Cows with ketosis frequently exhibit liver injury, primarily driven by increased influx of nonesterified fatty acids (NEFA) into the liver. This excess NEFA induces lipotoxicity, which triggers hepatic inflammatory responses and ultimately results in liver damage, further exacerbating ketosis. Neural precursor cell expressed developmentally downregulated 4 (NEDD4), an E3 ubiquitin ligase, plays a crucial role in regulating anti-inflammatory processes, yet its role in the hepatic injury in ketotic cows remains unclear. This study aimed to investigate the link between NEDD4 and hepatic inflammatory damage in ketotic cows. Liver biopsies and blood samples were collected from healthy (n = 10) and clinically ketotic (n = 10) cows at 3 to 9 DIM. Primary bovine hepatocytes isolated from healthy one-day-old calves were treated with NEFA (0, 0.6, 1.2, or 2.4 mM, 12 h) or transfected with adenovirus-expressed NEDD4 (Ad-NEDD4) or NEDD4 small interfering RNA (48 h), followed by treatment with 1.2 mM NEFA (12 h). For degradation pathway experiment, Ad-NEDD4 transfected hepatocytes were exposed to chloroquine (50 µM, 6 h) or MG132 (10 µM, 10 h). For molecular cloning, HA-tagged NEDD4 (HA-NEDD4) and flag-tagged bovine receptor-interacting protein kinase 1 (Flag-RIPK1) plasmids were expressed in 293T cells for 48 h, then treated with MG132 (10 µM, 10 h). Compared with the control group, ketotic cows showed higher serum concentrations of BHB, NEFA, TNF-α, and high mobility group box 1 (HMGB1), along with elevated activities of aspartate aminotransferase and alanine aminotransferase, but lower DMI, milk yield, and glucose. Histology analyses revealed inflammatory cell infiltration and pronounced necrosis. Hepatic IL-1β and the ratios of phosphorylated (p-) NF-κB to NF-κB and p-RIPK1 to RIPK1 were elevated. In vitro, the exogenous addition of 1.2 mM and 2.4 mM NEFA significantly reduced the abundance of NEDD4 in bovine hepatocytes, and increased the proportion of propidium iodide-positive hepatocytes. Concurrently, the concentrations of TNF-α and HMGB1 in the culture medium were elevated. Additionally, the protein expression levels of IL-1β and p-RIPK1 in bovine hepatocytes, as well as the ratio of p-NF-κB/NF-κB, were significantly increased. These findings indicate that NEFA can induce inflammatory injury in bovine hepatocytes in a dose-dependent manner. NEDD4 overexpression alleviated these NEFA-induced effects by reducing the protein RIPK1 expression and phosphorylation, whereas NEDD4 silencing exacerbated them. Co-immunoprecipitation demonstrated that NEDD4 interacted with RIPK1, promoting its ubiquitination and degradation of RIPK1 and suppressing phosphorylation. In summary, these data indicate that NEDD4 protects bovine hepatocytes from NEFA-induced inflammatory injury by regulating RIPK1 stability and activity, providing insights into its protective role in ketosis-related liver damage.

Prepartum obesity predisposes dairy cows to a higher risk of postpartum metabolic disorder. Volatile fatty acids (VFA) produced through ruminal microbial fermentation of feed substrates serve as a key form of energy for dairy cows. However, the precise mechanisms through which the rumen microbiota promote adipocyte lipid accumulation in obese dairy cows remain to be elucidated. Thus, the aim of this study was to investigate the mechanisms by which rumen microbiota regulates prepartum obesity in dairy cows. Plasma glucose, insulin, triglyceride, and free fatty acids were greater in obese dairy cows. In the adipose tissue, the triglyceride content and expression of genes involved in lipid synthesis were higher in obese dairy cows. In the liver, the expression of genes involved in gluconeogenesis and lipid synthesis was higher in obese dairy cows. The ruminal total VFA, acetate, and propionate were higher in obese dairy cows compared to normal cows. The 16S rRNA gene analysis revealed that rumen bacteria, including Tidjanibacter inops_A, Rikenella massiliensis, Papillibacter cinnamivorans, and Parabacteroides merdae, were enriched in the rumen of obese dairy cows. Enrichment of these bacteria promoted carbohydrate degradation and VFA production. The metabolome analysis showed that obese dairy cows had elevated citric acid level in the rumen, which was positively associated with body condition score, body weight, adipocyte diameter, ruminal VFA concentration, and the abundance of VFA-producing bacteria. Our results suggest that rumen bacterial flora in prepartum obese dairy cows supply more VFA to the host, which may induce lipid deposition in adipocytes.

