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 weeks. 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 euthanized, 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 (On d 20: diet, P < 0.001; time, P = 0.02. On d 21: diet, P < 0.001; time, P = 0.002). 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 (P = 0.016), interleukin 1 ß (IL-1ß; P < 0.01), and apoptosis-related cysteine protease 1 (caspase-1; P < 0.01) and the activity of alanine aminotransferase (P = 0.026) and aspartate aminotransferase (P = 0.002) in the blood plasma of the HG group were all significantly increased. Hepatic caspase-1 activity (P < 0.001) was increased in the livers of the HG group. The increased expression levels of pyroptosis- and NLRP3 inflammasome-related genes IL1B (P = 0.002), IL18 (P < 0.001), gasdermin D (GSDMD; P = 0.001), apoptosis-associated speck-like protein containing a card (ASC; P = 0.001), NLR family pyrin domain-containing 3 (NLRP3; P = 0.002), and caspase-1 (CASP1; P < 0.001) 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; P = 0.006), IL-1ß (P < 0.001), IL-18 (P = 0.076), cleaved-caspase-1 (P = 0.001), ASC (P = 0.016), NLRP3 (P = 0.017), and cleaved-caspase-11 (P < 0.001) and upregulated expression of mitophagy-related proteins microtubule-associated protein 1 light chain 3 II (MAP1LC3-II; P = 0.001), beclin 1 (BECN1; P = 0.001), Parkin (P < 0.001), and PTEN induced kinase 1 (PINK1; P = 0.006) 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.

Conjugated Linoleic Acid Ameliorates Hydrogen Peroxide-Induced Mitophagy and Inflammation via the DRP1-mtDNA-STING Pathway in Bovine Hepatocytes.

Oxidative stress is tightly associated with liver dysfunction and injury in dairy cows. Previous studies have shown that cis-9, trans-11 conjugated linoleic acid (CLA) possesses anti-inflammatory and antioxidative abilities. In this study, the bovine hepatocytes were pretreated with CLA for 6 h, followed by treatment with hydrogen peroxide (H2O2) for another 6 h to investigate the antioxidative effect of CLA and uncover the underlying mechanisms. The results demonstrated that H2O2 treatment elevated the level of mitophagy, promoted mitochondrial DNA (mtDNA) leakage into the cytosol, and activated the stimulator of interferon genes (STING)/nuclear factor kappa B (NF-κB) signaling pathway to trigger an inflammatory response in bovine hepatocytes. In addition, the dynamin-related protein 1(DRP1)-mtDNA-STING-NF-κB axis contributed to the H2O2-induced oxidative injury of bovine hepatocytes. CLA could reduce mitophagy and the inflammatory response to attenuate oxidative damage via the DRP1/mtDNA/STING pathway in bovine hepatocytes. These findings offer a theoretical foundation for the hepatoprotective effect of CLA against oxidative injury in dairy cows.

PEB-DDI: A Task-Specific Dual-View Substructural Learning Framework for Drug-Drug Interaction Prediction.

Adverse drug-drug interactions (DDIs) pose potential risks in polypharmacy due to unknown physicochemical incompatibilities between co-administered drugs. Recent studies have utilized multi-layer graph neural network architectures to model hierarchical molecular substructures of drugs, achieving excellent DDI prediction performance. While extant substructural frameworks effectively encode interactions from atom-level features, they overlook valuable chemical bond representations within molecular graphs. More critically, given the multifaceted nature of DDI prediction tasks involving both known and novel drug combinations, previous methods lack tailored strategies to address these distinct scenarios. The resulting lack of adaptability impedes further improvements to model performance. To tackle these challenges, we propose PEB-DDI, a DDI prediction learning framework with enhanced substructure extraction. First, the information of chemical bonds is integrated and synchronously updated with the atomic nodes. Then, different dual-view strategies are selected based on whether novel drugs are present in the prediction task. Particularly, we constructed Molecular fingerprint-Molecular graph view for transductive task, and Bipartite graph-Molecular graph view for inductive task. Rigorous evaluations on benchmark datasets underscore PEB-DDI's superior performance. Notably, on DrugBank, it achieves an outstanding accuracy rate of 98.18% when predicting previously unknown interactions among approved drugs. Even when faced with novel drugs, PEB-DDI consistently exhibits outstanding generalization capabilities with an accuracy rate of 88.06%, attributing to the proper migrating of molecular basic structure learning.

