Cadmium aggravates liver injury by activating ferroptosis and neutrophil extracellular traps formation in Nile tilapia (Oreochromis niloticus).

Cadmium (Cd) is a pervasive environmental contaminant and a significant risk factor for liver injury. The present study was undertaken to evaluate the involvement of ferroptosis and neutrophil extracellular traps (NETs) in Cd-induced liver injury in Nile tilapia (Oreochromis niloticus), and to explore its underlying mechanism. Cd-induced liver injury was associated with increased total iron, malondialdehyde (MDA), and Acyl-CoA synthetase long-chain family member 4 (ACSL4), together with reduced levels of glutathione, glutathione peroxidase-4a (Gpx4a), and solute carrier family 7 member 11 (SLC7A11), which are all hallmarks of ferroptosis. Moreover, liver hyperemia, neutrophil infiltration, increased inflammatory factors and myeloperoxidase, as well as elevated serum DNA content in Cd-stimulated Nile tilapia suggested that a considerable number of neutrophils were recruited to the liver. Furtherly, in vitro experiments demonstrated that Cd induced the formation of NETs, and the possible mechanism was related to the generation of reactive oxygen species and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, along with the P38 and extracellular regulated protein kinase (ERK) signaling pathways. We concluded that ferroptosis and NETs are the critical mechanisms contributing to Cd-induced liver injury in Nile tilapia. These findings will contribute to Cd toxicological studies in aquatic animals.

The release of zearalenone-induced heterophil extracellular traps in chickens is associated with autophagy, glycolysis, PAD enzyme, and P2X1 receptor.

Zearalenone (ZEA) is produced mainly by fungi belonging to genus Fusarium in foods and feeds. Heterophil extracellular traps (HETs) are a novel defense mechanism of chicken innate immunity involving activated heterophils. However, the conditions and requirements for ZEA-triggered HET release remain unknown. In this study, immunostaining analysis demonstrated that ZEA-triggered extracellular fibers were composed of histone and elastase assembled on DNA skeleton, showing that ZEA can induce the formation of HETs. Further experiments indicated that ZEA-induced HET release was concentration-dependent (ranging from 20 to 80 µM ZEA) and time-dependent (ranging from 30 to 180 min). Moreover, in 80 µM ZEA-exposed chicken heterophils, reactive oxygen species (ROS) level, catalase (CAT), superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and glutathione (GSH) content were increased. Simultaneously, ZEA at 80 µM activated ERK and p38 MAPK signaling pathways by increasing the phosphorylation level of ERK and p38 proteins. Pharmacological inhibition assays revealed that blocking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, ERK, and p38 mitogen-activated protein kinase (MAPK) reduced ZEA-induced ROS levels but had no impact on HET formation. Furthermore, immunostaining analysis indicated that the heterophil underwent the formation of autophagosome based on being stained with LC3B. The pharmacological inhibition assays demonstrated that rapamycin-, wortmannin-, and 3-methyladenine (3-MA)-treatments modulated ZEA-triggered HET formation, indicating that heterophil autophagy played a key role in ZEA-induced HET formation. Further studies on energy metabolism showed that inhibition of lactate/glucose transport, hexokinase-2 (HK-2), fructose-2,6-biphosphatase 3 (PFKFB3) in glycolysis abated ZEA-induced HETs, implying that glycolysis was one of the factors influencing the ZEA-induced HET formation. Besides, inhibition of the peptidylarginine deiminase (PAD) enzyme and P2X1 significantly reduced the ZEA-induced HET formation. In conclusion, we demonstrated that ZEA-triggered HET formation, which was associated with glycolysis, autophagy, PAD enzyme, and P2X1 receptor activation, providing valuable insight into the negative effect of ZEA on chicken innate immunity.

Exposure to ZnO nanoparticles induced blood-milk barrier dysfunction by disrupting tight junctions and cell injury.

Zinc oxide nanoparticles (ZnO-NPs) are one of the most widely used nanomaterials with excellent chemical and biological properties. However, their widespread application has led to increased risk to the natural environment and public health. A growing number of studies have shown that ZnO-NPs deposited in target organs interact with internal barriers to trigger injurious responses. The underlying mechanism of ZnO-NPs on the blood-milk barrier dysfunction remains to be understood. Our results revealed that excessive accumulation of ZnO-NPs induced histopathological injuries in the mammary gland, leading to the distribution of ZnO-NPs in the milk of lactating mice. A prominent diffusion of blood-milk barrier permeability marker, albumin-fluorescein isothiocyanate conjugate (FITC-albumin) was observed at cell-cell junction after ZnO-NPs exposure. Meanwhile, ZnO-NPs weakened the blood-milk barrier function by altering the expression of tight junction proteins. The excessive accumulation of ZnO-NPs also induced inflammatory response by activating the NF-κB and MAPK signaling pathways, leading to the dysfunctional blood-milk barrier. Furthermore, we found that ZnO-NPs led to increased iron accumulation and lipid oxidation, thus increasing oxidative injury and ferroptosis in mammary glands. These results indicated that ZnO-NPs weaken the integrity of the blood-milk barrier by directly affecting tight junctions and cellular injury in different ways.

