Colostrum-derived extracellular vesicles: potential multifunctional nanomedicine for alleviating mastitis.

Bovine mastitis is an infectious disease that causes substantial economic losses to the dairy industry worldwide. Current antibiotic therapy faces issues of antibiotic misuse and antimicrobial resistance, which has aroused concerns for both veterinary and human medicine. Thus, this study explored the potential of Colo EVs (bovine colostrum-derived extracellular vesicles) to address mastitis. Using LPS-induced murine mammary epithelial cells (HC11), mouse monocyte macrophages (RAW 264.7), and a murine mastitis model with BALB/C mice, we evaluated the safety and efficacy of Colo EVs, in vivo and in vitro. Colo EVs had favorable biosafety profiles, promoting cell proliferation and migration without inducing pathological changes after injection into murine mammary glands. In LPS-induced murine mastitis, Colo EVs significantly reduced inflammation, improved inflammatory scores, and preserved tight junction proteins while protecting milk production. Additionally, in vitro experiments demonstrated that Colo EVs downregulated inflammatory cytokine expression, reduced inflammatory markers, and attenuated NF-κB pathway activation. In summary, we inferred that Colo EVs have promise as a therapeutic approach for mastitis treatment, owing to their anti-inflammatory properties, potentially mediated through the NF-κB signaling pathway modulation.

Nuclear Factor Erythroid 2-Related Factor 1 Regulates the Expression of Proteasomal Genes in Ketotic Cows and Protects Mammary Cells Against Free Fatty Acid-Induced Endoplasmic Reticulum Stress.

Ketosis is a common metabolic disorder in high-yielding cows and is characterized by high concentrations of BHB and free fatty acids (FFA). High concentrations of FFA induce endoplasmic reticulum (ER) stress in multiple organs including mammary tissue, and result in reduced milk production and lower milk quality. In non-ruminants, loss of nuclear factor erythroid 2-related factor 1 (NFE2L1) results in ER stress. The physiological functions and molecular mechanisms controlled by NFE2L1 in bovine mammary tissue are poorly understood. Thus, the present study aimed to elucidate the role of the NFE2L1 on proteasomal homeostasis and ER stress in mammary tissue from early-lactation (DIM 6 to 14) healthy cows (CON, blood concentration of BHB <1.2 mM, n = 10) and cows with clinical ketosis (CK blood concentration of BHB >3 mM, n = 10). Compared with CON, serum concentration of glucose was lower due to CK, while serum concentrations of BHB and FFA were greater. Protein and mRNA abundance of NFE2L1 along with abundance of proteasomal subunits (PSMD1, PSMD14, PSMA1, PSMB1, and PSMB5 genes and PSMB4 and PSMB6 proteins) were lower in cows with CK, indicating that expression of NFE2L1 and proteasomal homeostasis was impaired by ketosis. In vitro, primary bovine mammary epithelial cells were exposed to various concentrations of FFA (0, 0.3, 0.6, or 1.2 mM). Compared with the 0 mM FFA, the ratio of phosphorylated (p)-protein kinase R-like ER kinase (PERK)/PERK along with the expression of inositol-requiring enzyme 1 (IRE1) α, activating transcription factor 6 (ATF6), glucose regulated protein 78 (GRP78), and C/EBP homologous protein (CHOP) was higher with 1.2 mM FFA. A similar response was observed for ER stress-associated genes (CHOP, GRP78, and XBP1) indicating that high concentrations of FFA induced ER stress. In line with in vivo results, 1.2 mM FFA downregulated the protein and mRNA abundance of NFE2L1, the abundance of PSMB6 protein, and PSM genes (PSMC1, PSMC3, and PSMD1), and increased the accumulation of ubiquitin. This suggested a marked negative effect of high FFA on NFE2L1 and proteasomal homeostasis. Silencing of NFE2L1 triggered upregulation of ER stress-associated genes as well as protein abundance of GRP78 and CHOP. Further, compared with CON-siRNA, the abundance of PSM genes was downregulated in the NFE2L1-siRNA group. In contrast, abundance of markers of ER stress and PSM genes and proteins indicated that overexpression of NFE2L1 relieved the FFA-induced ER stress and improved 26S proteasome homeostasis. Our data suggested that the mammary gland experiences ER stress during ketosis partly due to disruption of proteasomal homeostasis from the excess FFA. As such, NFE2L1 could represent a target for potential therapeutic applications in the field to alleviate the accumulation of malformed proteins that may impair the long-term lactogenic capacity of the udder.

Role of diacylglycerol O-acyltransferase 1 (DGAT1) in lipolysis and autophagy of adipose tissue from ketotic dairy cows.

High-yielding dairy cows in early lactation often encounter difficulties in meeting the energy requirements essential for maintaining milk production. This is primarily attributed to insufficient dry matter intake, which consequently leads to sustained lipolysis of adipose tissue. Fatty acids released by lipolysis can disrupt metabolic homeostasis. Autophagy, an adaptive response to intracellular environmental changes, is considered a crucial mechanism for regulating lipid metabolism and maintaining a proper cellular energy status. Despite its close relationship with aberrant lipid metabolism and cytolipotoxicity in animal models of metabolic disorders, the precise function of diacylglycerol o-acyltransferase 1 (DGAT1) in bovine adipose tissue during periods of negative energy balance is not fully understood, particularly regarding its involvement in lipolysis and autophagy. The objective of the present study was to assess the effect of DGAT1 on both lipolysis and autophagy in bovine adipose tissue and isolated adipocytes. Adipose tissue and blood samples were collected from cows diagnosed as clinically ketotic (n = 15) or healthy (n = 15) following a veterinary evaluation based on clinical symptoms and serum concentrations of BHB, which were 3.19 mM (interquartile range = 0.20) and 0.50 mM (interquartile range = 0.06), respectively. Protein abundance of DGAT1 and phosphorylation levels of unc-51-like kinase 1 (ULK1), were greater in adipose tissue from cows with ketosis, whereas phosphorylation levels of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) were lower. Furthermore, when adipocytes isolated from the harvested adipose tissue of 15 healthy cows were transfected with DGAT1 overexpression adenovirus or DGAT1 small interfering RNA followed by exposure to epinephrine (EPI), it led to greater ratios and protein abundance of phosphorylated hormone-sensitive triglyceride lipase (LIPE) to total LIPE and adipose triglyceride lipase (ATGL), while inhibiting the protein phosphorylation levels of ULK1, PI3K, AKT, and mTOR. Overexpression of DGAT1 in EPI-treated adipocytes reduced lipolysis and autophagy, whereas silencing DGAT1 further exacerbated EPI-induced lipolysis and autophagy. Taken together, these findings indicate that upregulation of DGAT1 may function as an adaptive response to suppress adipocytes lipolysis, highlighting the significance of maintaining metabolic homeostasis in dairy cows during periods of negative energy balance.