Metabolic dysfunction-associated steatohepatitis (MASH) is emerging as a leading cause of chronic liver disease. MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) is a potential therapeutic target, whereas suppression of total MTORC1 activity can lead to unwanted effects. Here, we found that byakangelicin (Bya), a natural compound, selectively inhibited MTORC1-mediated phosphorylation of TFEB (transcription factor EB), without affecting canonical MTORC1 substrates. Knockout of hepatic Tfeb blocked the alleviation effects of Bya on hepatic steatosis, inflammation, insulin resistance, and fibrosis in mice, while reintroduction of TFEB restored these effects. We identified Bya directly bound to MET370 and PHE552 of FLCN (folliculin), suppressing the function of the FLCN-FNIP1 (folliculin interacting protein 1)/FNIP2 complex, which in turn inhibited MTORC1-mediated cytoplasmic sequestration of TFEB. Mutation of FLCN (M370A and F552A) in the liver abolished Bya-induced protection against MASH. Thus, Bya is a promising therapeutic natural compound for MASH, and selective inhibition of MTORC1 is a potential approach to treat this disease.Abbreviations: aa, amino acids; AAV, adeno-associated virus; Bio, biotin; Bio-Bya, biotin-conjugated Bya; BSA, bovine serum albumin; BW, body weight; Bya, byakangelicin; CETSA, cellular thermal shift assay; CHIP-atlas, chromatin immunoprecipitation atlas; Cmax, maximum concentration; CQ, chloroquine; DARTS; drug affinity responsive target stability assay; EIF4EBP1/4E-BP1, eukaryotic translation initiation factor 4E binding protein 1; FBS, fetal bovine serum; FDA, food and drug administration; FIMO-JASPAR, find individual motif occurrences-JASPAR; FLCN, folliculin; FNIP1, folliculin interacting protein 1; GAP, GTPase-activating protein; GOT1/AST, glutamic-oxaloacetic transaminase 1; GPT/ALT, glutamic-pyruvic transaminase; GTRD, gene transcription regulatory database; GTT, glucose tolerance test; H&E, hematoxylin and eosin; Hbonds, hydrogen bonds; HFD, high-fat diet; HFHC, high-fat and high-cholesterol; HOMA-IR, homeostatic model assessment of insulin resistance; HSCs, hepatic stellate cells; IP, immunoprecipitation; ITT, insulin tolerance test; KD, dissociation constant; KEGG, kyoto encyclopedia of genes and genomes; KPBS, potassium phosphate-buffered saline; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LW/BW, liver-to-body weight ratio; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MASH, metabolic dysfunction-associated steatohepatitis; MASLD, metabolic dysfunction-associated steatotic liver disease; MCD, methionine and choline deficient; MST, microscale thermophoresis assay; MTOR, mechanistic target of rapamycin kinase; MTORC1, MTOR complex 1; ND, normal diet; NFKB/NF-κB, nuclear factor kappa B; NFKBIA/IKBA, NFKB inhibitor alpha; OP, oleate acid and palmitate acid; PBS, phosphate-buffered saline; PCA, principal component analysis; qRT-PCR, real-time quantitative PCR; RELA/p65, RELA proto-oncogene, NF-kB subunit; Res, resmetirom; Rg, radius of gyration; RMSD, root-mean-square deviation; RMSF, root-mean-square fluctuation; RPS3, ribosomal protein S3; RPS6KB1/S6K1, ribosomal protein S6 kinase B1; RRAGC, ras related GTP binding C; SASA, solvent-accessible surface area; SNRPD2, small nuclear ribonucleoprotein D2 polypeptide; SQSTM1/p62, sequestosome 1; T1/2, half-life; TFE3, transcription factor binding to IGHM enhancer 3; TFEB, transcription factor EB; TMEM192, transmembrane protein 192; VIM, vimentin; WT, wild-type.

Subclinical ketosis in dairy cows is accompanied by adaptive changes in the secretory function of the mammary gland. However, the molecular basis underlying this adaptation remains unclear. The secretory capacity of the mammary gland is closely linked to the activation of the unfolded protein response (UPR) and endoplasmic reticulum (ER) biogenesis, particularly the inositol-requiring enzyme 1α (IRE1α) pathway. Thus, this study aims to investigate whether BHB activates the IRE1α-XBP1 signaling axis to promote ER biogenesis, thereby sustaining casein synthesis. In this study, MAC-T cells were treated with 1.8 mM BHB for 0, 12, 24, or 48 h to mimic subclinical ketotic conditions. We first observed that BHB activated all 3 branches of the UPR without inducing the proapoptotic element of the UPR. Meanwhile, MAC-T cells treated with BHB show a significant increase in ER tracker staining and upregulated mRNA levels of ER biogenesis-related genes, such as choline kinase alpha (CHKA), choline-phosphate cytidylyltransferase alpha (PCYT1A), and Surfeit 4 (SURF4). Subsequently, BHB upregulated the mRNA abundance of genes related to ribosome biogenesis and the proregenerative phenotype in MAC-T cells. The MAC-T cells treated with 1.8 mM BHB also displayed increased protein abundance of β-casein, along with the casein beta (CSN2), casein kappa (CSN3), casein alpha S1 (CSN1S1), and casein alpha S2 (CSN1S2) milk protein genes. Compared with the 1.8 mM BHB group, cotreatment with BHB and KIRA6 significantly suppressed the BHB-induced increase in ER tracker fluorescence, ER biogenesis-related gene expression, and β-casein protein abundance. Silencing XBP1 via small interfering RNA inhibited BHB-induced ER biogenesis and β-casein synthesis, whereas the overexpression of XBP1 alleviated these effects. Furthermore, under BHB treatment, overexpression of XBP1 partially attenuated the suppressive effects of IRE1α inhibition on ER biogenesis and β-casein synthesis. Overall, our data demonstrate that BHB promotes ER biogenesis and milk protein synthesis in mammary epithelial cells through activation of the IRE1α-XBP1 signaling pathway, suggesting a protective and adaptive role for BHB in maintaining mammary gland function under subclinical ketotic conditions.