Sequence Alignment/Map format: a comprehensive review of approaches and applications.

The Sequence Alignment/Map (SAM) format file is the text file used to record alignment information. Alignment is the core of sequencing analysis, and downstream tasks accept mapping results for further processing. Given the rapid development of the sequencing industry today, a comprehensive understanding of the SAM format and related tools is necessary to meet the challenges of data processing and analysis. This paper is devoted to retrieving knowledge in the broad field of SAM. First, the format of SAM is introduced to understand the overall process of the sequencing analysis. Then, existing work is systematically classified in accordance with generation, compression and application, and the involved SAM tools are specifically mined. Lastly, a summary and some thoughts on future directions are provided.

Subclinical ketosis leads to lipid metabolism disorder by downregulating the expression of acetyl-coenzyme A acetyltransferase 2 in dairy cows.

Ketosis is a metabolic disease that often occurs in dairy cows postpartum and is a result of disordered lipid metabolism. Acetyl-coenzyme A (CoA) acetyltransferase 2 (ACAT2) is important for balancing cholesterol and triglyceride (TG) metabolism; however, its role in subclinical ketotic dairy cows is unclear. This study aimed to explore the potential correlation between ACAT2 and lipid metabolism disorders in subclinical ketotic cows through in vitro and in vivo experiments. In the in vivo experiment, liver tissue and blood samples were collected from healthy cows (CON, n = 6, ß-hydroxybutyric acid [BHBA] concentration <1.0 mM) and subclinical ketotic cows (subclinical ketosis [SCK], n = 6, BHBA concentration = 1.2-3.0 mM) to explore the effect of ACAT2 on lipid metabolism disorders in SCK cows. For the in vitro experiment, bovine hepatocytes (BHEC) were used as the model. The effects of BHBA on ACAT2 and lipid metabolism were investigated via BHBA concentration gradient experiments. Subsequently, the relation between ACAT2 and lipid metabolism disorder was explored by transfection with siRNA of ACAT2. Transcriptomics showed an upregulation of differentially expression genes during lipid metabolism and significantly lower ACAT2 mRNA levels in the SCK group. Compared with the CON group in vivo, the SCK group showed significantly higher expression levels of peroxisome proliferator-activated receptor γ (PPARγ) and sterol regulator element binding protein 1c (SREBP1c) and significantly lower expression levels of peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyl-transferase 1A (CPT1A), sterol regulatory element binding transcription factor 2 (SREBP2), and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). Moreover, the SCK group had a significantly higher liver TG content and significantly lower plasma total cholesterol (TC) and free cholesterol content. These results were indicative of TG and cholesterol metabolism disorders in the liver of dairy cows with SCK. Additionally, the SCK group showed an increased expression of perilipin-2 (PLIN2), decreased expression of apolipoprotein B, and decreased plasma concentration of very low-density lipoproteins (VLDL) and low-density lipoproteins cholesterol (LDL-C) by downregulating ACAT2, which indicated an accumulation of TG in liver. In vitro experiments showed that BHBA induced an increase in the TG content of BHEC, decreased content TC, increased expression of PPARγ and SREBP1c, and decreased expression of PPARα, CPT1A, SREBP2, and HMGCR. Additionally, BHBA increased the expression of PLIN2 in BHEC, decreased the expression and fluorescence intensity of ACAT2, and decreased the VLDL and LDL-C contents. Furthermore, silencing ACAT2 expression increased the TG content; decreased the TC, VLDL, and LDL-C contents; decreased the expression of HMGCR and SREBP2; and increased the expression of SREBP1c; but had no effect on the expression of PLIN2. These results suggest that ACAT2 downregulation in BHEC promotes TG accumulation and inhibits cholesterol synthesis, leading to TG and cholesterol metabolic disorders. In conclusion, ACAT2 downregulation in the SCK group inhibited cholesterol synthesis, increased TG synthesis, and reduced the contents of VLDL and LDL-C, eventually leading to disordered TG and cholesterol metabolism.

Butyrate Protects against γ-d-Glutamyl-meso-diaminopimelic Acid-Induced Inflammatory Response and Tight Junction Disruption through Histone Deacetylase 3 Inhibition in Bovine Mammary Epithelial Cells.

