Long-term Cu exposure alters CYP450s activity and induces jejunum injury and apoptosis in broilers.

Copper (Cu) is an essential trace element that plays a crucial role in numerous physiopathological processes related to human and animal health. In the poultry industry, Cu is used to promote growth as a feed supplement, but excessive use can lead to toxicity on animals. Cytochrome P450 enzymes (CYP450s) are a superfamily of proteins that require heme as a cofactor and are essential for the metabolism of xenobiotic compounds. The purpose of this study was to explore the influence of exposure to Cu on CYP450s activity and apoptosis in the jejunum of broilers. Hence, we first simulated the Cu exposure model by feeding chickens diets containing different amounts of Cu. In the present study, histopathological observations have revealed morphological damage to the jejunum. The expression levels of genes and proteins of intestinal barrier markers were prominently downregulated. While the mRNA expression level of the gene associated with CYP450s was significantly increased. Additionally, apoptosis-related genes and proteins (Bak1, Bax, Caspase-9, Caspase-3, and CytC) were also significantly augmented by excessive Cu, while simultaneously decreasing the expression of Bcl-2. It can be concluded that long-term Cu exposure affects CYP450s activity, disrupts intestinal barrier function, and causes apoptosis in broilers that ultimately leads to jejunum damage.

Rapid detection of SARS-CoV-2 with CRISPR-Cas12a.

The recent outbreak of betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is responsible for the Coronavirus Disease 2019 (COVID-19) global pandemic, has created great challenges in viral diagnosis. The existing methods for nucleic acid detection are of high sensitivity and specificity, but the need for complex sample manipulation and expensive machinery slow down the disease detection. Thus, there is an urgent demand to develop a rapid, inexpensive, and sensitive diagnostic test to aid point-of-care viral detection for disease monitoring. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) 12a-based diagnostic method that allows the results to be visualized by the naked eye. We also introduced a rapid sample processing method, and when combined with recombinase polymerase amplification (RPA), the sample to result can be achieved in 50 minutes with high sensitivity (1-10 copies per reaction). This accurate and portable detection method holds a great potential for COVID-19 control, especially in areas where specialized equipment is not available.

miR-181b-1-3p affects the proliferation and differentiation of chondrocytes in TD broilers through the WIF1/Wnt/ß-catenin pathway.

Thiram is a plant fungicide, its excessive use has exceeded the required environmental standards. It causes tibial dyschondroplasia (TD) in broilers which is a common metabolic disease that affects the growth plate of tibia bone. It has been studied that many microRNAs (miRNAs) are involved in the differentiation of chondrocytes however, their specific roles and mechanisms have not been fully investigated. The selected features of tibial chondrocytes of broilers were studied in this experiment which included the expression of miR-181b-1-3p and the genes related to WIF1/Wnt/ß-catenin pathway in chondrocytes through qRT-PCR, western blot and immunofluorescence. The correlation between miR-181b-1-3p and WIF1 was determined by dual luciferase reporter gene assay whereas, the role of miR-181b-1-3p and WIF1/Wnt/ß-catenin in chondrocyte differentiation was determined by mimics and inhibitor transfection experiments. Results revealed that thiram exposure resulted in decreased expression of miR-181b-1-3p and increased expression of WIF1 in chondrocytes. A negative correlation was also observed between miR-181b-1-3p and WIF1. After overexpression of miR-181b-1-3p, the expression of ACAN, ß-catenin and Col2a1 increased but the expression of GSK-3ß decreased. It was observed that inhibition of WIF1 increased the expression of ALP, ß-catenin, Col2a1 and ACAN but decreased the expression of GSK-3ß. It is concluded that miR-181b-1-3p can reverse the inhibitory effect of thiram on cartilage proliferation and differentiation by inhibiting WIF1 expression and activating Wnt/ß-catenin signaling pathway. This study provides a new molecular target for the early diagnosis and possible treatment of TD in broilers.

Exposure to copper activates mitophagy and endoplasmic reticulum stress-mediated apoptosis in chicken (Gallus gallus) cerebrum.