During the periparturient period, negative energy balance (NEB) in dairy cows leads to increased concentrations of non-esterified fatty acids (NEFA) in the blood, which can induce fatty liver disease and ketosis. Forkhead box protein A3 (FOXA3) is a key transcription factor that regulates liver metabolism; however, its specific role in the pathogenesis of fatty liver in dairy cows remains unclear. This study aimed to investigate the mechanism by which FOXA3 regulates hepatic lipid metabolism. We collected liver samples from dairy cows with fatty liver (n = 10) and from healthy cows (n = 10). Bovine primary hepatocytes were isolated from the liver tissue of calves (n = 5), followed by NEFA treatment, and we utilized FOXA3 overexpression, immunofluorescence, and RNA sequencing (RNA-seq) to conduct our analysis. Our results demonstrated that FOXA3 expression in the livers of cows with fatty liver was significantly lower than in healthy cows. NEFA treatment resulted in the downregulation of FOXA3 protein levels in hepatocytes, promoting triacylglycerol (TAG) accumulation and the expression of lipogenesis-related genes. Conversely, FOXA3 overexpression mitigated NEFA-induced lipid accumulation, inhibited the expression of lipogenesis-related genes and proteins-particularly SREBP1-and affected cell proliferation, and the intracellular localization of FOXA3 and SREBP1. RNA-seq analysis suggested that FOXA3 may influence hepatic lipogenesis through pathways such as PI3K-Akt and the cell cycle. In summary, FOXA3 mitigates NEFA-induced hepatic lipid accumulation through a dual mechanism: regulating SREBP1 expression and inhibiting cellular proliferation. These findings highlight FOXA3's potential as a novel target for the prevention and treatment of fatty liver disease in dairy cows.

Excessive nonesterified fatty acids (NEFA) during the transition period impose metabolic stress on the bovine mammary gland. High NEFA levels induce oxidative stress and trigger apoptosis in bovine mammary epithelial cells. Prohibitin 2 (PHB2) is a mitochondrial inner-membrane scaffolding protein known for its role in maintaining mitochondrial cristae integrity and regulating apoptosis. However, the specific effect of PHB2 on apoptosis in response to NEFA-induced stress in bovine mammary epithelial cells remains unclear. Therefore, this study aimed to investigate the regulatory role of PHB2 in high-NEFA-induced bovine mammary epithelial cells apoptosis, as well as the underlying mechanisms involved. First, mammary gland tissue from healthy (n = 15, BHB < 0.6 mM) and ketotic cows (n = 15, BHB > 3.0 mM) were collected. Compared with healthy cows, ketotic cows exhibited higher protein abundances of cleaved caspase-3 and cleaved PARP, lower protein abundances of PHB2 and long-form OPA1 (L-OPA1) in mammary glands, increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) labeling in mammary tissues, and elevated lactate dehydrogenase activity in milk. Second, MAC-T cells were treated with NEFA (0, 0.3, 0.6, or 1.2 mM). MAC-T treated with 1.2 mM NEFA displayed increased protein abundance of cleaved caspase-3, cleaved PARP and cytoplasmic cytochrome c (Cyt C), and decreased protein abundance of PHB2, L-OPA1, and mitochondrial Cyt C. Additionally, MAC-T treated with 1.2 mM NEFA had increased apoptosis rate and TUNEL labeling. Third, MAC-T cells were transfected with small interfering RNA to silence PHB2 or plasmid for overexpression of PHB2, followed by treatment with or without NEFA. PHB2 silencing aggravated NEFA-induced apoptosis in MAC-T cells, and PHB2 overexpression alleviated this effect. Fourth, MAC-T cells were transfected with noncleavable OPA1 mutant overexpression adenovirus. Overexpression of noncleavable OPA1 mutant ameliorates NEFA-induced mitochondrial dysfunction and apoptosis. In conclusion, PHB2 alleviates NEFA-induced mitochondrial dysfunction and apoptosis by preventing OPA1 cleavage.