The present study was conducted to evaluate the regulatory actions and underlying mechanisms of butyrate on the inflammatory response and tight junction (TJ) disruption in bovine mammary epithelial cells (BMECs). Results showed that butyrate declined histone deacetylase 3 (HDAC3) expression, blocked NF-κB activation, and thus suppressed inflammatory cytokine production in γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP)-triggered BMECs. Butyrate also depressed the protein abundance of myosin light chain kinase (MLCK), elevated the expression of TJ proteins, and restored the cellular distribution of TJ proteins and the barrier function of epithelial cells. HDAC3 overexpression abolished the protective effects of butyrate. In conclusion, butyrate alleviated the iE-DAP-induced inflammatory response and TJ injury by blocking NF-κB activation and decreasing inflammatory cytokine production and MLCK expression in a HDAC3-dependent manner. Our finding provides a mechanistic basis for further exploring the regulatory effects of butyrate on the mammary inflammatory response.

A high-concentrate diet induces colonic inflammation and barrier damage in Hu sheep.

Long-term feeding of a high-concentrate diet can induce subacute ruminal acidosis (SARA) in ruminants, which further leads to systemic inflammatory response. However, few studies have examined the effects of feeding a high-concentrate diet on the hindgut of ruminants. The purpose of this study was to investigate the effects of a high-concentrate diet on the composition of gut microbiota in colonic contents, inflammatory response, and barrier damage in the colon tissue of ruminants. A total of 12 healthy multiparous lactating Hu sheep were randomly allotted into the following 2 groups: a high-concentrate (HC) group (concentrate:forage = 7:3) and a low-concentrate (LC) group (concentrate:forage = 3:7). All sheep were fitted with ruminal fistulas. The formal feeding experiment lasted for 8 wk. After the feeding experiment, rumen fluid, portal vein blood, hepatic vein blood, colonic contents, and colon tissue samples were collected. The results showed that feeding the HC diet induced SARA in Hu sheep and significantly reduced pH in the colonic contents. The abundances of Firmicutes, Verrucomicrobiota, and Actinobacteriota decreased significantly, whereas those of Bacteroidota, Spirochaetota, and Fibrobacterota significantly increased in colonic contents. At the genus level, the relative abundances of 29 genera were significantly altered depending on the different type of diets. Analysis of the 10 bacterial genera with high relative abundance revealed that feeding the HC diet significantly reduced the abundance of UCG-005, Christensenellaceae R-7 group, UCG-010-norank, Monoglobus, [Eubacterium] coprostanoligenes group_norank, and Alistipes, whereas the abundances of Rikenellaceae RC9 gut group, Treponema, Bacteroides, and Prevotella increased. Compared with the LC group, feeding the HC diet significantly increased the concentration of LPS in rumen fluid, portal vein blood, hepatic vein blood, and colonic contents, and significantly upregulated the mRNA expression levels of proinflammatory cytokines in colon tissue, including TNF-α, IL-1ß, IL-6, and IL-8, indicating the occurrence of inflammatory response in the colon tissue. In addition, the structure of colonic epithelial cells was loose, the intercellular space became larger, epithelial cells were exfoliated, and the mRNA and protein abundances of ZO-1, occludin, claudin-1, claudin-3, and claudin-4 were significantly decreased in the HC group, which was consistent with the results of immunohistochemistry. Furthermore, feeding the HC diet increased the ratios of DNA methylation and chromatin compaction in the promoter regions of occludin and claudin-1, which in turn inhibited their transcriptional expression. Therefore, the present study demonstrated that feeding an HC diet induced SARA in Hu sheep, altered the composition and structure of the microbial community in the colonic contents, induced an inflammatory response, and disrupted the intestinal mucosal barrier in the colonic tissue.

Glutamine alleviates Lipopolysaccharide-induced corneal epithelial inflammation and oxidative stress in dogs.