A large amount of copper (Cu) used in production activities can lead to the enrichment of Cu in the environment, which can cause toxicity to animals. However, the toxicity mechanism of Cu on the cerebrum is still uncertain. Hence, a total of 240 chickens were separated into four groups in this study to reveal the potential connection between mitophagy and endoplasmic reticulum (ER) stress-mediated apoptosis in the chicken cerebrum in the case of excess Cu exposure. The cu exposure situation was simulated by diets containing various levels of copper (11 mg/kg, control group; 110 mg/kg, group I; 220 mg/kg, group II and 330 mg/kg, group III) for 49 days. The results of histology showed that vacuolar degeneration was observed in the treated groups, and the mitochondria swell and autophagosomes formation were found under excess Cu treatment. Additionally, the expression of mitophagy (PINK1, Parkin, LC3I, LC3II and p62) and ER stress (GRP78, PERK, ATF6, IRE1α, XBP1, CHOP, and JNK) indexes were significantly upregulated under excess Cu exposure. Furthermore, the mRNA and protein expression of Bcl-2 were decreased, while Bak1, Bax, Caspase12, and Caspase3 were increased compared to the control group. In summary, this study demonstrated that an overdose of Cu could induce mitophagy and ER stress-mediated apoptosis in the chicken cerebrum. These findings revealed an important potential connection between Cu toxicity and cerebrum damage, which provided a new insight into Cu neurotoxicity.

Arsenic causes mitochondrial biogenesis obstacles by inhibiting the AMPK/PGC-1α signaling pathway and also induces apoptosis and dysregulated mitophagy in the duck liver.

Arsenic is a dangerous metalloid-material which is known to cause liver injury in many animals and humans. However, little is known about the underlying mechanism of arsenic-induced hepatotoxicity in poultry. This study was executed to systematically investigate the potential role of mitochondrial biogenesis, mitophagy and apoptosis in duck hepatotoxicity caused by arsenic. Results showed that the body weight and liver coefficient of duck had distinct changed after arsenic-exposure, and the arsenic content in serum and liver also increased significantly in a dose-dependent manner. Meanwhile, histopathological examination and metabolomics results showed that arsenic-exposure caused severe steatosis and metabolism disorder in liver tissues. Furthermore, arsenic-exposure significantly inhibited AMPK/PGC-1α-mediated mitochondrial biogenesis, determined by the ultrastructure observation and down-regulation of p-AMPKα/AMPKα, PGC-1α, NRF1, NRF2, TFAM, TFB1M, TFB2M and COX-Ⅳ expression levels. Besides, arsenic-treatment obviously increased the levels of mitophagy (PINK1, Parkin, LC3, P62) and pro-apoptotic (Caspase-3, Caspase-9, Cleaved Caspase-3, Cytc, Bax, P53) indexes, and simultaneously resulted in reductions in anti-apoptosis index (Bcl-2). Overall, our findings provided evidences that arsenic-induced duck hepatotoxicity may be caused by a combination of impaired mitochondrial biosynthesis, mitophagy, and mitochondrial-dependent apoptosis. To our knowledge, this is the first report to systematically investigate the potential mechanism of arsenic-induced hepatotoxicity in poultry.

Copper induces mitochondria-mediated apoptosis via AMPK-mTOR pathway in hypothalamus of Pigs.

Copper (Cu), one of the heavy metals, is far beyond the carrying capacity of the environment with Cu mining, industrial wastewater discharging and the use of Cu-containing pesticides. Intaking excess Cu can cause toxic effects on liver, kidney, heart, but few studies report Cu toxicity on brain tissue. It is noteworthy that most toxicity tests are based on rodent models, but large mammals chosen as animal models has no reported. To explore the relationship of the Cu toxicity and mitochondria-mediated apoptosis on hypothalamus in pigs, the content of Cu, histomorphology, mitochondrial related indicators, apoptosis, and AMPK-mTOR signaling pathway were detected. Results showed that Cu could accumulate in hypothalamus and lead to mitochondrial dysfunction, evidenced by the decrease of ATP production, activities of respiratory chain complex I-IV, and mitochondrial respiratory function in Cu-treated groups. Additionally, the genes and proteins expression of Bax, Caspase-3, Cytc in treatment group were higher than control group. Furthermore, the protein level of p-AMPK was enhanced significantly and p-mTOR was declined, which manifested that AMPK-mTOR signaling pathway was activated in Cu-treated groups. In conclusion, this study illuminated that the accumulation of Cu could cause mitochondrial dysfunction, induce mitochondria-mediated apoptosis and activate AMPK-mTOR pathway in hypothalamus.

