells pretreated with VPA (0.5 mmol/L) for 6 h followed by 10 µg/mL of iE-DAP for 6 h (VD group). The results showed that the level of  interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the culture medium increased in the iE-DAP-treated cells and that pretreatment with VPA reversed this increase. iE-DAP increased both mRNA and protein expression levels of nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and receptor-interacting protein kinas (RIPK2) and activated inhibitor of NF-κB (IκB) and nuclear factor-kappa B p65 (NF-κB p65) through phosphorylation. Upon activation of the NF-κB pathway, the expression of the pro-inflammatory cytokines IL-6, interleukin-8 (IL-8) and interleukin-1ß (IL-1ß), the acute phase protein serum amyloid A 3 (SAA3) and the lingual antimicrobial peptide (LAP) but not  haptoglobi (HP) or bovine neutrophil beta defensing 5 (BNBD5) were increased in the DAP group. The VPA pretreatment induced the acetylation of signal transducers and activators of transcription(STAT1) and histone 3 (H3) by inhibiting histone deacetylase (HDAC) and then suppressed the NF-κB pathway. Moreover, VPA induced autophagy and reduced apoptosis in BMECs in the VD group. These results suggested that VPA treatment can attenuate the inflammatory response induced by iE-DAP.

Sodium Butyrate Inhibits the Inflammation of Lipopolysaccharide-Induced Acute Lung Injury in Mice by Regulating the Toll-Like Receptor 4/Nuclear Factor κB Signaling Pathway.

Bacterial pneumonia is a common disease in dairy herds worldwide, which brings great economic losses to farmers. Sodium butyrate (SB), an inhibitor of histone deacetylase, plays an important role in limiting inflammation. The purpose of this study was to investigate the protective effects of SB on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and explore the potential mechanism of SB protection. A total of 30 ICR mice were randomly divided into three groups ( n = 10): a phosphate-buffered saline (PBS) intratracheal instillation group, a LPS intratracheal instillation group, and a SB gavage group (SB was given 1 h before the LPS stimulation). After 12 h, samples of the blood and lung tissue were collected from the mice for experimental analysis. The results showed that the concentration of inflammatory cytokines [interleukin 1ß (IL1ß) and tumor necrosis factor α (TNF-α)], myeloperoxidase (MPO) activity in the lung tissue and blood, protein abundance of toll-like receptor 4 (TLR4), nuclear factor κB (NF-κB, p65), phosphorylated p65 (p-p65), inhibitor κBα (IκBα), and phosphorylated IκBα (p-IκBα), and relative mRNA expression of genes associated with inflammation, such as TLR4, NF-κB, IL1ß, interleukin 6 (IL6), and TNF-α, were significantly upregulated in the LPS group compared to the PBS group. However, the SB addition markedly downregulated the levels of these parameters in the LSB group compared to those in the LPS group. Furthermore, the structure of the lung tissue from the LPS group was severely disrupted in comparison to that of the PBS group. However, with SB administration, the severe structural disruption was relieved. In addition, an immunohistochemical analysis showed that positive immunoreactions to TLR4, p65, and TNF-α were significant in the LPS group; however, SB addition markedly attenuated this phenomenon. In conclusion, the ALI mouse model was successfully established with an intratracheal instillation of LPS. Furthermore, gavage with SB inhibited inflammation in LPS-induced ALI.

cis-9, trans-11-Conjugated Linoleic Acid Exerts an Anti-inflammatory Effect in Bovine Mammary Epithelial Cells after Escherichia coli Stimulation through NF-κB Signaling Pathway.

The anti-inflammatory effects of cis-9, trans-11-conjugated linoleic acid ( cis-9, trans-11-CLA) in diverse cells have been demonstrated in recent studies. The present study was conducted to observe the anti-inflammatory effects and involved mechanisms of CLA in bovine mammary epithelial cells (BMECs) exposed to Escherichia coli. According to the gene expression of IL-6, to optimize the treatment period and dose of CLA, 50 and 100 µM CLA were chosen to pretreat the cells for a period of 48 h. BMECs were exposed to 1 × 107 /mL E. coli for 6 h (ECO group), and cells were pretreated with 50 and 100 µM CLA for 48 h followed by E. coli challenge (C50 and C100 groups). After E. coli challenge, compared with that in the CON group (control group), the gene expressions of pro-inflammatory cytokines (IL-1ß and IL-6), chemokines (IL-8 and CCL-20), and antimicrobial peptide BNBD5 were increased, while the gene expression of the anti-inflammatory cytokine IL-10 was decreased significantly; CLA reversed this inflammation effect. Pretreatment with CLA also repressed the secretion of IL-6, IL-8, and TNF-α from BMECs in the culture medium following E. coli challenge. Therefore, cis-9, trans-11-CLA exerted anti-inflammatory effects in BMECs. The cells that were pretreated with CLA expressed remarkably lower levels of phospho-p65, phospho-IκB, and TLR4 and a higher level of PPARγ after E. coli challenge at the gene and protein levels. Compared to that in the ECO group, the nuclear translocation of phospho-p65 was suppressed when CLA was added. Combined with the above results, 50 µM CLA showed a better anti-inflammatory effect. In conclusion, CLA can reduce inflammation caused by E. coli in bovine mammary epithelial cells, and this effect is mediated through the TLR4-NF-κB pathway and PPARγ participation.

Sodium Butyrate Supplementation Alleviates the Adaptive Response to Inflammation and Modulates Fatty Acid Metabolism in Lipopolysaccharide-Stimulated Bovine Hepatocytes.