Pseudomonas aeruginosa is a common pathogenic bacteria in canine ophthalmology. Lipopolysaccharide (LPS), a component in the cell wall of gram-negative bacteria, is released following bacterial lysis and causes pathology and inflammation of the cornea. Antibiotics are used to treat bacterial keratitis, and the reuse of antibiotics can easily cause bacterial resistance. Research has shown that glutamine (GLN) has anti-inflammatory and antioxidant biological functions. Herein, we explored the effects and underlying mechanisms of GLN and established an LPS-induced cornea inflammation model. Treatment groups comprised: control check (CK), LPS, LPS + GLN, and Sham groups. Topical GLN treatment alleviated corneal opacity, reduced corneal injury, and accelerated corneal wound healing. Furthermore, GLN treatment altered the uniform distribution of corneal epithelial cells and transformed the healing approach of these cells in the corneal wound from crawling to filling. The expression of Toll-like receptor 4 (TLR4), IL-6, TNF-α, and p-p65 and the activity of myeloperoxidase and superoxide dismutase decreased while the content of malondialdehyde increased in the LPS + GLN group compared with those in the LPS group. Thus, our study suggests that LPS-induced inflammation and oxidative stress may be suppressed via the TLR4/NF-κB signaling pathway by GLN and that GLN could be used as an adjunct therapy to reduce antibiotic use.

Dietary disodium fumarate supplementation alleviates subacute ruminal acidosis (SARA)-induced liver damage by inhibiting pyroptosis via mitophagy-NLRP3 inflammasome pathway in lactating Hu sheep.

Liver damage is common in ruminants with subacute ruminal acidosis (SARA). Disodium fumarate (DF) could regulate rumen microbial community and neutralize ruminal organic acids. This study aimed to evaluate the effect of dietary DF supplementation on SARA-induced liver damage and investigate the underlying mechanism. The results showed that feeding a high-concentrate diet induced decreased rumen fluid pH and increased ruminal LPS. The rumen fluid pH in the HC group was less than 5.6 at 4 time points, indicating that SARA was successfully induced. The histopathological analysis showed that in the HC group, hemorrhage and inflammatory cell infiltration were observed in liver tissue. Using ELISA kits and biochemical analyzer, we identified that the contents of interleukin 1beta (IL-1ß), interleukin 18 (IL-18), caspase-1, and the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in hepatic vein were elevated in the HC group. However, DF supplementation increased rumen fluid pH value, decreased ruminal LPS, attenuated hemorrhage and inflammatory cell infiltration in the liver tissue, and decreased contents of IL-1ß, IL-18, caspase-1, AST, and ALT in the hepatic vein. Real-time PCR and western blot analysis displayed that SARA-induced increased expression of pyroptosis-related proteins (GSDMD-NT) was attenuated in the HCDF group. Meanwhile, SARA induced increased expression of mitophagy and inflammasome-related proteins (MAP1LC3-II, PINK1, Parkin, cleaved-caspase-11, cleaved-caspase-1, NLRP3, and ASC) and elevated expression of inflammasome-related genes (NLRP3, CASP1, and ASC), which was reversed by DF supplementation. Moreover, SARA activated toll-like receptor 4 (TLR4)-nuclear factor kappa B (NF-κB) signaling pathway and inhibited the entry of forkhead box A2 (FOXA2) into the nucleus, which was reversed by DF supplementation. Collectively, our data suggest that dietary DF supplementation inhibited hepatocyte pyroptosis by regulating the mitophagy-NLRP3 inflammasome pathway and the NF-κB signaling pathway, thus alleviating SARA-induced liver damage in Hu sheep.

ß-carotene targets IP3R/GRP75/VDAC1-MCU axis to renovate LPS-induced mitochondrial oxidative damage by regulating STIM1.

Endoplasmic reticulum (ER) and mitochondria are the main sites for the storage and regulation of Ca2+ homeostasis. An imbalance of Ca2+ homeostasis can cause ER stress and mitochondrial dysfunction, thereby inducing apoptosis. The store-operated calcium entry (SOCE) is the main channel for extracellular calcium influx. Mitochondria-associated endoplasmic reticulum (MAM) is an important agent for Ca2+ transfer from the ER to the mitochondria. Therefore, regulation of SOCE and MAMs has potential therapeutic value for disease prevention and treatment. In this study, bovine mammary epithelial cells (BMECs) and mice were used as models to explore the mechanisms of ß-carotene to relieve ER stress and mitochondrial dysfunction. BAPTA-AM, EGTA (Ca2+ inhibitor), and BTP2 (SOCE channel inhibitor) alleviated ER stress and mitochondrial oxidative damage induced by increased intracellular Ca2+ levels after lipopolysaccharide (LPS) stimulation. Furthermore, inhibition of ER stress by 4-PBA (ER stress inhibitor), 2-APB (IP3R inhibitor), and ruthenium red (mitochondrial calcium uniporter (MCU) inhibitor) restored mitochondrial function by reducing mitochondrial ROS. Our data also confirm that ß-carotene targeted STIM1 and IP3R channels to repair LPS-induced ER stress and mitochondrial disorders. Consistent with the in vitro study, in vito experiments in mice further showed that ß-carotene attenuated LPS-induced ER stress and mitochondrial oxidative damage by inhibiting the expression of STIM1 and ORAI1, and reducing the level of Ca2+ in mouse mammary glands. Therefore, ER stress-mitochondrial oxidative damage mediated by the STIM1-ER-IP3R/GRP75/VDAC1-MCU axis plays an vital role in the development of mastitis. Our results provided novel ideas and therapeutic targets for the prevention and treatment of mastitis.