Metabolomics analysis reveals the effect of copper on autophagy in myocardia of pigs.

Among different synthetic compounds copper (Cu) is persistently and frequently used as growth promoter, antibacterial, antifungal and antiparasitic agent and has become common environmental pollutant. Therefore, this study explores the cardio-toxic effects of control group (10 mg/kg bw Cu) and treatment group (125 and 250 mg/kg bw Cu), and it association with process of autophagy and metabolomics in myocardium of pigs kept in three different experimental treatments for a period of 80 days. The results of serum biochemical parameters showed a significantly increase in creatinine kinase (CK), creatine kinase-MB (CK-MB), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C) and aspartate aminotransferase (AST) in pigs exposed to 125 mg/kg bw and 250 mg/kg bw Cu. Meanwhile, the severe structural abnormalities in cardiomyocytes were found when exposed to 250 mg/kg Cu at day 80. In addition, the mRNA and proteins (Beclin1, ATG5 and LC3II) expression levels were significantly increased and p62 was significantly decreased in cardiomyocytes exposed to 250 mg/kg Cu at day 80 of the trial. Further, UPLC-QTOF/MS technique showed that 7 metabolites were up-regulated and 37 metabolites were down-regulated in cardiomyocytes after 250 mg/kg Cu treatment, with a principal impact on the metabolic pathways including glycerophospholipid metabolism, one carbon pool by folate, fatty acid elongation and fatty acid degradation, which were related to autophagy. Overall, our study identified the autophagy processes and metabolites in metabolic pathways in Cu-induced myocardium injury, which provided useful evidence of myocardium toxicity caused by Cu exposure via metabolomics and multiple bioanalytic methods.

Long-term exposure to the fluoride blocks the development of chondrocytes in the ducks: The molecular mechanism of fluoride regulating autophagy and apoptosis.

Long-term exposure to excessive fluoride causes chronic damage in the body tissues and could lead to skeletal and dental fluorosis. Cartilage damage caused by excessive fluoride intake has gained wide attention, but how fluoride accumulation blocks the development of chondrocytes is still unclear. Here, we report a negative correlation between the length and growth plate width after NaF treatments via apoptosis and autophagy, with shrinkage of cells, nuclear retraction, dissolution of chondrocytes. Whereas, fluoride exposure had no significant effect on the number and distribution of the osteoclasts which were well aligned. More importantly, fluoride exposure induced apoptosis of tibial bone through CytC/Bcl-2/P53 pathways via targeting Caspase3, Caspase9, Bak1, and Bax expressions. Meanwhile, the Beclin1, mTOR, Pakin, Pink, and p62 were elevated in NaF treatment group, which indicated that long-term excessive fluoride triggered the autophagy in the tibial bone and produced the chondrocyte injury. Altogether, fluoride exposure induced the chondrocyte injury by regulating the autophagy and apoptosis in the tibial bone of ducks, which demonstrates that fluoride exposure is a risk factor for cartilage development. These findings revealed the essential role of CytC/Bcl-2/P53 pathways in long-term exposure to fluoride pollution and block the development of chondrocytes in ducks, and CytC/Bcl-2/P53 can be targeted to prevent fluoride induced chondrocyte injury.

Arsenic (III) and/or Antimony (III) induced disruption of calcium homeostasis and endoplasmic reticulum stress resulting in apoptosis in mice heart.

Arsenic (As) and antimony (Sb) are known as an environmental contaminant with cardiotoxicity properties. The endoplasmic reticulum (ER) is the largest calcium reservoir in the cell, and its calcium homeostasis disorder plays a vital role in endoplasmic reticulum stress (ERS) and apoptosis. The objective of this study was to investigate whether As and Sb induced apoptosis via endoplasmic reticulum stress (ERS) linked to calcium homeostasis disturbance. In this study, thirty-two adult mice were gavage-fed daily with As2O3 (4 mg/kg), SbCl3 (15 mg/kg) and co-treat with SbCl3 (15 mg/kg) and As2O3 (4 mg/kg) daily for 60 days. It was observed that As or/and Sb caused histopathological lesions and ER expansion of the heart. Meanwhile, the gene expression of ER Ca2+ release channels (RyR2 and IP3R) and calmodulin-dependent protein kinase II (CaMKII) increased while the levels of mRNA and protein of ER Ca2+ uptake channel (SERCA2) downregulated significantly compared to the controls. Then, As or/and Sb induced ERS and triggered the ER apoptotic pathway by activating unfolded protein response (UPR)-associated genes ((PERK, ATF6, IRE1, XBP1, JNK, GRP78), and apoptosis-related genes (Caspase12, Caspase3, p53, CHOP). Above indicators in As + Sb group became more severe than that of As group and Sb group. Overall, our results proved that the cardiotoxicity caused by As or/and Sb might be concerning disturbing calcium homeostasis, which induced apoptosis through the ERS pathway.