Caveolin 1 in bovine liver is associated with fatty acid-induced lipid accumulation and the ER unfolded protein response: role in fatty liver development.

Disruption of endoplasmic reticulum (ER) homeostasis, i.e., ER stress, is intrinsically linked with lipid metabolism disorders in dairy cows. Caveolin 1 (CAV1) is a ubiquitously-expressed membrane-associated scaffolding protein involved in regulating the secretory pathway within the ER. Whether inhibiting the activity of CAV1 affects the ER and its potential role in hepatic lipid deposition in dairy cows is unknown. Biopsies of liver tissue from Holstein cows (days in milk: median = 13 d, range = 5 to 21) diagnosed as healthy (n = 6, hepatic TAG levels < 1%, milk production: median = 38.9 kg/day, Interquartile range = 38.0 and 40.8) or suffering from fatty liver (n = 6, hepatic TAG levels > 5%, milk production: median = 36.6 kg/day, Interquartile range = 35.7 and 38.1) revealed that fatty liver was associated with lower abundance of CAV1 gene and protein, higher phosphorylation (p) levels of PERK and IRE1α, and increased abundance of ATF6, GRP78, CHOP protein, and several unfolded protein response (UPR) genes (ATF4, sXBP1, and GRP78). Proteins related to de novo fatty acid synthesis, including ACC1, SREBP-1c, PPARγ, and downstream targets genes of SREBP1 (ACACA and FASN) also had greater abundance. This in vivo analysis highlighted a mechanistic link between CAV1 protein abundance, ER stress, and lipid metabolism during fatty liver. A mechanistic study was then performed in vitro with primary hepatocytes isolated from 5 healthy calves (weight, 40-45 kg; 1 d old). Initially, hepatocytes were treated with FFA (1.2 mM) for 1, 3, 6, or 12 h. FFA treatment reduced CAV1 protein abundance linearly while it reduced abundance of ER stress-related proteins, p-IRE1α, p-PERK, GRP78, ATF6, and CHOP. Proteins related to de novo fatty acid synthesis (ACC1, SREBP-1c, PPARγ) also increased linearly, and lipid droplets accumulated progressively over time following FFA treatment. Subsequently, to assess the role of CAV1 in FFA-induced ER stress and de novo fatty acid synthesis, hepatocytes were transfected with pCMV-CAV1 (cattle)-3 × FLAG-Neo (pc-CAV1) plasmid to overexpress CAV1 or with siRNA to silence CAV1 (siCAV1) transcription. Overexpression of CAV1 alleviated ER stress by reducing levels of p-PERK and p-IRE1α, as well as the protein abundance of ATF6, GRP78, CHOP, and several UPR genes (GRP78, ATF4, and sXBP1). Similarly, CAV1 overexpression decreased protein abundance of ACC1, SREBP-1c, PPARγ, and downstream targets genes of SREBP1 (ACACA and FASN). Conversely, silencing CAV1 exacerbated FFA-induced ER stress and de novo fatty acid synthesis. Considering the negative role of FFA-induced ER stress on lipid accumulation in hepatocytes, a second in vitro experiment involved hepatocytes treated with 0.5 µg/mL tunicamycin (TM, a typical ER stress inducer) for 24 h with or without overexpressing CAV1 (pc-CAV1). Overexpressing caveolin 1 reversed TM-induced increases in mRNA and protein associated with ER stress and de novo fatty acid synthesis. Furthermore, use of hepatocytes transfected with pc-CAV1 for 48 h and subjected to co-immunoprecipitation revealed that CAV1 interacts with IRE1α and ATF6. Overall, the data suggest that CAV1 may help reduce hepatic ER stress and mitigate fatty acid synthesis by binding to and inhibiting IRE1α and ATF6 signaling.

Diacylglycerol O-acyltransferase (DGAT) isoforms play a role in peridroplet mitochondrial fatty acid metabolism in bovine liver.

Hepatocellular lipid accumulation characterizes fatty liver in dairy cows. Lipid droplets (LD), specialized organelles that store lipids and maintain cellular lipid homeostasis, are responsible for the ectopic storage of lipids associated with several metabolic disorders. In recent years, non-ruminant studies have reported that LD-mitochondria interactions play an important role in lipid metabolism. Due to the role of diacylglycerol acyltransferase isoforms (DGAT1 and DGAT2) in LD synthesis, we explored mechanisms of mitochondrial fatty acid transport in ketotic cows using liver biopsies and isolated primary hepatocytes. Compared with healthy cows, cows with fatty liver had massive accumulation of LD and high protein expression of the triglyceride (TAG) synthesis-related enzymes DGAT1 and DGAT2, LD synthesis-related proteins perilipin 2 (PLIN2) and perilipin 5 (PLIN5), and the mitochondrial fragmentation-related proteins dynamin-related protein 1 (DRP1) and fission 1 (FIS1). In contrast, factors associated with fatty acid oxidation, mitochondrial fusion and mitochondrial electron transport chain complex were lower compared with those in the healthy cows. In addition, transmission electron microscopy revealed significant contacts between LD-mitochondria in liver tissue from cows with fatty liver. Compared with isolated cytoplasmic mitochondria, expression of carnitine palmitoyl transferase 1A (CPT1A) and DRP1 was lower, but mitofusin 2 (MFN2) and mitochondrial electron transport chain complex was greater in isolated peridroplet mitochondria from hepatic tissue of cows with fatty liver. In vitro data indicated that exogenous free fatty acids (FFA) induced hepatocyte LD synthesis and mitochondrial dynamics consistent with in vivo results. Furthermore, DGAT2 inhibitor treatment attenuated the FFA-induced upregulation of PLIN2 and PLIN5 and rescued the impairment of mitochondrial dynamics. Inhibition of DGAT2 also restored mitochondrial membrane potential and reduced hepatocyte reactive oxygen species production. The present in vivo and in vitro results indicated there are functional differences among different types of mitochondria in the liver tissue of dairy cows with ketosis. Activity of DGAT2 may play a key role in maintaining liver mitochondrial function and lipid homeostasis in dairy cows during the transition period.

Role of hypoxia-inducible-factor-1α (HIF-1α) in ferroptosis of adipose tissue during ketosis.