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.

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.

A high-concentrate diet induces inflammatory injury via regulating Ca2+/CaMKKß-mediated autophagy in mammary gland tissue of dairy cows.

Introduction: Calmodulin-dependent protein kinase ß (CaMKKß) is closely related to Ca2+ concentration. An increase in Ca2+ concentration in the cytoplasm activates CaMKKß, and activated CaMKKß affects the activities of AMPK and mTOR and induces autophagy. A high-concentrate diet leads to Ca2+ disorder in mammary gland tissue. Objectives: Therefore, this study mainly investigated the induction of mammary gland tissue autophagy by a high-concentrate diet and the specific mechanism of lipopolysaccharide (LPS)-induced autophagy in bovine mammary epithelial cells (BMECs). Material and Methods: Twelve mid-lactation Holstein dairy cows were fed with a 40% concentrate diet (LC) and a 60% concentrate diet (HC) for 3 weeks. At the end of the trial, rumen fluid, lacteal vein blood, and mammary gland tissue were collected. The results showed that the HC diet significantly decreased rumen fluid pH, with a pH lower than 5.6 for more than 3 h, indicating successfully induction of subacute rumen acidosis (SARA). The mechanism of LPS-induced autophagy in BMECs was studied in vitro. First, the cells were divided into a Ctrl group and LPS group to study the effects of LPS on the concentration of Ca2+ and autophagy in BMECs. Then, cells were pretreated with an AMPK inhibitor (compound C) or CaMKKß inhibitor (STO-609) to investigate whether the CaMKKß-AMPK signaling pathway is involved in LPS-induced BMEC autophagy. Results: The HC diet increased the concentration of Ca2+ in mammary gland tissue and pro-inflammatory factors in plasma. The HC diet also significantly increased the expression of CaMKKß, AMPK, and autophagy-related proteins, resulting in mammary gland tissue injury. In vitro cell experiments showed that LPS increased intracellular Ca2+ concentration and upregulated protein expression of CaMKKß, AMPK, and autophagy-related proteins. Compound C pretreatment decreased the expression of proteins related to autophagy and inflammation. In addition, STO-609 pretreatment not only reversed LPS-induced BMECs autophagy but also inhibited the protein expression of AMPK, thereby alleviating the inflammatory response in BMECs. These results suggest that inhibition of the Ca2+/CaMKKß-AMPK signaling pathway reduces LPS-induced autophagy, thereby alleviating inflammatory injury of BMECs. Conclusion: Therefore, SARA may increase the expression of CaMKKß by increasing Ca2+ levels and activate autophagy through the AMPK signaling pathway, thereby inducing inflammatory injury in mammary gland tissue 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.

Effects of dietary disodium fumarate supplementation on muscle quality, chemical composition, oxidative stress and lipid metabolism of Hu sheep induced by high concentrate diet.

Long-term feeding of high-concentrate (HC) diet causes the decrease of rumen pH, and induces subacute rumen acidosis (SARA), which results in metabolic disorders in sheep. This not only reduces animal performance, but also increases the risk of oxidative stress and inflammatory reaction. Disodium fumarate can improve the rumen buffering capacity and increase rumen pH. This experiment was conducted to investigate the effects of high concentrate diet on muscle quality, chemical composition, oxidative damage and lipid metabolism of Hu sheep, and the regulating effect of disodium fumarate. The results showed that HC diet induced SARA by reducing rumen pH value, thus causing oxidative stress and lipid metabolism disorder in longissimus lumborum (LL) muscle of Hu sheep, which also reduced meat quality by increasing shear force, drip loss, cooking loss, chewiness and hardness, and reducing the contents of crude fat and crude protein in LL muscle. However, disodium fumarate can improve meat quality of SARA Hu sheep by regulating rumen pH, inhibiting muscle oxidative stress and promoting lipid metabolism.