Portal fibrosis, a determinant of progression in virtually all chronic liver diseases, prototypically develops in biliary diseases. Using single-cell RNA sequencing and genetic cell fate tracing in mouse models, we identify Clec3b⁺ fibroblasts as a distinct subset of portal fibroblasts, which rapidly expand after biliary injury and give rise to the bulk of portal myofibroblasts. Mechanistic analyses reveal that Clec3b⁺ portal fibroblasts activation is governed by a Krüppel-like factor 4 (KLF4)/periostin (POSTN) axis, i.e., KLF4 directly binds to the Postn promoter and represses its transcription in quiescent fibroblasts, whereas after injury, KLF4 is downregulated, which allows POSTN, acting via αvβ5 integrin, to drive portal fibroblast activation and portal fibrosis. Our findings identify Clec3b+ portal fibroblasts as the primary effectors of portal fibrosis and demonstrate that the KLF4/POSTN signaling axis regulates their activation, offering potential therapeutic targets for inhibiting fibrosis in biliary diseases.

The ketogenic diet (KD), an emerging nutritional intervention for cancer, reprograms cellular energy metabolism from glucose to ketone bodies, including acetoacetate (AcAc), acetone (Ac), and β-hydroxybutyrate (BHB). However, the mechanisms connecting ketone body signal sensing to tumor growth suppression remain elusive. Here, we show that RagC, a key component of mTORC1 pathway, senses BHB but not AcAc and Ac, to dictate tumor suppression. KD-derived BHB inhibits mTORC1 activity by promoting β-hydroxybutyrylation (Kbhb) of RagC at lysine 349 (K348 in mice). Mechanistically, RagC-K349bhb is dynamically catalyzed by p300 and erased by SIRT1, disrupting RagC interaction with Raptor/mTOR and blocking mTORC1 recruitment to lysosomes. Clinically, BHB-mediated RagC-K349bhb suppresses colorectal cancer (CRC) growth via mTORC1 inhibition in both RagC-K348R knockin mice and CRC patient-derived samples. Thus, we identify a BHB sensing mechanism by mTORC1 and highlight the potential role of RagC-K349bhb as a therapeutic target for BHB-based CRC treatment.

The aim of this study was to evaluate the effect of Fructus Ligustri Lucidi (FLL) supplementation on energy metabolism, immunity, and the rumen microbiome in peripartum cows. Twenty healthy multiparous Holstein dairy cows were enrolled in the trial based on expected calving date, parity, and previous lactation milk yield. They were randomly assigned to either a control diet (Con; n = 10) or the control diet supplemented with 150 g/d per cow of FLL (FLL; n = 10). The trial spanned 6 wk, from 3 wk prepartum to 3 wk postpartum. Cows were milked thrice daily at 0500, 1100, and 1700 h, with milk yield and SCC recorded. Blood samples were collected from each cow at -3, -2, -1, 0, 1, 2, and 3 weeks relative to calving to evaluate the metabolic, inflammatory, and oxidative profiles. Peripheral blood neutrophils were isolated to measure reactive oxygen species (ROS), phagocytic activity, and neutrophil extracellular traps (NET). Rumen liquid was obtained via esophageal tubing at -3, 0, and 3 wk to investigate microbial biodiversity. Results indicated that FLL supplementation did not significantly affect the milk yield or SCC within 3 wk postpartum. Notably, FLL ameliorated negative energy balance from 1 wk to 3 wk, mitigated inflammation from calving day to 3 wk, and reduced oxidative stress at both -1 and 1 wk. The FLL treatment enhanced neutrophil phagocytosis from calving day to 1 wk, increased NET release at -1 wk, and reduced neutrophil ROS levels on calving day. Moreover, FLL increased rumen microbial α-diversity on calving day. At the phylum level, Firmicutes abundance was higher in the Con group on calving day compared with 3 wk pre- and postpartum, whereas Bacteroidota exhibited the opposite trend; FLL maintained stable Firmicutes and Bacteroidota abundances throughout the peripartum period. At the genus level, Prevotella abundance decreased in both groups on calving day but rebounded by 3 wk, with significantly higher Prevotella levels in the FLL group on calving day. The level 2 Kyoto Encyclopedia of Genes and Genomes pathway prediction further indicated divergent metabolic profiles: lipid metabolism pathways were less active in the FLL group than in the Con group on calving day and at 3 wk, whereas immune-related pathways were enriched in the FLL group. Energy metabolism pathway activity was higher in the FLL group on calving day but lower by 3 wk compared with the Con group. Correlation analysis indicated that rumen microbiome shifts were associated with energy metabolism, immunity, and oxidative stress. In conclusion, this study elucidates the dynamic changes in rumen microbiome during the peripartum period and highlights the beneficial effects of FLL supplementation on energy metabolism, immune function, and rumen microbial homeostasis in peripartum dairy cows.