Postpartum cows experience lipolysis in adipose tissue due to negative energy balance (NEB), and accumulation of free fatty acids (FFA) leads to metabolic stress in adipose tissue. Ferroptosis is a type of cell death triggered by excessive buildup of iron-dependent lipid peroxides, which is involved in the occurrence and development of various metabolic diseases in nonruminants. However, it is still unclear whether ferroptosis occurs in the adipose tissue of ketotic cows and the regulatory mechanisms behind ferroptosis. Despite multiple studies demonstrating the significant involvement of hypoxia-inducible-factor-1α (HIF-1α) in regulating cellular dysfunction, its specific function in adipose tissue of ketotic dairy cows remains uncertain, particularly its regulation of oxidative stress and ferroptosis. The study aimed to explore the impact of HIF-1α on oxidative stress and ferroptosis in bovine subcutaneous adipose tissue and isolated adipocytes. The adipose tissue of clinical ketosis cows (n = 15) with a serum BHB concentration of 3.13 mM (interquartile range = 0.14) and healthy cows (n = 15) with a serum BHB concentration of and 0.58 mM (interquartile range = 0.13) was collected. The results showed that the concentrations of lipid peroxidation malondialdehyde (MDA), reactive oxygen species (ROS), Fe2+ and total iron were increased in adipose tissue of cows with ketosis, while the contents of glutathione (GSH) were reduced. Furthermore, the protein levels of HIF-1α, heme oxygenase 1 (HMOX1), catalase (CAT), superoxide dismutase 1 (SOD1), acyl-CoA synthetase 4 (ACSL4), and nuclear factor erythroid-derived 2-like 2 (NFE2L2) exhibited higher abundance in adipose tissue obtained from cows with ketosis, whereas the protein abundance of solute carrier family 7 member 11 (SLC7A11), glutamate cysteine ligase catalytic subunit (GCLC), kelch-like ECH-associated protein 1 (KEAP1), glutamate cysteine ligase regulatory subunit (GCLM) and glutathione peroxidase 4 (GPX4) were lower. To simulate the ferroptosis state of adipose tissue in ketotic cows, primary bovine adipocytes were isolated from the adipose tissue of healthy cows and cultured with erastin to construct ferroptosis model. Adipocytes were cultured with either an adenovirus overexpressing HIF-1α or small interfering RNA targeting HIF-for 48 h, followed by exposure to erastin (1 µM) for 24 h. Treatment with erastin led to higher protein abundance of CAT, SOD1, NFE2L2 and HMOX1, while it inhibited the protein expression levels of GCLC, SLC7A11, GCLM, GPX4 and KEAP1. Furthermore, erastin treatment elevated the levels of ROS, MDA, Fe2+, total iron and reduced the content of GSH. The overexpression of HIF-1α reversed the erastin-induced decreases in the protein abundance of GPX4 and SLC7A11, as well as the levels of MDA, ROS, Fe2+ and total iron, while significantly increasing protein abundance and content of CAT, SOD1, NFE2L2, HMOX1, GCLC, GCLM, GPX4, SLC7A11 and GSH. Conversely, the silencing of HIF-1α further exacerbated the erastin-induced levels of MDA, ROS, Fe2+ and total iron, while inhibiting the upregulation of SOD1, CAT, NFE2L2 and HMOX1. Collectively, these findings suggest that activation of HIF-1α may function as an adaptive mechanism to mitigate ferroptosis and alleviate oxidative stress in adipose tissue.

Fatty acids promote M1 polarization of monocyte-derived macrophages in healthy or ketotic dairy cows and a bovine macrophage cell line by impairing mTOR-mediated autophagy.

Excessive concentrations of free fatty acids (FFA) are the main factors causing immune dysfunction and inflammation in dairy cows with ketosis. Polarization of macrophages (the process of macrophages freely switching from one phenotype to another) into M1 or M2 phenotypes is an important event during inflammation induced by environmental stimuli. In nonruminants, mammalian target of rapamycin (mTOR)-mediated autophagy (a major waste degradation process) regulates macrophage polarization. Thus, our objective was to unravel the role of mTOR-mediated autophagy on macrophage polarization in ketotic dairy cows. We performed 4 experiments: (1) In vitro differentiated monocyte-derived macrophages from healthy dairy cows or dairy cows with clinical ketosis (CK) were treated for 24 h with 100 ng/mL LPS and 100 ng/mL IFN-γ or with 10 ng/mL IL4 and 10 ng/mL IL10; (2) Immortalized bovine macrophages were treated for 24 h with 0, 0.3, 0.6, or 1.2 mM FFA, LPS, and IFN-γ, or with IL4 and IL10; (3) Macrophages were pretreated with 2 µM 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine (MHY1485) for 30 min before treatment with LPS and IFN-γ or IL4 and IL10; (4) Macrophages were pretreated with 100 nM rapamycin (RAPA) for 2 h before treatment with LPS and IFN-γ or IL4 and IL10. Compared with healthy cows, cows with CK had a greater mean fluorescence intensity (MFI) of CD86+, but lower MFI of CD206+ and lower number of autophagosomes and autolysosomes in macrophages. Exogenous FFA treatment upregulated protein abundance of inducible nitric oxide synthase (iNOS) and the MFI of CD86, whereas it downregulated the protein abundance of arginase 1 and the MFI of CD206. In addition, FFA increased the p-p65/p65 protein abundance and tumor necrosis factor α, IL1B, and IL6 mRNA abundance, but decreased LC3-phosphatidylethanolamine conjugate protein abundance and the number of autophagosomes and autolysosomes number. Pretreatment with MHY1485 promoted macrophage M1 polarization and inhibited macrophage M2 polarization via decreased mTOR-mediated autophagy. Activation of mTOR-mediated autophagy by pretreatment with RAPA attenuated the upregulation of inflammation in M1 macrophages that was induced by FFA. These data revealed that high concentrations of FFA promote macrophage M1 polarization in ketotic dairy cows by impairing mTOR-mediated autophagy.

Regulation of cholesterol metabolism during high fatty acid-induced lipid deposition in calf hepatocytes.