Copper exposure induces mitochondrial dynamic disorder and oxidative stress via mitochondrial unfolded protein response in pig fundic gland.

Cu is a metallic element that widely spread over in the environment, which have raised wide concerns about the potential toxic effects and public health threat. The objective of this study aimed to investigate the impression of copper (Cu)-triggered toxicity on mitochondrial dynamic, oxidative stress, and unfolded protein response (UPRmt) in fundic gland of pigs. Weaned pigs were randomly distributed into three groups, fed with different Cu of 10 mg/kg (control group), 125 mg/kg (group I), and 250 mg/kg (group Ⅱ). The trial persisted for 80 days and the fundic gland tissues were collected for further researches. Moreover, the markers participated to mitochondrial dynamic, UPRmt，and oxidative stress in fundic gland were determined. Results revealed that vacuolar degeneration were observed in the treated groups contrast with control group, and the Cu level was boosted with the increasing intake of Cu. Besides that, the levels of CAT, TRX, H2O2, and G6PDH were reduced in group Ⅰ and group Ⅱ, the mRNA levels of NRF2, HO-1, SOD-1, CAT, SOD-2, GSR, GPX1, GPX4, and TRX in the treated groups were promoted contrast to control group. Furthermore, the protein expression of KEAP1 was dramatically decreased, and the protein expression of NRF2, TRX and HO-1 were markedly enhanced in group Ⅰ and Ⅱ at 80 days. Moreover, the mRNA and protein expression levels of MFN1, MFN2, and OPA1 down-regulated and protein level of DRP1 was increased with the adding levels of Cu. Nevertheless, the UPRmt-related mRNA levels of CLPP, HTRA-2, CHOP, HSP10, and HSP60 were enhanced dramatically in Cu treatment group compared with control group. In general, our current study demonstrated that excessive absorption of Cu in fundic gland were related with stimulating UPRmt, oxidative stress, and the NRF2 interceded antioxidant defense. These results could afford an updated evidence on molecular theory of Cu-invited toxicity.

Metabolomics and transcriptomics indicated the molecular targets of copper to the pig kidney.

Copper poses huge environmental and public health concerns due to its widespread and persistent use in the past several decades. Although it is well established that at higher levels copper causes nephrotoxicity, the exact mechanisms of its toxicity is not fully understood. Therefore, this experimental study for the first time investigates the potential molecular mechanisms including transcriptomics, metabolomics, serum biochemical, histopathological, cell apoptosis and autophagy in copper-induced renal toxicity in pigs. A total of 14 piglets were randomly assigned to two group (7 piglets per group) and treated with a standard diet (11 mg CuSO4 per kg of feed) and a high copper diet (250 mg CuSO4 per kg of feed). The results of serum biochemical tests and renal histopathology suggested that 250 mg/kg CuSO4 in the diet significantly increased serum creatinine (CREA) and induced renal tubular epithelial cell swelling. Results on transcriptomics and metabolomics showed alteration in 804 genes and 53 metabolites in kidneys of treated pigs, respectively. Combined analysis of transcriptomics and metabolomics indicated that different genes and metabolism pathways in kidneys of treated pigs were involved in glycerophospholipids metabolism and glycosphingolipid metabolism. Furthermore, copper induced mitochondrial apoptosis characterized by increased bax, bak, caspase 3, caspase 8 and caspase 9 expressions while decreased bcl-xl and bcl2/bax expression. Exposure to copper decreased the autophagic flux in terms of increased number of autophagosomes, beclin1 and LC3b/LC3a expression and p62 accumulation. These results indicated that the imbalance of glycosphingolipid metabolism, the impairment of autophagy and increase mitochondrial apoptosis play an important role in copper induced renal damage and are useful mechanisms to understand the mechanisms of copper nephrotoxicity.