Quercetin Alleviates Deoxynivalenol-Induced Intestinal Damage by Suppressing Inflammation and Ferroptosis in Mice.

Deoxynivalenol (DON), one of the most prevalent mycotoxins found in food and feed, can cause gastrointestinal inflammation and systemic immunosuppression, presenting a serious hazard to human and animal health. Quercetin (QUE) is a plant polyphenol with anti-inflammatory and antioxidant properties. In this research, we investigated the potential function of QUE as a treatment for DON-induced intestinal damage. Thirty male specific-pathogen-free BALB/c mice were randomly allocated to treatment with QUE (50 mg/kg) and/or DON (0, 0.5, 1, and 2 mg/kg). We found that QUE attenuated DON-induced intestinal damage in mice by improving jejunal structural injury and changing tight junction proteins (claudin-1, claudin-3, ZO-1, and occludin) levels. QUE also suppressed DON-triggered intestinal inflammation by inhibiting the TLR4/NF-κB signaling pathway. Meanwhile, QUE decreased the oxidative stress caused by DON by enhancing the concentrations of SOD and GSH, while diminishing the contents of MDA. In particular, QUE reduced DON-induced intestinal ferroptosis. DON-induced intestinal damage elevated TfR and 4HNE levels, along with transcription levels of ferroptosis-related genes (PTGS2, ACSL4, and HAMP1) while diminishing mRNA levels of FTH1, SLC7A11, GPX4, FPN1, and FSP1, all of which were reversed by QUE treatment. Our findings imply that QUE alleviates DON-induced intestinal injury in mice by inhibiting the TLR4/NF-κB signaling pathway and ferroptosis. In this study, we elucidate the toxicological mechanism of DON, provide a basic foundation or theory for future DON prevention and treatment, and explore strategies to prevent and alleviate DON's hazardous effects.

Investigations into ferroptosis in methylmercury-induced acute kidney injury in mice.

Methylmercury (MeHg) is a highly poisonous form of mercury and a risk factor for kidney impairment in humans that currently has no effective means of therapy. Ferroptosis is a non-apoptotic metabolic cell death linked to numerous diseases. It is currently unknown whether ferroptosis takes part in MeHg-induced kidney damage. Here, we established a model of acute kidney injury (AKI) in mice by gavage with different doses of MeHg (0, 40, 80, 160 µmol/kg). Serological analysis revealed elevated levels of UA, UREA, and CREA; H&E staining showed variable degrees of renal tubule injury; qRT-PCR detection displayed increased expression of KIM-1 and NGAL in the groups with MeHg treatment, indicated that MeHg successfully induced AKI. Furthermore, MDA levels enhanced in renal tissues of mice with MeHg exposure whereas GSH levels decreased; ACSL4 and PTGS2 nucleic acid levels elevated while SLC7A11 levels reduced; transmission electron microscopy illustrated that the density of the mitochondrial membrane thickened and the ridge reduced considerably; protein levels for 4HNE and TfR1 improved since GPX4 levels declined, all these results implying the involvement of ferroptosis as a result of MeHg exposure. Additionally, the observed elevation in the protein levels of NLRP3, p-p65, p-p38, p-ERK1/2, and KEAP1 in tandem with downregulated Nrf2 expression levels indicate the involvement of the NF-κB/NLRP3/MAPK/Nrf2 pathways. All the above findings suggested that ferroptosis and the NF-κB/NLRP3/MAPK/Nrf2 pathways are implicated in MeHg-induced AKI, thereby providing a theoretical foundation and reference for future investigations into the prevention and treatment of MeHg-induced kidney injury.

The release of FB1-induced heterophil extracellular traps in chicken is dependent on autophagy and glycolysis.

Fumonisin B1 (FB1), a worldwide contaminating mycotoxin produced by Fusarium, poses a great threat to the poultry industry. It was reported that extracellular traps could be induced by FB1 efficiently in chickens. However, the relevance of autophagy and glycolysis in FB1-triggered heterophil extracellular trap (HET) formation is unclear. In this study, immunostaining revealed that FB1-induced HETs structures were composed of DNA coated with histones H3, and elastase, and that heterophils underwent LC3B-related autophagosome formation assembly driven by FB1. Western blotting showed that FB1 downregulated the phosphorylated phosphoinositide 3-kinase3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin complex 1 (mTORC1) axis and raised the AMP-activated kinase α (AMPKα) activation protein. Furthermore, rapamycin- and 3-Methyladenine (3-MA)-treatments modulated FB1-triggered HET formation according to the pharmacological analysis. Further studies on energy metabolism showed that glucose/lactate transport and glycolysis inhibitors abated FB1-induced HETs. These results showed that FB1-induced HET formation might interact with the autophagy process and relied on glucose/monocarboxylic acid transporter 1 (MCT1) and glycolysis, reflecting chicken's early innate immune responses against FB1 intake.