Cholesterol in the circulation is partly driven by changes in feed intake, but aspects of cholesterol metabolism during development of fatty liver are not well known. The objective of this study was to investigate mechanisms of cholesterol metabolism in calf hepatocytes challenged with high concentrations of fatty acids (FA). To address mechanistic insights regarding cholesterol metabolism, liver samples were collected from healthy control dairy cows (n = 6; 7-13 d in milk) and cows with fatty liver (n = 6; 7-11 d in milk). In vitro, hepatocytes isolated from 3 healthy female calves (1 d old) were challenged with or without a mix of 1.2 mM FA to induce metabolic stress. In addition, hepatocytes were processed with 10 µmol/L of the cholesterol synthesis inhibitor simvastatin or 6 µmol/L of the cholesterol intracellular transport inhibitor U18666A with or without the 1.2 mM FA mix. To evaluate the role of cholesterol addition, hepatocytes were treated with 0.147 mg/mL methyl-ß-cyclodextrin (MßCD + FA) or 0.147 mg/mL MßCD with or without 10 and 100 µmol/L cholesterol before incubation with FA (CHO10 + FA and CHO100 + FA). In vivo data from liver biopsies were analyzed by 2-tailed unpaired Student's t-test. Data from in vitro calf hepatocytes were analyzed by one-way ANOVA. Compared with healthy cows, blood plasma total cholesterol and plasma low-density lipoprotein cholesterol content in cows with fatty liver was markedly lower, whereas the hepatic total cholesterol content did not differ. In contrast, compared with healthy controls, the triacylglycerol content in the liver and the content of FA, ß-hydroxybutyrate, and aspartate aminotransferase in the plasma of cows with fatty liver were greater. The results revealed that both fatty liver in vivo and challenge of calf hepatocytes with 1.2 mM FA in vitro led to greater mRNA and protein abundance of sterol regulatory element binding transcription factor 1 (SREBF1) and fatty acid synthase (FASN). In contrast, mRNA and protein abundance of sterol regulatory element binding transcription factor 2 (SREBF2), acyl coenzyme A-cholesterol acyltransferase, and ATP-binding cassette subfamily A member 1 (ABCA1) were lower. Compared with the FA group, the cholesterol synthesis inhibitor simvastatin led to greater protein abundance of microsomal triglyceride transfer protein and mRNA abundance of SREBF2, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), ACAT2, and lower ABCA1 and FASN protein abundance. In contrast, compared with the FA group, the cholesterol intracellular transport inhibitor U18666A + FA led to greater total cholesterol concentration and greater protein and mRNA abundance of FASN. Compared with the MßCD + FA group, the addition of 10 µmol/L cholesterol led to greater concentration of cholesteryl ester and excretion of apolipoprotein B100, and greater protein and mRNA abundance of ABCA1 and microsomal triglyceride transfer protein, and lower concentration of malondialdehyde. Overall, a reduction in cholesterol synthesis promoted FA metabolism in hepatocytes likely to relieve the oxidative stress caused by the high FA load. The data suggest that maintenance of normal cholesterol synthesis promotes very low-density lipoprotein excretion and can reduce lipid accumulation and oxidative stress in dairy cows that experience fatty liver.

Sirtuin 3 relieves inflammatory responses elicited by lipopolysaccharide via the PGC1α-NFκB pathway in bovine mammary epithelial cells.

Excessive inflammation in bovine mammary endothelial cells (BMEC) due to mastitis leads to disease progression and eventual culling of cattle. Sirtuin 3 (SIRT3), a mitochondrial deacetylase, downregulates pro-inflammatory cytokines in BMEC exposed to high concentrations of nonesterified fatty acids by blunting nuclear factor-κB (NFκB) signaling. In nonruminants, SIRT3 is under the control of PGC1α, a transcriptional cofactor. Specific aims were to study (1) the effect of SIRT3 on inflammatory responses of lipopolysaccharide (LPS)-challenged bovine mammary epithelial cells (bovine mammary alveolar cells-T, MAC-T) models, and (2) the role of PGC1α in the attenuation of NFκB signaling via SIRT3. To address these objectives, first, MAC-T cells were incubated in triplicate with 0, 50, 100, 150, or 200 µg/mL LPS (derived from Escherichia coli O55:B5) for 12 h with or without a 2-h incubation of the NFκB inhibitor ammonium pyrrolidine dithiocarbamate (APDC, 10 µM). Second, SIRT3 was overexpressed using adenoviral expression (Ad-SIRT3) at different multiplicity of infection (MOI) for 6 h followed by a 12 h incubation with 150 µg/mL LPS. Third, cells were treated with the PGC1α agonist ZLN005 (10 µg/mL) for 24 h and then challenged with 150 µg/mL LPS for 12 h. Fourth, cells were initially treated with the PGC1α inhibitor SR-18292 (100 µM) for 6 h followed by a 6-h culture with or without 50 MOI Ad-SIRT3 and a challenge with 150 µg/mL LPS for 12 h. Data were analyzed using one-way ANOVA with subsequent Bonferroni correction. Linear and quadratic contrasts were used to determine dose-responses to LPS. There were linear and quadratic effects of LPS dosage on cell viability. Incubation with 150 and 200 µg/mL LPS for 12 h decreased cell viability to 78.6 and 34.9%, respectively. Compared with controls, expression of IL1B, IL6, and TNFA was upregulated by 5.2-, 5.9-, and 2.7-fold with 150 µg/mL LPS; concentrations of IL-1ß, IL-6, and tumor necrosis factor-α (TNF-α) in cell medium also increased. Compared with the LPS group, LPS+APDC increased cell viability and reversed the upregulation of IL1B, IL6, and TNFA expression. However, mRNA and protein abundance of SIRT3 decreased linearly with increasing LPS dose. Ad-SIRT3 infection (50 MOI) reduced IL1B, IL6, and TNFA expression and also their concentrations in cell medium, and decreased pNFκB P65/NFκB P65 ratio and nuclear abundance of NFκB P65. The PGC1α agonist increased SIRT3 expression, whereas it decreased cytokine expression, pNFκB P65/NFκB P65 ratio, and prevented NFκB P65 nuclear translocation. Contrary to the agonist, the PGC1α inhibitor had opposite effects, and elevated the concentrations of IL-1ß, IL-6, and TNF-α in cell medium. Overall, data suggested that SIRT3 activity could attenuate LPS-induced inflammatory responses in mammary cells via alterations in the PGC1α-NFκB pathway. As such, there may be potential benefits for targeting SIRT3 in vivo to help prevent or alleviate negative effects of mastitis.

Integrated meta-omics analyses reveal a role of ruminal microorganisms in ketone body accumulation and ketosis in lactating dairy cows.

