CuO-NPs-triggered heterophil extracellular traps exacerbate liver injury in chicks by promoting oxidative stress and inflammatory responses.

With the widespread use of copper oxide nanoparticles (CuO-NPs), their potential toxicity to the environment and biological health has attracted close attention. Heterophil extracellular traps (HETs) are an innate immune mechanism of chicken heterophils against adverse stimuli, but excessive HETs cause damage. Here, we explored the effect and mechanism of CuO-NPs on HETs formation in vitro and further evaluated the potential role of HETs in chicken liver and kidney injury. Heterophils were exposed to 5, 10, and 20 µg/mL of CuO-NPs for 2 h. The results showed that CuO-NPs induced typical HETs formation, which was dependent on NADPH oxidase, P38 and extracellular regulated protein kinases (ERK1/2) pathways, and glycolysis. In in vivo experiments, fluorescence microplate and morphological analysis showed that CuO-NPs elevated the level of HETs in chicken serum and caused liver and kidney damage. Meanwhile, CuO-NPs caused hepatic oxidative stress (MDA, SOD, CAT, and GSH-PX imbalance), and also induced an increase in mRNA expression of their inflammatory and apoptosis-related factors (IL-1ß, IL-6, TNF-α, COX-2, iNOS, NLRP3, and Caspase-1, 3, 11). However, these results were significantly altered by DNase I (HETs degradation reagent). In conclusion, the present study demonstrates for the first time that CuO-NPs induce the formation of HETs and that HETs exacerbate pathological damage in chicken liver and kidney by promoting oxidative stress and inflammation, providing insights into immunotoxicity and potential prevention and treatment targets caused by CuO-NPs overexposure.

Nanosilver-stimulated heterophil extracellular traps promoted liver and kidney injury in chicken.

Nanosilver is a metallic silver monomer with a diameter of <100 nm, which has excellent antibacterial activity and is widely used in the fields of medicine and sanitation, disinfection of drinking water in daily life and feed additives in livestock and poultry farming. Heterophil extracellular traps (HETs) are an important part of innate immunity in chickens and have an excellent antimicrobial effect, but their excessive release caused tissue damage. Nanosilver overdose caused toxic effects in chickens, while immunotoxic effects of nanosilver on chickens have not been reported. In this study, we explored the effects of nanosilver-induced HETs on chicken liver and kidney damage and further investigated the molecular mechanism of nanosilver-induced HETs release. The results showed that nanosilver significantly upregulated serum HETs and caused liver and kidney damage. The classical structure of nanosilver-induced HETs was also observed, and nanosilver-induced HETs were dependent on reactive oxygen species (ROS), extracellular regulatory protein kinase (ERK)1/2, p38 and glycolysis pathways. In summary, this research suggests that nanosilver induced HETs release, but excessive HETs release also caused damage to chicken. It also helps to understand the importance of moderate application of nanosilver, which may improve animal immunity but avoid negative effects in safeguarding the economic efficiency of poultry farming.

Alumina nanoparticles-induced heterophil extracellular traps exacerbate liver injury by regulating oxidative stress and inflammation in chickens.

Widely used alumina nanoparticles (Al2O3 NPs) exposed to the environment pose a serious threat to human and animal health. The formation of heterophil extracellular traps (HETs) is a mechanism of innate immune defense against infection, but excessive HETs cause pathological damage. Here, we aim to explore the influence and mechanism of Al2O3 NPs on the formation of HETs in vitro, and further investigate the role of HETs release in histopathological damage after Al2O3 NPs treatment. Immunofluorescence analysis showed that Al2O3 NPs induced the formation of HETs, which was characterized by modified histones and elastase in the DNA backbone. Fluorescence microplate analysis showed that HETs formation was dependent on NADPH oxidase, P38, extracellular regulated protein kinases (ERK1/2) pathways and glycolysis. In vivo investigation showed that Al2O3 NPs significantly caused HETs release and liver damage. Biochemical analysis showed that Al2O3 NPs inhibited the activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX). Real-time fluorescence quantification results showed that Al2O3 NPs caused the overexpression of inflammation-related molecules interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), caspase-1 and caspase-11. All these changes were significantly changed by DNase I (Degradation reagent for HETs). Together, these suggest that Al2O3 NPs-induced HETs exacerbate liver injury by regulating oxidative stress and inflammatory responses, which provide a new perspective and potential prophylaxis and treatment targets for Al2O3 NPs toxicological research.

Aflatoxin B1-activated heterophil extracellular traps result in the immunotoxicity to liver and kidney in chickens.