Copper induces energy metabolic dysfunction and AMPK-mTOR pathway-mediated autophagy in kidney of broiler chickens.

To explore the effects of copper (Cu) on energy metabolism and AMPK-mTOR pathway-mediated autophagy in kidney, a total of 240 one-day-old broiler chickens were randomized into four equal groups and fed on the diets with different levels of Cu (11, 110, 220, and 330 mg/kg) for 49 d. Results showed that excess Cu could induce vacuolar degeneration and increase the number of autophagosomes in kidney, and the adenosine triphosphate (ATP) level and mRNA levels of energy metabolism-related genes were decreased with the increasing dietary Cu level. Moreover, immunohistochemistry and immunofluorescence showed that the positive expressions of Beclin1 and LC3-II were mainly located in cytoplasm of renal tubular epithelial cells and increased significantly with the increasing levels of Cu. The mRNA levels of Beclin1, Atg5, LC3-I, LC3-II, Dynein and the protein levels of Beclin1, Atg5, LC3-II/LC3-I and p-AMPKα1/AMPKα1 were markedly elevated in treated groups compared with control group (11 mg/kg Cu). However, the mRNA and protein levels of p62 and p-mTOR/mTOR were significantly decreased with the increasing levels of Cu. These results suggest that impaired energy metabolism induced by Cu may lead to autophagy via AMPK-mTOR pathway in kidney of broiler chickens.

Copper induces oxidative stress with triggered NF-κB pathway leading to inflammatory responses in immune organs of chicken.

Copper (Cu) is a necessary trace mineral due to its biological activity. Excessive Cu can induce inflammatory response in humans and animals, but the underlying mechanism is still unknown. Here, 240 broilers were used to study the effects of excessive Cu on oxidative stress and NF-κB-mediated inflammatory responses in immune organs. Chickens were fed with diet containing different concentrations of Cu (11, 110, 220, and 330 mg of Cu/kg dry matter). The experiment lasted for 49 days. Spleen, thymus, and bursa of Fabricius (BF) on day 49 were collected for histopathological observation and assessment of oxidative stress status. Additionally, the mRNA and protein levels of NF-κB and inflammatory cytokines were also analyzed. The results indicated that excess Cu could increase the number and area of splenic corpuscle as well as the ratio of cortex and medulla in thymus and BF. Furthermore, excessive Cu intake could decrease activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px); but increase contents of malondialdehyde (MDA), TNF-α, IL-1, IL-1ß; up-regulate mRNA levels of TNF-α, IFN-γ, IL-1, IL-1ß, IL-2, iNOS, COX-2, NF-κB and protein levels of TNF-α, IFN-γ, NF-κB, p-NF-κB in immune organs. In conclusion, excessive Cu could cause pathologic changes and induce oxidative stress with triggered NF-κB pathway, and might further regulate the inflammatory response in immune organs of chicken.

Long-term exposure to copper induces autophagy and apoptosis through oxidative stress in rat kidneys.

Copper (Cu) is an essential trace element for most organisms. However, excessive Cu can be highly toxic. The purpose of this study was to elucidate the mechanism underlying Cu toxicity in the kidneys of rats after treatment with CuCl2 (15 [control], 30, 60, or 120 mg/kg in the diet) for 180 days. Histological and ultrastructural changes, antioxidant enzyme activity, and the mRNA and protein levels of apoptosis and autophagy-related genes were measured. The results showed that Cu exposure led to significant accumulation of copper in kidneys and disorganized kidney morphology. The activities of total anti-oxidation capacity (T-AOC) and superoxide dismutase (SOD) in the kidneys decreased significantly, while the malondialdehyde (MDA) content increased. Furthermore, excessive Cu markedly upregulated the expression of autophagy and apoptosis-related genes (LC3A, LC3B, ATG-5, Beclin-1, Caspase3, CytC, P53, Bax), but downregulated the expression of P62, mTOR and BCL-2. Moreover, the LC3B/LC3A, ATG-5, Beclin-1, P53, Caspase3 proteins were up-regulated while P62 was down-regulated in the kidney tissues of the treatment groups. Overall, these findings provide strong evidence that excess Cu can trigger autophagy and apoptosis via the mitochondrial pathway by inducing oxidative stress in rat kidneys.