The extent to which a nutrition-related disorder such as ketosis alters the ruminal microbiota or whether microbiota composition is related to ketosis and potential associations with host metabolism is unknown. We aimed to evaluate variations occurring in the ruminal microbiota of ketotic and nonketotic cows in the early postpartum period, and how those changes may affect the risk of developing the disease. Data on milk yield, dry matter intake (DMI), body condition score, and blood ß-hydroxybutyrate (BHB) concentrations at 21 d postpartum were used to select 27 cows, which were assigned (n = 9 per group) to a clinical ketotic (CK, 4.10 ± 0.72 mmol BHB/L, DMI 11.61 ± 0.49 kg/d, ruminal pH 7.55 ± 0.07), subclinical ketotic (SK, 1.36 ± 0.12 mmol BHB/L, DMI 15.24 ± 0.34 kg/d, ruminal pH 7.58 ± 0.08), or control (NK, 0.88 ± 0.14 mmol BHB/L, DMI 16.74 ± 0.67/d, ruminal pH 7.61 ± 0.03) group. Cows averaged 3.6 ± 0.5 lactations and a body condition score of 3.11 ± 0.34 at the time of sampling. After blood serum collection for metabolomics analysis (1H nuclear magnetic resonance spectra), 150 mL of ruminal digesta was collected from each cow using an esophageal tube, paired-end (2 × 300 bp) sequencing of isolated DNA from ruminal digesta was performed via Illumina MiSeq, and sequencing data were analyzed using QIIME2 (v 2020.6) to measure the ruminal microbiota composition and relative abundance. Spearman correlation coefficients were used to evaluate relationships between relative abundance of bacterial genera and concentrations of serum metabolites. There were more than 200 genera, with approximately 30 being significant between NK and CK cows. Succinivibrionaceae UCG 1 taxa decreased in CK compared with NK cows. Christensenellaceae (Spearman correlation coefficient = 0.6), Ruminococcaceae (Spearman correlation coefficient = 0.6), Lachnospiraceae (Spearman correlation coefficient = 0.5), and Prevotellaceae (Spearman correlation coefficient = 0.6) genera were more abundant in the CK group and were highly positively correlated with plasma BHB. Metagenomic analysis indicated a high abundance of predicted functions related to metabolism (37.7%), genetic information processing (33.4%), and Brite hierarchies (16.3%) in the CK group. The 2 most important metabolic pathways for butyrate and propionate production were enriched in CK cows, suggesting increased production of acetyl coenzyme A and butyrate and decreased production of propionate. Overall, the combined data suggested that microbial populations may be related to ketosis by affecting short-chain fatty acid metabolism and BHB accumulation even in cows with adequate feed intake in the early postpartum period.

Store-operated Ca2+ entry-sensitive glycolysis regulates neutrophil adhesion and phagocytosis in dairy cows with subclinical hypocalcemia.

Hypocalcemia in dairy cows is associated with a decrease of neutrophil adhesion and phagocytosis, an effect driven partly by changes in the expression of store-operated Ca2+ entry (SOCE)-related molecules. It is well established in nonruminants that neutrophils obtain the energy required for immune function through glycolysis. Whether glycolysis plays a role in the acquisition of energy by neutrophils during hypocalcemia in dairy cows is unknown. To address this relationship, we performed a cohort study and then a clinical trial. Neutrophils were isolated at 2 d postcalving from lactating Holstein dairy cows (average 2.83 ± 0.42 lactations, n = 6) diagnosed as clinically healthy (CON) or with plasma concentrations of Ca2+ <2.0 mmol/L as a criterion for diagnosing subclinical hypocalcemia (HYP, average 2.83 ± 0.42 lactations, n = 6). In the first experiment, neutrophils were isolated from blood of CON and HYP cows and used to analyze aspects of adhesion and phagocytosis function through quantitative reverse-transcription PCR along with confocal laser scanning microscopy, mRNA expression of the glycolysis-related gene hexokinase 2 (HKII), and components of the SOCE moiety ORAI calcium release-activated calcium modulator 1 (ORAI1, ORAI2, ORAI3, stromal interaction molecule 1 [STIM1], and STIM2). Results showed that adhesion and phagocytosis function were reduced in HYP cows. The mRNA expression of adhesion-related syndecan-4 (SDC4), integrin ß9 (ITGA9), and integrin ß3 (ITGB3) and phagocytosis-related molecules complement component 1 R subcomponent (C1R), CD36, tubulinß1 (TUBB1) were significantly decreased in the HYP group. In the second experiment, to address how glycolysis affects neutrophil adhesion and phagocytosis, neutrophils isolated from CON and HYP cows were treated with 2 µM HKII inhibitor benserazide-d3 or 1 µM fructose-bisphosphatase 1 (FBP1) inhibitor MB05032 for 1 h. Results revealed that the HKII inhibitor benserazide-d3 reduced phagocytosis and the mRNA abundance of ITGA9, and CD36 in the HYP group. The FBP1 inhibitor MB05032 increased adhesion and phagocytosis and increased mRNA abundance of HKII, ITGA9, and CD36 in the HYP group. Finally, to investigate the mechanism whereby SOCE-sensitive glycolysis affects neutrophil adhesion and phagocytosis, isolated neutrophils were treated with 1 µM SOCE activator thapsigargin or 50 µM inhibitor 2-APB for 1 h. Results showed that thapsigargin increased mRNA abundance of HKII, ITGA9, and CD36, and increased adhesion and phagocytosis in the HYP group. In contrast, 2-APB decreased mRNA abundance of HKII and both adhesion and phagocytosis of neutrophils in the CON group. Overall, the data indicated that SOCE-sensitive intracellular Ca2+ levels affect glycolysis and help regulate adhesion and phagocytosis of neutrophils during hypocalcemia in dairy cows.

Prediction and Verification of Curcumin as a Potential Drug for Inhibition of PDCoV Replication in LLC-PK1 Cells.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus (CoV) that causes lethal watery diarrhea in neonatal pigs and poses economic and public health burdens. Currently, there are no effective antiviral agents against PDCoV. Curcumin is the active ingredient extracted from the rhizome of turmeric, which has a potential pharmacological value because it exhibits antiviral properties against several viruses. Here, we described the antiviral effect of curcumin against PDCoV. At first, the potential relationships between the active ingredients and the diarrhea-related targets were predicted through a network pharmacology analysis. Twenty-three nodes and 38 edges were obtained using a PPI analysis of eight compound-targets. The action target genes were closely related to the inflammatory and immune related signaling pathways, such as the TNF signaling pathway, Jak-STAT signaling pathway, and so on. Moreover, IL-6, NR3C2, BCHE and PTGS2 were identified as the most likely targets of curcumin by binding energy and 3D protein-ligand complex analysis. Furthermore, curcumin inhibited PDCoV replication in LLC-PK1 cells at the time of infection in a dose-dependent way. In poly (I:C) pretreated LLC-PK1 cells, PDCoV reduced IFN-ß production via the RIG-I pathway to evade the host's antiviral innate immune response. Meanwhile, curcumin inhibited PDCoV-induced IFN-ß secretion by inhibiting the RIG-I pathway and reduced inflammation by inhibiting IRF3 or NF-κB protein expression. Our study provides a potential strategy for the use of curcumin in preventing diarrhea caused by PDCoV in piglets.

Ethyl Gallate Inhibits Bovine Viral Diarrhea Virus by Promoting IFITM3 Expression, Lysosomal Acidification and Protease Activity.