Ketotic cows undergo severe negative energy balance, in which increased circulating non-esterified fatty acids, specifically palmitic acid (PA), serve as primary drivers of liver injury. Yet, effective strategies for alleviating this lipotoxic injury remain limited. Although tryptophan metabolite indole-3-acetic acid (3-IAA) has been documented to exert hepatoprotective effects in non-ruminants, its role in alleviating lipotoxicity-induced injury in bovine hepatocytes remains unclear. In the present study, ketotic cows exhibited increased serum beta-hydroxybutyrate and non-esterified fatty acid concentrations and hepatic triacylglycerol accumulation and decreased serum glucose and 3-IAA concentrations. Notably, serum 3-IAA concentration negatively correlated with the hepatic triacylglycerol content. In primary bovine hepatocytes, treatment with 200 mu mol/L 3-IAA improved viability and reduced cytotoxicity under PA challenge. Transcriptomic analysis further indicated that 3-IAA mainly regulated pathways related to lipid metabolism and endoplasmic reticulum (ER) stress. Further analysis showed that 3-IAA attenuated PA-induced lipid accumulation and downregulated sterol regulatory element-binding protein 1c protein abundance and its downstream target gene expression, while upregulating the expression of fatty acid oxidation-related molecules, indicating reduced lipogenesis and enhanced fatty acid oxidation. In addition, 3-IAA markedly alleviated PA-induced ER stress, as evidenced by reduced expression of ER stress-related markers. Notably, 3-IAA increased sirtuin 3 (SIRT3) protein and mRNA expression in PA-treated hepatocytes, whereas SIRT3 knockdown partially abrogated the protective effects of 3-IAA. Collectively, these findings demonstrate that 3-IAA mitigates PA-induced lipid accumulation and ER stress in bovine hepatocytes, at least in part through a SIRT3-dependent mechanism, highlighting its potential as an endogenous regulator of hepatic homeostasis in ketotic cows.

Heat stress severely impairs immune function and threatens dairy cow health and productivity. Although heat stress suppresses bovine neutrophil (PMN) phagocytosis, its effects governing PMN survival remain unclear. L-arginine (L-Arg) exerts immunomodulatory and cytoprotective effects, yet its role in repairing heat-damaged PMN has not been defined. In this study, an in vitro heat stress model (42 °C for 2.5 h) was established. Heat stress significantly reduced cell viability, induced abnormal nuclear morphology, and triggered apoptotic signaling, accompanied by severe oxidative stress (elevated reactive oxygen species (ROS)/Malondialdehyde (MDA), decreased SOD/glutathione peroxidase (GSH-Px)). The ROS scavenger N-Acetylcysteine (NAC) mitigated both oxidative stress and apoptosis, confirming oxidative stress as a core mediator of PMN apoptosis induced by heat stress. Pretreatment with 4 mmol/L L-Arg significantly alleviated heat-induced oxidative damage and apoptosis. Mechanistically, in bovine PMN, we first demonstrated that heat stress blocks nuclear factor erythroid 2-related factor 2 (NFE2L2) nuclear translocation; inhibition of NFE2L2 (ML385) abolished L-Arg’s protection, verifying the NFE2L2-dependent ROS scavenging pathway. In conclusion, this study reveals that heat stress induces bovine PMN oxidative injury and apoptosis by suppressing NFE2L2-mediated ROS scavenging, and L-Arg restores PMN viability and immune resilience by reactivating the NFE2L2 antioxidant pathway. These findings provide a targeted L-Arg supplement strategy to improve PMN survival and disease resistance in heat-stressed dairy cows.