Aflatoxin B1 (AFB1) is a mycotoxin with strong toxicity and play a large proportion in aspergillosis. Heterophil extracellular traps (HETs) was considered as an innate immune response of chickens to resist pathogens. AFB1 has been reported to trigger macrophages extracellular traps (METs) in THP-1 cells and RAW264.7 cells, but whether AFB1 could also activate HETs release, and the mechanism underlying AFB1-activated HETs in chicken remains unclear. In this study, we confirmed that AFB1could induce HETs release, which was a network of DNA-based structures consist of citrullinated histone 3 (citH3) and elastase. Meanwhile, AFB1-activated HETs rely on the glycolytic process to provide energy, NADPH oxidase and p38 signaling pathway. Moreover, it has been verified that AFB1-activated HETs release could significantly increase the biochemical indexes of liver (ALT and AST) and kidney (CRE and BUN) in serum. In addition, histopathological observation showed that AFB1 caused swelling, necrosis and vacuolation of hepatocytes in liver, and necrosis, exfoliated of nephrocyte in kidney. Further investigation demonstrated that AFB1 significantly decreased the levels of SOD and GSH-PX but increased the level of MDA, and meanwhile induced the mRNA expressions of TNF-α, IL-6 and IL-1ß, iNOS, COX-2, NLRP3, caspase-1, caspase-3 and caspase-11. However, all these AFB1-induced biochemical indexes and histopathological changes were effectively alleviated by DNase I (the standard degradant for HETs). In conclusion, it has preliminary confirmed that AFB1-activated HETs formation contributed to the immunotoxicity in chicken and provide new strategies for the therapy in aspergillosis.

Morin alleviates aflatoxin B1-induced liver and kidney injury by inhibiting heterophil extracellular traps release, oxidative stress and inflammatory responses in chicks.

Aflatoxin B1 (AFB1) is a secondary metabolite produced by Aspergillus flavus and parasitic aspergillus, mainly existing in cereals, peanuts, corn, and other crops, which seriously endanger poultry, human health, and environment. Morin, a flavonoid compound extracted from moraceae plants, possess antioxidant, antibacterial, and anti-inflammatory effects. However, whether morin has a protective effect on AFB1-induced liver and kidney damage in chicks has not been specifically reported. In this study, we mainly confirmed the protective effect of morin on AFB1-induced liver and kidney damage in chicks and clarified its mechanism. It was found that morin can significantly reduce the liver biochemical indicators of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and kidney indicators of creatinine (CRE) and urea nitrogen (BUN) levels. Meanwhile, histopathological examination showed that morin effectively relieved AFB1-caused liver damage, including hepatocyte disruption, swelling, and inflammatory cell infiltration, and effectively relieved kidney damage, including renal cell necrosis, exfoliation, and vacuolization. Further investigation of its mechanism demonstrated that morin significantly inhibited AFB1-induced heterophil extracellular traps (HETs) release, and decreased the level of malondialdehyde (MDA) but increased the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in vivo. Moreover, quantitative real-time PCR (qRT-PCR) analysis showed that morin also significantly decreased AFB1-induced mRNA expressions of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), caspase-1, caspase-3, and caspase-11. In conclusion, all results confirmed that morin could protect AFB1-caused liver and kidney damage by inhibiting HETs release, regulating oxidative stress, and inhibiting inflammatory response, suggesting that morin can be utilized as a potential drug for prevention and treatment of aflatoxicosis in poultry breeding industry.

Baicalin protects against zearalenone-induced chicks liver and kidney injury by inhibiting expression of oxidative stress, inflammatory cytokines and caspase signaling pathway.

Zearalenone (ZEA) is a secondary metabolite produced by fungi such as Fusarium and Fusarium flavum, which is classified as a mycotoxin. Crops and feed in a humid surrounding are widely polluted by ZEA, which further endangering the healthful aquaculture of poultry and even human health. Up to now, prevention and cure of mycotoxicosis is still a crucial subject of poultry husbandry. Baicalin (BAI) is a flavonoid refined from dried roots of Scutellaria baicalensis possessing the function of hepatoprotective, anti-inflammatory, anti-oxidant, and anti-atherosclerotic efficacies.etc. But whether Baicalin also has a protective effect against ZEA intoxication is unclear. Therefore, the aim of this study was to establish a model of ZEA-induced toxic injury in chicks, and then to investigate the way in which Baicalin plays a protective role in the mechanism of ZEA-induced liver and kidney injury in chicks. The results exhibit that Baicalin could not only significantly decrease aspartate aminotransferase (AST) , alanine aminotransferase (ALT) and creatinine (Cre) levels in serum, but also ameliorate ZEA-induced pathologic changes of liver and kidney. Baicalin could also significantly regulate ZEA-induced the changes of catalase (CAT) , malondialdehyde (MDA) , total sulfhydryl group , except for glutathione peroxidase (GSH-px) , and inhibit the mRNA levels of inflammatory cytokines tumor necrosis factor-α (TNF-α) , interleukin-1ß (IL-1ß) and cyclooxygenase-2 (COX-2) with caspase-3 and caspase-11 in the caspase signaling pathway , meanwhile inhibit the cell apoptosis in immunohistochemistry. In summary, we successfully established a model of ZEA-induced liver injury in chicks, and confirm that Baicalin can reduce ZEA-induced liver and kidney injury in chicks. The mechanism of these effects is via inhibiting inflammation, oxidative stress and apoptosis, which also indicates the potential applicability of Baicalin for the prevention and treatment of ZEA-induced toxicity in chicks.