Bovine viral diarrhea virus (BVDV) is a highly contagious viral disease which causes economic losses to the cattle industry. Ethyl gallate (EG) is a phenolic acid derivative which has various potentials to modulate the host response to pathogens, such as via antioxidant activity, antibacterial activity, inhibition of the production of cell adhesion factors, and so on. This study aimed to evaluate if EG influences BVDV infection in Madin-Darby Bovine Kidney (MDBK) cells, and to understand the antiviral mechanism. Data indicated that EG effectively inhibited BVDV infection by co-treatment and post-treatment in MDBK cells with noncytotoxic doses. In addition, EG suppressed BVDV infection at an early stage of the viral life cycle by blocking entry and replication steps but not viral attachment and release. Moreover, EG strongly inhibited BVDV infection by promoting interferon-induced transmembrane protein 3 (IFITM3) expression, which localized to the cytoplasm. The protein level of cathepsin B was significantly reduced by BVDV infection, whereas with treatment with EG, it was significantly enhanced. The fluorescence intensities of acridine orange (AO) staining were significantly decreased in BVDV-infected cells but increased in EG-treated cells. Finally, Western blot and immunofluorescence analyses demonstrated that EG treatment significantly enhanced the protein levels of autophagy markers LC3 and p62. Chloroquine (CQ) significantly increased IFITM3 expression, and Rapamycin significantly decreased it. Thus, EG may regulate IFITM3 expression through autophagy. Our results showed that EG could have a solid antiviral activity on BVDV replication in MDBK cells via increased IFITM3 expression, lysosomal acidification, protease activity, and regulated autophagy. EG might have value for further development as an antiviral agent.

Chitosan and anisodamine enhance the immersion immune efficacy of inactivated Elizabethkingia miricola vaccine in black spotted frogs.

Black spotted frogs have rich nutrition and delicious meat, and its market consumption has increased year by year. However, outbreaks of the diseases have caused huge losses to the breeding industry. The crooked head disease caused by Elizabethkingia miricola (E. miricola) is highly contagious and lethal, and there is no effective treatment method. Vaccination is the most promising strategy to prevent infectious diseases. Immersion vaccination has attracted many researchers because of its simplicity of operation in preventing infectious diseases. In addition, immersion vaccines can be more effective when used with adjuvants. In this study, we prepared inactivated E. miricola with 0.3% formaldehyde, and the black spotted frogs were vaccinated by soaking in inactivated E. miricola vaccine, anisodamine + vaccine mixture, ß-glucan + vaccine mixture, chitosan + vaccine mixture for 60 min. PBS was used as a control. After being challenged by E. miricola, the survival rate of anisodamine + vaccine (57%) and chitosan + vaccine group (63%) was significantly higher than that of the control group (17%). By analyzing pathological sections, we found that the chitosan + vaccine and anisodamine + vaccine groups protected the brain, eye, liver and kidney tissues of the black spotted frogs compared to the control group, which was consistent with the trend of survival rate. In addition, chitosan + vaccine and anisodamine + vaccine groups had better effects on LZM, TSOD and C3 in serum than control group. Meanwhile, the numbers of the percentage of leukocytes/haemocytes in the peripheral blood of immunized black spotted frogs increased. The anisodamine + vaccine group (5.3%) and chitosan + vaccine (5.38%) group were significantly higher than the blank control group (2.24%), which indicate that the two groups induced a more significant immune response and were more resistant to bacterial invasion. The tissue bacterial loads in liver, brain, kidney and eye were significantly lower in the anisodamine + vaccine and chitosan + vaccine groups than that of the control group. This study explored and demonstrated the good efficiency of chitosan and anisodamine as adjuvants for immunization by immersion and provided a reference for improving the efficiency of immunization by immersion.

Dietary supplementation with nanoparticle CMCS-20a enhances the resistance to GCRV infection in grass carp (Ctenopharyngodon idella).

Combination of antimicrobial proteins and nanomaterials provides a platform for the development of immunopotentiators. Oral administration of immunopotentiators can significantly enhance the immunity of organisms, which provides ideas for disease prevention. In this study, we confirmed that nanoparticles CMCS-20a can efficiently prevent grass carp reovirus (GCRV) infection. Firstly, we verified that CiCXCL20a is involved in the immune responses post GCRV challenge in vivo and alleviates the cell death post GCRV challenge in CIK cells. Then, we prepared nanoparticles CMCS-20a using carboxymethyl chitosan (CMCS) loaded with grass carp (Ctenopharyngodon idella) CXCL20a (CiCXCL20a). Meanwhile, we confirmed nanoparticles CMCS-20a can alleviate the degradation in intestine. Subsequently, we added it to the feed by low temperature vacuum drying method and high temperature spray drying method, respectively. Grass carp were oral administration for 28 days and challenged by GCRV. Low temperature vacuum drying group (LD-CMCS-20a) significantly improve grass carp survival rate, but not high temperature spray drying group (HD-CMCS-20a). To reveal the mechanisms, we investigated the serum biochemical indexes, intestinal mucus barrier, immune gene regulation and tissue damage. The complement component 3 content, lysozyme and total superoxide dismutase activities are highest in LD-CMCS-20a group. LD-CMCS-20a effectively attenuates the damage of GCRV to the number of intestinal villous goblet cells and mucin thickness. LD-CMCS-20a effectively regulates mRNA expressions of immune genes (IFN1, Mx2, Gig1 and IgM) in spleen and head kidney tissues. In addition, LD-CMCS-20a obviously alleviate tissue lesions and viral load in spleen. These results indicated that the nanoparticles CMCS-20a can enhance the disease resistance of fish by improving their immunity, which provides a new perspective for fish to prevent viral infections.

Role of sortilin 1 (SORT1) on fatty acid-mediated cholesterol metabolism in primary calf hepatocytes.

Ketosis is a common metabolic disorder in peripartal dairy cows that is caused by excessive mobilization of fat and incomplete hepatic metabolism of fatty acids (FFA). Recent data in nonruminant models revealed that sortilin 1 (SORT1) is involved in a variety of lipid metabolism-related diseases. It plays important roles in the regulation of triglyceride (TAG) and total cholesterol (TC) levels. In this study, we first used liver biopsies from healthy cows (serum ß-hydroxybutyrate concentration <0.6 mM) and cows diagnosed with clinical ketosis (serum ß-hydroxybutyrate concentration >3.0 mM) to assess alterations in cholesterol synthesis, transport, and excretion. Then, to assess mechanistic links between SORT1 and fatty acid-mediated cholesterol metabolism, hepatocytes isolated from 4 healthy female calves (1 d old, 35-45 kg) were challenged with or without a mixture of free fatty acids (FFA; 1.2 mM) to induce metabolic stress. Hepatocytes were then treated with empty adenovirus vectors (with green fluorescent protein; Ad-GFP) or with SORT1-overexpressing adenovirus (Ad-SORT1) for 6 h or with SORT1 inhibitor (SORT1i) for 2 h, followed by a challenge with (Ad-GFP+FFA, Ad-SORT1+FFA, or SORT1i+FFA) or without (Ad-GFP, Ad-SORT1, or SORT1i) 1.2 mM FFA mixture for 12 h. Data analysis of calf hepatocyte treatment comparisons were assessed by 2-way ANOVA, and multiplicity for each experiment was adjusted using the Bonferroni procedure. Expression levels of factors related to cholesterol synthesis, transport, and excretion in liver tissue of cows with ketosis was lower. Hepatocytes challenged with FFA had lower concentrations of TC and mRNA and protein abundances of sterol regulatory element-binding protein 2 (SREBF2), acetyl acyl coenzyme A-cholesterol acyltransferase 2 (ACAT2), ATP-binding cassette transporter A1 (ABCA1), ABC subfamily G member 5 (ABCG5), and ABC subfamily G member 8 (ABCG8). Compared with FFA challenge alone, SORT1i + FFA led to greater protein abundance of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR), ACAT2, and ABCG5, and greater mRNA abundance of ABCG5. Compared with FFA challenge alone, SORT1 overexpression led to lower protein abundance of SREBF2. In contrast, protein abundance of ABCA1 was greater. Overall, our data suggested that exogenous FFA induced abnormal cholesterol metabolism in hepatocytes, whereas a high abundance of SORT1 affected cholesterol esterification and potentially influx into bile. Thus, downregulation of hepatic SORT1 might be a cholesterol-regulated protective mechanism in the presence of a marked increase in FFA.