High nonesterified fatty acid (NEFA) concentrations in cows with clinical ketosis lead to metabolic dysfunction in mammary cells, resulting in oxidative stress. Studies have shown that autophagy is impaired in the mammary glands of ketotic cows, and enhancing autophagy mitigates oxidative stress in these animals. Cullin 3 (CUL3), an E3 ubiquitin ligase, is integral for maintaining cellular homeostasis, particularly regulation of oxidative stress and autophagy. Whether CUL3 is involved in mitigating NEFA-induced oxidative stress is unknown. This study aimed to investigate the protective effects and underlying mechanisms whereby CUL3 mitigates NEFA-induced oxidative stress in mammary epithelial cells. First, mammary gland tissue and blood samples were collected from healthy cows (n = 12, BHB <0.6 mM) and cows with clinical ketosis (n = 12, BHB >3.0 mM). Compared with healthy cows, cows with clinical ketosis had reduced productive performance, decreased CUL3 expression, impaired autophagic activity, and increased oxidative stress status in mammary tissue. In vitro, incubating the immortalized bovine mammary epithelial cell line (MAC-T) with 1.2 mM NEFA downregulated CUL3 expression, impaired autophagy, and increased oxidative stress. Adenovirus-mediated overexpression of CUL3 attenuated NEFA-induced accumulation of peroxides and reactive oxygen species, whereas silencing of CUL3 via small interfering RNA exacerbated these effects. Even when nuclear factor erythroid 2 related factor 2 (NFE2L2) expression was reduced by overexpression of CUL3, there was no worsening of NEFA-induced reductions in mRNA levels of NFE2L2 downstream target genes (NADPH quinone oxidoreductase 1 [NQO1], heme oxygenase-1 [HMOX1], glutamate-cysteine ligase catalytic subunit [GCLC)], and glutamate-cysteine ligase modifier subunit [GCLM]). The reduction in NEFA-induced oxidative stress by CUL3 was diminished upon autophagy related 5 (ATG5) silencing suggesting that CUL3 alleviates NEFA-induced oxidative stress via autophagy. Additionally, CUL3 overexpression aggravated the NEFA-induced decrease in BCL2 apoptosis regulator (BCL2) expression along with alleviating the NEFA-induced decrease in Beclin1 (BECN1) expression. Under NEFA treatment, overexpression of BCL2 partly mitigated the CUL3-induced elevation in BECN1. Overall, oxidative stress and impaired autophagy are characterized in the mammary tissue of cows with clinical ketosis. CUL3 activation, likely through the BCL2-BECN1 pathway, enhances autophagy and mitigates NEFA-induced oxidative stress in MAC-T cells. Thus, targeting CUL3-mediated autophagy could be a promising therapeutic strategy to reduce oxidative stress-induced damage in bovine mammary epithelial cells.

High nonesterified fatty acid (NEFA) concentrations in cows with clinical ketosis lead to metabolic dysfunction in mammary cells, resulting in oxidative stress. Studies have shown that autophagy is impaired in the mammary glands of ketotic cows, while enhancing autophagy mitigate oxidative stress in these animals. Cullin3 (CUL3), an E3 ubiquitin ligase, is integral for maintaining cellular homeostasis, particularly regulation of oxidative stress and autophagy. Whether CUL3 is involved in mitigating NEFA-induced oxidative stress is unknown. This study aimed to investigate the protective effects and underlying mechanisms whereby CUL3 mitigates NEFA-induced oxidative stress in mammary epithelial cells. First, mammary gland tissue and blood samples were collected from healthy cows [n = 12, β-hydroxybutyrate (BHB) < 0.6 mM] and cows with clinical ketosis (n = 12, BHB > 3.0 mM). Compared with healthy cows, cows with clinical ketosis had reduced productive performance, decreased CUL3 expression, impaired autophagic activity, and increased oxidative stress status in mammary tissue. In vitro, incubating MAC-T with 1.2 mM NEFA downregulated CUL3 expression, impaired autophagy, and increased oxidative stress. Adenovirus-mediated overexpression of CUL3 attenuated NEFA-induced accumulation of peroxides and reactive oxygen species (ROS), while silencing of CUL3 via siRNA exacerbated these effects. Even when nuclear factor erythroid 2 related factor 2 (NFE2L2) expression was reduced by overexpression of CUL3, there was no worsening of NEFA-induced reductions in mRNA levels of NFE2L2 downstream target genes [NADPH quinone oxidoreductase 1 (NQO1), Heme oxygenase-1 (HMOX1), Glutamate-cysteine ligase catalytic (GCLC) and Glutamate cysteine ligase modifier subunit (GCLM)]. The reduction in NEFA-induced oxidative stress by CUL3 was diminished upon autophagy related 5 (ATG5) silencing suggesting that CUL3 alleviates NEFA-induced oxidative stress via autophagy. Additionally, CUL3 overexpression aggravated the NEFA-induced decrease in BCL2 apoptosis regulator (BCL2) expression while alleviating the NEFA-induced decrease in beclin1(BECN1) expression. Under NEFA treatment, overexpression of BCL2 partly mitigated the CUL3-induced elevation in BECN1. Overall, oxidative stress and impaired autophagy characterize mammary tissue of cows with clinical ketosis. CUL3 activation, likely through the BCL2-BECN1 pathway, enhances autophagy and mitigates NEFA-induced oxidative stress in MAC-T cells. Thus, targeting CUL3-mediated autophagy could be a promising therapeutic strategy to reduce oxidative stress-induced damage in bovine mammary epithelial cells.