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.

Disodium Fumarate Alleviates Endoplasmic Reticulum Stress, Mitochondrial Damage, and Oxidative Stress Induced by the High-Concentrate Diet in the Mammary Gland Tissue of Hu Sheep.

The long-term feeding of the high-concentrate diet (HC) reduced rumen pH and induced subacute rumen acidosis (SARA), leading to mammary gland tissue damage among ruminants. Disodium fumarate enhanced rumen bufferation and alleviated a decrease in rumen pH induced by the HC diet. Therefore, the purpose of this study was to investigate whether disodium fumarate could alleviate endoplasmic reticulum (ER) stress, mitochondrial damage, and oxidative stress induced by the high-concentrate diet in the mammary gland tissue of Hu sheep. In this study, 18 Hu sheep in mid-lactation were randomly divided into three groups: one fed with a low-concentrate diet (LC) diet, one fed with a HC diet, and one fed with a HC diet with disodium fumarate (AHC). Each sheep was given an additional 10 g of disodium fumarate/day. The experiment lasted for eight weeks. After the experiment, rumen fluid, blood, and mammary gland tissue were collected. The results show that, compared with the LC diet, the HC diet could reduce rumen pH, and the pH below 5.6 was more than 3 h, and the LPS content of blood and rumen fluid in HC the diet was significantly higher than in the LC diet. This indicates that the HC diet induced SARA in Hu sheep. However, the supplementation of disodium fumarate in the HC diet increased the rumen pH and decreased the content of LPS in blood and rumen fluid. Compared with the LC diet, the HC diet increased Ca2+ content in mammary gland tissue. However, the AHC diet decreased Ca2+ content. The HC diet induced ER stress in mammary gland tissue by increasing the mRNA and protein expressions of GRP78, CHOP, PERK, ATF6, and IRE1α. The HC diet also activated the IP3R-VDAC1-MCU channel and lead to mitochondrial damage by inhibiting mitochondrial fusion and promoting mitochondrial division, while disodium fumarate could alleviate these changes. In addition, disodium fumarate alleviated oxidative stress induced by the HC diet by activating Nrf2 signaling and reducing ROS production in mammary gland tissue. In conclusion, the supplementation of disodium fumarate at a daily dose of 10 g/sheep enhanced rumen bufferation by maintaining the ruminal pH above 6 and reduced LPS concentration in ruminal fluid and blood. This reaction avoided the negative effect observed by non-supplemented sheep that were fed with a high-concentrate diet involving endoplasmic reticulum stress, oxidative stress, and mitochondrial damage in the mammary gland tissue of Hu sheep.

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

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

High concentrate diet induced inflammatory response and tight junction disruption in the mammary gland of dairy cows.

This study aimed to investigate the effect and mechanism of a high concentrate (HC) diet on the inflammatory response and cellular tight junctions (TJs) in the mammary gland of dairy cows. Twelve lactating Holstein dairy cows were randomly assigned into low concentrate (LC) and HC groups (n = 6), which were fed with LC diet and HC diet respectively for 3 weeks. The HC diet lead to subacute ruminant acidosis with a rumen pH < 5.6 more than 3 h daily. The HC diet triggered an inflammatory response with increased levels of inflammatory cytokines in the lacteal vein, upregulated expression of inflammation-related genes, elevated activity of myeloperoxidase, and inflammatory cells infiltration in the mammary gland. Furthermore, the HC diet induced the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways with enhanced phosphorylation ratios of NF-κB P65, inhibitor of NF-κB (IκB), P38 and extracellular signal-regulated kinase 1/2 (ERK1/2) as well as decreased ratios of DNA methylation and chromatin compaction of genes coding for proinflammatory cytokines, which contributed to the upregulation of proinflammatory cytokine expression. The HC diet also destroyed the integrity of TJ with discontinuous and decreased expression levels of zonula occludens-1, Occludin, Claudin-4 and increased expression level of Claudin-1 in the mammary epithelial cells compared with LC group. Conclusively, the HC diet induced the activation of NF-κB and MAPK signaling pathways and epigenetic modifications, promoted the transcription of proinflammatory cytokines, and finally caused inflammatory response and TJ disruption in the mammary gland of dairy cows.

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

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.