Effects of diacylglycerol O-acyltransferase 1 (DGAT1) on endoplasmic reticulum stress and inflammatory responses in adipose tissue of ketotic dairy cows.

Adipose tissue of ketotic dairy cows exhibits greater lipolytic rate and signs of inflammation, which further aggravate the metabolic disorder. In nonruminants, the endoplasmic reticulum (ER) is a key organelle coordinating metabolic adaptations and cellular functions; thus, disturbances known as ER stress lead to inflammation and contribute to metabolic disorders. Enhanced activity of diacylglycerol O-acyltransferase 1 (DGAT1) in murine adipocytes undergoing lipolysis alleviated ER stress and inflammation. The aim of the present study was to investigate the potential role of DGAT1 on ER stress and inflammatory response of bovine adipose tissue in vivo and in vitro. Adipose tissue and blood samples were collected from cows diagnosed as clinically ketotic (n = 15) or healthy (n = 15) following a veterinary evaluation based on clinical symptoms and serum concentrations of ß-hydroxybutyrate, which were 4.05 (interquartile range = 0.46) and 0.52 mM (interquartile range = 0.14), respectively. Protein abundance of DGAT1 was greater in adipose tissue of ketotic cows. Among ER stress proteins measured, ratios of phosphorylated PKR-like ER kinase (p-PERK) to PERK and phosphorylated inositol-requiring enzyme 1 (p-IRE1) to IRE1, and protein abundance of cleaved ATF6 protein were greater in adipose tissue of ketotic cows. Furthermore, ratios of phosphorylated RELA subunit of NF-κB (p-RELA) to RELA and phosphorylated c-jun N-terminal kinase (p-JNK) to JNK were greater, whereas protein abundance of NF-κB inhibitor α (NFKBIA) was lower in adipose tissue of ketotic cows. In addition, mRNA abundance of proinflammatory cytokines including TNF and IL-6 was greater in adipose tissue of ketotic cows. To better address mechanistic aspects of these responses, primary bovine adipocytes isolated from the harvested adipose tissue of healthy cows were subjected to lipolysis-stimulating conditions via incubation with 1 µM epinephrine (EPI) for 2 h. In another experiment, adipocytes were cultured with DGAT1 overexpression adenovirus and DGAT1 small interfering RNA for 48 h, respectively, followed by EPI (1 µM) exposure for 2 h. Treatment with EPI led to greater ratios of p-PERK to PERK, p-IRE1 to IRE1, p-RELA to RELA, p-JNK to JNK, and cleaved ATF6 protein, whereas EPI stimulation inhibited protein abundance of NFKBIA. Furthermore, treatment with EPI upregulated the secretion of proinflammatory cytokines into culture medium, including TNF-α and IL-6. Overexpression of DGAT1 in EPI-treated adipocytes attenuated ER stress, the activation of NF-κB and JNK signaling pathways, and the secretion of inflammatory cytokines. In contrast, silencing DGAT1 further aggravated EPI-induced ER stress and inflammatory responses. Overall, these data indicated that activation of DGAT1 may act as an adaptive mechanism to dampen metabolic dysregulation in adipose tissue. As such, it contributes to relief from ER stress and inflammatory responses.

Role of sortilin 1 (SORT1) on lipid metabolism in bovine liver.

High circulating concentrations of fatty acids cause triacylglycerol (TAG) accumulation in hepatocytes of dairy cows, a common metabolic disorder after calving. Low secretion of apolipoprotein B (APOB) and very low density lipoprotein (VLDL) are thought to be the major factors for TAG accumulation in hepatocytes. Recent data in nonruminant models revealed that sortilin 1 (SORT1) is a key regulator of VLDL secretion in part due to its ability to bind APOB. Thus, SORT1 could play a role in the susceptibility of dairy cows to develop fatty liver. To gain mechanistic insights in vivo and in vitro, we performed experiments using liver biopsies or isolated primary hepatocytes. For the in vivo study, blood and liver samples were collected from healthy multiparous dairy cows (n = 6; 9.0 ± 2.1 d in milk) and cows with fatty liver (n = 6; 9.7 ± 2.2 d in milk). In vitro, hepatocytes isolated from 4 healthy female calves (1 d old, 42-51 kg) were challenged with (fatty acids) or without (control) a 1.2 mM mixture of fatty acids in an attempt to induce metabolic stress. Furthermore, hepatocytes were treated with empty adenovirus vectors (Ad-GFP) or SORT1 overexpressing adenovirus (Ad-SORT1) for 6 h, or SORT1 inhibitor for 2 h followed by a challenge with (Ad-GFP + fatty acids, Ad-SORT1 + fatty acids, or SORT1 inhibitor + fatty acids) or without (Ad-GFP, Ad-SORT1, or SORT1 inhibitor) the 1.2 mM mixture of fatty acids for 12 h. Data from liver biopsies were compared using a 2-tailed unpaired Student's t-test. Data from calf hepatocytes were analyzed by one-way ANOVA. Data revealed that both fatty liver and in vitro challenge with fatty acids were associated with greater concentrations of TAG and mRNA and protein abundance of SORT1, SREBF1, FASN, and ACACA. In contrast, mRNA and protein abundance of CPT1A and APOB, and mRNA abundance of MTTP were markedly lower. Compared with fatty acid challenge alone, SORT1 overexpression led to greater concentration of TAG and mRNA abundance of SREBF1, FASN, ACACA, DGAT1, and DGAT2, and protein abundance of SREBF1, FASN, and ACACA. In contrast, concentration of secreted VLDL-APOB and mRNA abundance of APOB and MTTP, and protein abundance of CPT1A, APOB, and MTTP were lower. Compared with fatty acid challenge alone, SORT1 inhibitor + fatty acids led to lower concentrations of TAG and mRNA abundance of SREBF1, FASN, and DGAT2, and protein abundance of FASN, ACACA, and DGAT1. Concentrations of secreted VLDL-APOB and mRNA abundance of CPT1A and protein abundance of CPT1A and APOB were greater. Overall, in vitro data suggested that greater SORT1 abundance induced by exogenous fatty acids caused a reduction in VLDL-APOB secretion and increased hepatocyte TAG synthesis. Such mechanism was also apparent in tissue from cows with fatty liver. Thus, targeted downregulation of hepatic SORT1 could represent a viable mechanism to unload lipid during conditions where the influx of fatty acids increases markedly.