Subclinical ketosis (SCK), a common metabolic disorder in dairy cows during the peripartum period, is accompanied by systemic inflammation and elevated circulating BHB. β-Hydroxybutyrate contributes to the development of systemic inflammation by inhibiting the spontaneous apoptosis of neutrophils in SCK cows. However, the underlying molecular mechanism is still unclear. This study investigated the role of the degranulation-macrophage-1 antigen (Mac-1) axis in this process. The results demonstrated that BHB promoted the degranulation process and activated membrane Mac-1 (CD11b/CD18) on neutrophils both ex vivo and in vitro. Neutrophils were isolated from healthy cows and treated with 2 mM BHB, a specific degranulation inhibitor, or a Mac-1 blocking antibody in vitro. We found that the released granule contents during degranulation activated the intracellular Mac-1 signaling pathway, which was involved in BHB-mediated inhibition of bovine neutrophil apoptosis. Overall, our findings reveal that the degranulation-Mac-1 axis plays a critical role in regulating BHB-induced inhibition of spontaneous neutrophil apoptosis in dairy cows with SCK.

Immune dysregulation-induced inflammation serves as a driving force in the progression of metabolic dysfunction-associated steatohepatitis (MASH), while the underlying cellular and molecular mechanisms remain largely uncharted. A Western diet (WD) is employed to construct mouse models of metabolic dysfunction associated steatotic liver disease (MASLD) or MASH. Mass cytometry identifies a c-kit+ cDC1 subset whose frequency is reduced in the livers of mice and patients with MASH compared with healthy controls. Adoptive cell transfer of c-kit+ cDC1 protects the progression of MASH. Moreover, analysis of gut microbe sequence shows that WD-fed mice and MASLD/MASH patients exhibit gut microbiota dysbiosis, with an elevated abundance of H2S-producing Desulfovibrio_sp. Transplanting of MASH-derived fecal flora, Desulfovibrio_sp., or injecting H2S intraperitoneally into MASLD mice decreases the c-kit+cDC1 population and exacerbates liver inflammation. Mechanistically, H2S induces autophagic cell death of cDC1 in a c-kit-dependent manner in cDC-specific c-kit-/- and Atg5-/- mice. We thus uncover that microbiota-derived H2S triggers the autophagic cell death of c-kit+ cDC1 and ignites the liver inflammatory cascade in MASH.

Ketosis is a prevalent metabolic disorder in periparturient dairy cows and severely impairs production performance. It has been established that ketotic cows manifest hepatic oxidative damage; however, the precise molecular mechanisms underlying this pathology remain elusive. The present results demonstrate that ketotic dairy cows exhibit severe hepatic oxidative damage and ferroptosis. This pathological state is characterized by aberrant hepatic iron accumulation, driven by an imbalance between increased transferrin receptor 1 (TFR1)-mediated iron uptake and decreased ferroportin (FPN)-mediated iron export. Furthermore, we observed a substantial accumulation of lipid peroxides due to both an impaired nuclear factor erythroid 2-related factor 2 (NFE2L2)/glutathione peroxidase 4 (GPX4) antioxidant pathway and enhanced acyl-CoA synthetase long-chain family member 4 (ACSL4)-mediated lipid peroxidation. In vitro, β-hydroxybutyrate (BHB) induced oxidative injury and ferroptosis in bovine hepatocytes, and the molecular patterns were consistent with those in vivo. Mechanistically, BHB disrupted TFR1/FPN-mediated iron metabolism, impaired the NFE2L2-mediated GSH synthesis pathway, and promoted ACSL4-mediated lipid peroxidation. Notably, inhibition of ferroptosis can significantly alleviate BHB-caused oxidative damage in bovine hepatocytes. In summary, these data demonstrate that BHB-induced ferroptosis is mechanistically associated with hepatic oxidative injury in ketotic dairy cows. These findings not only contribute to mechanistic insights into hepatic oxidative injury of ketotic cows but also highlight the potential of targeting ferroptosis for therapeutic intervention in ketosis.

Deoxynivalenol (DON) and fumonisin B1 (FB1) are prevalent mycotoxins commonly found together in dairy cow feed, posing significant risks to cow health and food safety. This study investigates combined toxic effects of DON and FB1 on bovine mammary epithelial cells (BMECs), and role of AMPK-Caspase 6 axis in mycotoxin-induced apoptosis in BMECs and mouse model. We showed that even subcytotoxic doses of DON, combined with FB1, induced synergistic or additive toxic effects, reducing cell viability, increasing LDH release, and promoting apoptosis. This combination also impaired milk triglyceride synthesis and downregulated genes involved in milk fat and protein production. Silencing Caspase 6 via siRNA, inhibiting with Z-VEID-FMK, or activating AMPK with AICAR alleviated apoptosis. Conversely, overexpressing Caspase 6 diminished AMPK’s protective effects. In vivo, inhibiting Caspase 6 alleviated apoptosis in mammary tissues. These findings highlight therapeutic potential of targeting the AMPK-Caspase 6 pathway to mitigate combined toxicity of DON and FB1.