The protective effects of myricetin against acute liver failure via inhibiting inflammation and regulating oxidative stress via Nrf2 signaling.

This study aimed to investigate the protective effects and mechanisms of myricetin on acute liver failure in mice induced by lipopolysaccharide (LPS)/D-galactosamine (D-Gal). Our results showed myricetin (25, 50 and 100 mg/kg) pretreatment significantly improved the pathological changes of liver tissues, decreased serum ALT and AST (p < 0.001) induced by LPS/D-GalN. Moreover, MDA and MPO levels were reduced (p < 0.001), CAT and SOD activities were increased (p < 0.001) with myricetin (50 and 100 mg/kg) pretreatment. Likewise, inflammatory cytokines TNF-α and IL-6 mRNA in liver tissues were markedly decreased (p < 0.001) by myricetin. Besides, Nrf2 protein expression was drastically elevated (p < 0.001) by myricetin (25, 50 and 100 mg/kg). All these findings imply that myricetin may protect against acute liver failure by suppressing inflammation and regulating oxidative stress via Nrf2 signaling, and that it may be a possible strategy to avoid liver damage.

Citrinin stimulated heterophil extracellular trap formation in chickens.

Citrinin (CTN), a secondary fungal metabolite produced by several Aspergillus, Penicillium, and Monascus genera species, is a toxin with a wide range of biological activities. Neutrophil extracellular traps represent a novel potential mechanism of the neutrophil response to foreign matters, and chicken heterophils can release similar heterophil extracellular traps (HETs). In this study, we aimed to investigate the effect of CTN on HET formation. Density gradient centrifugation was used to isolate chicken peripheral blood heterophils, and then immunofluorescence was used to observe the effects of CTN on HET formation. The mechanisms of HET formation were analyzed using pharmacological inhibitors and quantification of extracellular DNA, and the production of reactive oxygen species was detected with a fluorescent probe. Our results revealed that CTN (50-400 µM) had no cytotoxic effect on heterophils. CTN exposure induced the release of HETs composed of chromatin decorated with histones and elastase, and CTN-triggered HETs were dose- and time-dependent to some extent. Furthermore, CTN increased ROS production and activated p38 and ERK1/2 signaling pathways in heterophils. However, inhibition of the p38 signaling pathway, ERK1/2 signaling pathway, and NADPH oxidase pathway did not block HET formation induced by CTN. Inhibition of peptidyl arginine deiminase 4 (PAD4) enzyme and P2×1 receptor decreased HET formation after CTN stimulation, suggesting that HET formation exposed to CTN was mediated by PAD4 and P2×1 receptor. In conclusion, these findings may suggest a canonical mechanism relevant to the innate immunity caused by mycotoxins in chickens.

Lysine specific demethylase 1 inhibitor alleviated lipopolysaccharide/D-galactosamine-induced acute liver injury.

Acute liver injury is a severe clinical syndrome with markedly high mortality and poor prognosis. An accumulating body of evidence has demonstrated that epigenetic mechanisms have essential roles in the pathogenesis of acute liver injury. Lysine-specific demethylase 1 (LSD1) belongs to the amine oxidase superfamily of flavin adenine dinucleotide (FAD)-dependent enzymes, specifically demethylates H3 lysine 4. In the study, we investigated the effects and mechanisms of LSD1 in lipopolysaccharide (LPS)/D-Galactosamine (D-Gal)-induced acute liver injury in mice. Western blot analysis showed that LSD1 phosphorylation and di-methylated histone H3 on lysine 4 (H3K4me2) protein expression were significantly increased after LPS/D-Gal treatment (2.3 and 2.4 times higher than control respectively). GSK-LSD1 2HCl is an irreversible and selective LSD1 inhibitor. Pre-treatment with LSD1 inhibitor alleviated LPS/D-Gal-induced liver damage, decreased serum levels of alanine transaminase and aspartate aminotransferase in mice. Moreover, the LSD1 phosphorylation level in low, medium, and high LSD1 inhibitor groups was lower by a factor of 1.6, 1.9, and 2.0 from the LPS/D-Gal group, respectively. Mechanistically, LSD1 inhibitor further inhibited NF-κB signaling cascades and subsequently inhibited the production of pro-inflammatory cytokine TNF-α, IL-6, and IL-1ß induced by LPS/D-Gal in liver tissues. Furthermore, LSD1 inhibitor upregulated the protein expression of Nrf2/HO-1 signaling pathways, and the activities of related antioxidant enzymes were enhanced. Collectively, our data demonstrated that LSD1 inhibitor protected against the LPS/D-Gal-induced acute liver injury via inhibiting inflammation and oxidative stress, and targeting the epigenetic marker may be a potent therapeutic strategy for acute liver injury.