Role of diacylglycerol O-acyltransferase (DGAT) isoforms in bovine hepatic fatty acid metabolism.

Fatty acid accumulation in hepatocytes induced by high concentrations of fatty acids due to lipolysis and the associated oxidative damage they cause occur most frequently after calving. Because of their role in esterification of fatty acids, diacylglycerol acyltransferase isoforms (DGAT1 and DGAT2) could play a role in the susceptibility of dairy cows to develop fatty liver. To gain mechanistic insights, we performed in vivo and in vitro analyses using liver biopsies or isolated primary hepatocytes. The in vivo study (n = 5 cows/group) involved healthy cows [average liver triacylglycerol (TAG) = 0.78%; 0.58 to 0.93%, ratio of triglyceride weight to wet liver weight] or cows diagnosed with fatty liver (average TAG = 7.60%; 5.31 to 10.54%). In vitro, hepatocytes isolated from 3 healthy female calves (1 d old, 44 to 53 kg) were challenged with (fatty acids) or without (control) a 1.2 mM mixture of fatty acids in an attempt to induce metabolic stress. Furthermore, hepatocytes were treated with DGAT1 inhibitor or DGAT2 inhibitor for 2 h followed by a challenge with (DGAT1 inhibitor + fatty acids or DGAT2 inhibitor + fatty acids) or without (DGAT1 inhibitor or DGAT2 inhibitor) the 1.2 mM mixture of fatty acids for 12 h. Data analysis of liver biopsies was compared using a 2-tailed unpaired Student's t-test. Data from calf hepatocyte treatment comparisons were assessed by one-way ANOVA, and multiplicity for each experiment was adjusted by the Holm's procedure. Data indicated that both fatty liver and in vitro challenge with fatty acids were associated with greater mRNA and protein abundance of SREBF1, FASN, DGAT1, and DGAT2. In contrast, mRNA and protein abundance of CPT1A and very low-density lipoprotein synthesis-related proteins MTTP and APOB were markedly lower. However, compared with fatty acid challenge alone, DGAT1 inhibitor + fatty acids led to greater mRNA and protein abundance of CPT1A and APOB, and greater mRNA abundance of SREBF1 and MTTP. Furthermore, this treatment led to lower mRNA abundance of FASN and DGAT2 and TAG concentrations. Compared with fatty acid challenge alone, DGAT2 inhibitor + fatty acids led to greater mRNA and protein abundance of CPT1A, MTTP, and APOB, and lower mRNA and protein abundance of SREBF1 and FASN. In addition, compared with control and fatty acids, there was greater protein abundance of GRP78 and PERK in both DGAT1 and DGAT2 inhibitor with or without fatty acids. Furthermore, compared with control and fatty acids, reactive oxygen species concentrations in the DGAT1 inhibitor with or without fatty acid group was greater. Overall, data suggested that DGAT1 is particularly relevant in the context of hepatocyte TAG synthesis from exogenous fatty acids. Disruption of both DGAT1 and DGAT2 altered lipid homeostasis, channeling fatty acids toward oxidation and generation of reactive oxygen species. Both DGAT isoforms play a role in promoting fatty acid storage into TAG and lipid droplets to protect hepatocytes from oxidative damage.

Role of ORAI calcium release-activated calcium modulator 1 (ORAI1) on neutrophil extracellular trap formation in dairy cows with subclinical hypocalcemia.

Hypocalcemia in dairy cows is associated with decreased neutrophil phagocytosis, adhesion capacity, migration, and reactive oxygen species (ROS) production through alterations in ORAI calcium release-activated calcium modulator 1 (ORAI1). Neutrophils can resist the invasion of pathogenic microorganisms by releasing neutrophil extracellular traps (NET). However, the mechanisms controlling NET formation during hypocalcemia are unknown. To address the role of ORAI1 in NET formation, neutrophils were isolated at 2 d postcalving from lactating Holstein dairy cows (n = 10 per group) diagnosed as clinically healthy (control) or with plasma concentrations of Ca2+ <2.0 mmol/L as a criterion for diagnosing subclinical hypocalcemia (SCH). A series of ex vivo experiments were conducted as follows: first, neutrophils isolated from both groups of cows were treated with phorbol 12-myristate 13-acetate (PMA) to stimulate NET formation; second, neutrophils from control and SCH were pretreated with or without the ROS scavenger N-acetylcysteine (NAC), the sarcoendoplasmic Ca2+ ATPase inhibitor thapsigargin, or ORAI1 blocker 2APB and then treated with PMA to stimulate NET formation; and third, neutrophils were transfected with small interfering (si)ORAI1 or nontarget siRNA (siNEG) and then stimulated with PMA to induce formation of NET. A one-way ANOVA was used for statistical analysis of individual experiments. In the first experiment, neutrophils from SCH cows formed NET with fewer DNA filaments, more diffused nuclei, and reduced translocation of myeloperoxidase (MPO) and neutrophil elastase (NE) to the nucleus. Neutrophils from SCH cows stimulated with PMA had a lower mitochondrial permeability, the state of mitochondrial permeability transition pore was open, ROS production was lower and there was increased mitochondrial damage. In the second experiment, in both control and SCH-PMA stimulated neutrophils, exogenous NAC decreased NET formation (assessed via Hoechst 33342 dye; Beyotime). Furthermore, following the challenge with PMA, thapsigargin increased NET formation and ROS production, but blocking ORAI1 with 2APB decreased NADPH oxidase activation, ROS production, and NET formation. In the third experiment, neutrophils transfected with siORAI1 before stimulation with PMA had lower intracellular concentrations of Ca2+, NET formation, and ROS production. Overall, the data indicated that SCH reduces NET formation in neutrophils partly due to damaged mitochondria. The reduction in ORAI1 abundance in neutrophils of dairy cows with hypocalcemia also decreases ROS production.