Hepatic triacylglycerol (TAG) metabolic dysregulation, characterized by concurrent enhancement of synthesis and attenuation of degradation, is fundamental to the pathogenesis of fatty liver in dairy cows. Patatin-like phospholipase domain containing 2 (PNPLA2) catalyzes the rate-limiting hydrolysis of TAG to free fatty acids (FFA). Meanwhile, peroxisome proliferator-activated receptor α/δ (PPARα/δ) promote fatty acid oxidation and inhibit inflammatory response, and are activated by FFA released during lipolysis. Given this regulatory interplay, the study investigates the hepatic status of PNPLA2, PPARα/δ signaling and PNPLA2-associated regulatory factors. The results showed that fatty liver cows exhibited a severe inflammatory response in the liver. In addition, hepatic mRNA and protein levels of PNPLA2, phosphorylated hormone-sensitive lipase, and monoacylglycerol lipase were reduced in the fatty liver cows compared with healthy cows, while those of G0/G1 switch protein 2 (G0S2) and abhydrolase domain-containing 5, lysophosphatidic acid acyltransferase (ABHD5) were not significantly different between the two groups. Notably, the hepatic transcriptional activities of PPARα and PPARδ were significantly lower in cows with fatty liver than in healthy cows. In summary, the present data indicated that fatty liver cows exhibited hepatic inhibition of the PNPLA2-PPARα/δ signaling axis and a marked inflammatory response, which may be a key factor triggering and exacerbating fatty liver. These findings not only contribute to the mechanistic insights into bovine fatty liver development but also highlight the PNPLA2-PPARα/δ signaling axis as a potential target for therapeutic intervention.

Dairy cows with ketosis frequently exhibit impaired liver function and inflammation. Necroptosis, a form of programmed cell death associated with cellular lysis, releases damage-associated molecular patterns (DAMP) into injured tissues, thereby amplifying inflammatory responses and tissue damage. However, the role of necroptosis in the liver of ketotic cows remains unknown. The present study aimed to investigate whether necroptosis is associated with hepatic damage in dairy cows with ketosis. Dairy cows were selected as either the clinically healthy control group (n = 15) or a ketotic group diagnosed with ketosis, and were matched for lactation numbers (median = 3, range = 2-4) and DIM (median = 6 d, range = 3-9 d). Liver tissue samples were collected via percutaneous needle biopsy, and blood samples were obtained by coccygeal venipuncture. Primary bovine hepatocytes were isolated from the liver of 1-d-old calves. In vivo experiments showed significant increases in the activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl-transferase (GGT), and glutamate dehydrogenase (GLDH) in the blood of ketotic cows. Concurrently, the mRNA abundance of IL-6 (IL6), tumor necrosis factor-α (TNF), and IL-1β (IL1B) in the liver of ketotic cows also significantly increased. Histopathological examination revealed significant cytoplasmic vacuolation, lytic necrosis, and inflammatory cell infiltration in the liver of ketotic cows, along with increased CD11b immunofluorescence intensity. Furthermore, the expression levels of necroptosis-related proteins, including the ratio of phosphorylated receptor-interacting protein kinase 1 (p-RIPK1)/receptor-interacting protein kinase 1 (RIPK1), phosphorylated receptor-interacting protein kinase 3 (p-RIPK3)/receptor-interacting protein kinase 3 (RIPK3), and phosphorylated mixed lineage kinase domain-like pseudokinase (p-MLKL)/mixed lineage kinase domain-like pseudokinase (MLKL) significantly increased in the liver of ketotic cows. The mRNA abundance of RIPK1, RIPK3, and MLKL also significantly increased. Immunohistochemical analysis confirmed elevated p-MLKL and p-RIPK3 expression in the liver of ketotic cows. In vitro experiments showed that compared with the control group, bovine hepatocytes treated with 10 ng/mL TNF-α significantly activated necroptosis signaling, as ascertained by an increase in ratios of p-RIPK1/RIPK1, p-RIPK3/RIPK3, and p-MLKL/MLKL, and in the mRNA expression of RIPK1, RIPK3, and MLKL. Moreover, TNF-α treatment significantly upregulated the mRNA abundance of inflammatory cytokines IL1B and IL6, as well as the activity of ALT, AST, GGT, and GLDH in the culture medium of hepatocytes. Importantly, treatment with 100 µM Necrostatin 1 or 3 µM GSK-872 notably attenuated TNF-α-induced necroptosis signaling, inflammatory cytokine expression, and the activity of ALT, AST, GGT, and GLDH. In conclusion, these data suggest that necroptosis is associated with hepatic damage, and may be a potential therapeutic target to ameliorate liver dysfunction in dairy cows with ketosis.