This study aimed to evaluate whether sodium butyrate (SB) attenuates the hepatic response to LPS-induced inflammation in bovine hepatocytes. Hepatocytes isolated from cows at ∼160 days in milk (DIM) were exposed to 0.5 mmol/L SB for 18 h as pretreatment. Cells pretreated with SB were used for the SB group, and those subjected to 4 µg/mL lipopolysaccharide (LPS) challenge for 6 h were used for the lipopolysaccharide pretreated with SB (LSB) group. The LPS-challenged hepatocytes showed increases in TNF-α and IL-6 production in culture medium (37 ± 11, P < 0.05); these increases were attenuated by pretreatment with SB in the LSB group (267 ± 4, P < 0.05). Compared to that in LPS-treated cells, the phospho-p65 and phospho-IκBα protein expression and nuclear translocation were suppressed when SB was added. Genes ( SREBP1c, SCD1, and DGAT1) and proteins (SREBP1c and SCD1) related to fatty acid metabolism were upregulated in LSB cells compared to those in LPS-treated cells ( P < 0.05). The ratios of phospho-AMPKα to AMPKα (0.32 ± 0.03 vs 0.70 ± 0.07) and phospho-ACCα to ACCα were decreased (0.81 ± 0.06 vs 2.06 ± 0.16) ( P < 0.05) in the LSB group. SB pretreatment reversed the histone H3 deacetylation that was increased by LPS stimulation in bovine hepatocytes (0.54 ± 0.02 vs 1.27 ± 0.11, P < 0.05). Our results suggest that SB pretreatment suppresses the hepatocyte changes that occur during the LPS-induced inflammatory response, which is accompanied by enhanced fatty acid synthesis, downregulated fatty acid oxidation, and histone H3 deacetylation, thus neutralizing the negative effects of infection.

Rumen-derived lipopolysaccharide provoked inflammatory injury in the liver of dairy cows fed a high-concentrate diet.

Rumen-derived lipopolysaccharide (LPS) is translocated from the rumen into the bloodstream when subacute ruminal acidosis (SARA) occurs following long-term feeding with a high-concentrate (HC) diet in dairy cows. The objective of this study was to investigate the mechanism of inflammatory responses in the liver caused by HC diet feeding. We found that SARA was induced in dairy cows when rumen pH below 5.6 lasted for at least 3 h/d with HC diet feeding. Also, the LPS levels in the portal and hepatic veins were increased significantly and hepatocytes were impaired as well as the liver function was inhibited during SARA condition. Meanwhile, the mRNA expression of immune genes including TNF receptor associated factor 6 (TRAF6), nuclear factor-kappa B (NF-κB), p38 mitogen-activated protein kinase (MAPK), extracellular regulated protein kinases (ERK) MAPK, Interleukin-1 (IL-1) and serum amyloid A (SAA) in the liver were significantly increased in SARA cows. Moreover, the phosphorylation level of NF-κB p65 and p38 MAPK proteins in the liver and the concentration of Tumor Necrosis Factor (TNF-α), Interleukin-1ß (IL-1ß) and Interleukin-6 (IL-6) in peripheral blood were obviously increased under SARA condition. In conclusion, the inflammatory injury in the liver caused by LPS that traveled from the digestive tract to the liver through the portal vein after feeding with a HC diet.

Lipopolysaccharide derived from the digestive tract activates inflammatory gene expression and inhibits casein synthesis in the mammary glands of lactating dairy cows.

To meet the nutrition requirements of lactation, dairy cows are usually fed a high concentrate diet (HC). However, high-grain feeding causes subacute ruminal acidosis (SARA), a metabolic disorder that causes milk protein depression. This study aimed to investigate the effect of lipopolysaccharide (LPS) released in the rumen on inflammatory gene expression and casein synthesis in mammary glands of lactating dairy cows fed a HC diet. We found that milk protein was significantly decreased in the HC group after 15 weeks of feeding. Overall, LPS concentrations in the rumen fluid, lacteal artery and vein were increased in the HC group. Transcriptome microarray was used to evaluate alterations in the signaling pathway in mammary glands. Signaling pathways involved in inflammatory responses were activated, whereas those involved in protein synthesis were inhibited in the HC group. mRNA expression involved in inflammatory responses, including that of TLR4, NF-κB and pro-inflammatory genes, was increased in the HC group, while αs1-casein (CSN1S1), ß-casein (CSN2), mTOR and S6K gene expression were decreased. Moreover, protein expression was consistent with the corresponding gene expression. After feeding with an HC diet, LPS derived from the rumen increased inflammatory gene expression and inhibited casein synthesis in the mammary glands of lactating dairy cows fed a HC diet.

Three promoters with different tissue specificity and pathogen inducibility express the toll-like-receptor 2 (TLR2)-encoding gene in cattle.

Toll-like-receptor 2 (TLR2) is a dominant receptor for perceiving presence of bacterial pathogens. The promoter controlling its tissue specific and infection induced expression in cattle was unknown. We structurally defined with 5'-RACE experiments three promoters (P1-3) controlling TLR2 expression in udder, liver and other tissues of cows suffering from E. coli mastitis. P1 is 5'-adjacent to exon 1 as defined by the prototypical TLR2 cDNA sequence. Exon 1 is spliced to the protein-encoding exon 2. P2 and P3 reside in intron 1, express exon 1A and exon 1B, respectively which are each spliced to exon 2. Infection induced massively (>30-fold) activity of P1 and P2, but not of P3 in udders and also somewhat in liver. However, the GC-rich housekeeping promoter P3 expressed exon1B in many tissues providing the wealth of TLR2-encoding transcripts. Similar induction data were obtained after challenging primary cultures of mammary epithelial cells (pbMEC) with E. coli. Reporter gene analyses in pbMEC and the liver cell line HepG2 collectively validated that P1 and constructs containing segments from P2/P3 are in principle capable to drive gene expression. Our structural data provide the basis for more detailed molecular analyses of the infection and tissue specific regulation of TLR2 expression.