Effect of a background Ca2+ entry pathway mediated by TRPC1 on myocardial damage of broilers with induced ascites syndrome.

Ascites syndrome (AS) in chickens is associated with profound vascular remodelling and increased pulmonary artery pressure as well as right ventricular hypertrophy. Classical transient receptor potential cation channels (TRPCs) are key regulators of cardiac hypertrophy that act via regulation of calcium influx in mammals. We investigated whether classical transient receptor potential channels in chickens with right ventricular hypertrophy still possess this mechanism for regulating Ca2+ flux. Intravenous injection of cellulose particles was successfully used to induce AS in chickens, and tissues were examined 22 days after treatment. The chickens in the test group showed cardiac hypertrophy with oedema of the cardiac muscle and disruption of myofilaments. The right-to-total ventricle weight ratio (RV/TV), the levels of serum aspartate aminotransferase (AST) and creatine kinase (CK) of the test group were significantly higher than in the control group. Intracellular calcium levels were significantly increased in cardiomyocytes from chickens in the test group. Gene expression of TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 in heart tissues from the test group showed no significant differences compared with controls. However, TRPC1 protein levels, as well as mRNA levels, were down-regulated in the heart muscle of AS chickens (P < 0.05). Although we observed an increase in calcium concentration, the expression of TRPC1 decreased in cardiac cells. We hypothesized that an increase in intracellular free calcium concentration could inversely regulate calcium channel expression. RESEARCH HIGHLIGHTS Intracellular Ca2+ levels were increased in the myocardium of AS broilers. Expression of TRPC1, which mediates calcium influx, was decreased in the myocardium of AS broilers. The relationship between intracellular Ca2+ levels and expression of TRPC1 requires further study.

Effects of copper on oxidative stress and autophagy in hypothalamus of broilers.

The purpose of this research was to discuss the effects of copper (Cu)-induced toxicity on oxidative stress and autophagy in hypothalamus of broilers. In this study, 240 one-day-old broilers were randomly divided into 4 groups and the contents of dietary Cu in 4 groups were 11 mg/kg (control group), 110 mg/kg (group I), 220 mg/kg (group II), and 330 mg/kg (group III). The experiment lasted for 49 days and the hypothalamus tissues were collected for histological observation and detection of Cu content. Additionally, the indicators related to oxidative stress in hypothalamus were determined. Moreover, the mRNA expression levels of autophagy-related genes and the protein expression levels of Beclin1, LC3-II/LC3-I, and p62 in hypothalamus were measured. Results showed that the treated groups were observed vacuolar degeneration in hypothalamus compared to control group, and the Cu content in hypothalamus was increased with the increase of dietary Cu. Furthermore, the activities of SOD, CAT, T-AOC were increased in group I and group II and then decreased in group III, and the content of MDA and the mRNA levels of Nrf2, HO-1, SOD-1, CAT, GCLC, GCLM, and GST in treated groups were elevated compared to control group. Moreover, the mRNA expression levels of Beclin1, Atg5, LC3-I, LC3-II and the protein expression levels of Beclin1 and LC3-II/LC3-I up-regulated significantly with the increasing levels of Cu. However, the mRNA expression levels of p62 and mTOR and the protein expression level of p62 down-regulated remarkably. Taken together, our present study evidenced that excessive intake of Cu could induce oxidative stress and autophagy in hypothalamus of broilers.

Inhibition of Caspase-1-dependent pyroptosis attenuates copper-induced apoptosis in chicken hepatocytes.

The purpose of this study was to investigate the effects of copper (Cu) on hepatocyte pyroptosis and the relationship between pyroptosis and apoptosis in the mechanisms of Cu toxicity. Primary chicken hepatocytes were cultured in different concentrations of Cu sulfate (CuSO4) (0, 10, 50, and 100 µM), N-acetylcysteine (NAC) (1 mM), and Z-YVAD-fluoromethylketone (Z-YVAD-FMK) (10 µM) for 24 h, and the combination of Cu and NAC or Z-YVAD-FMK for 24 h. Cellular morphology and function, cell viability, mitochondria membrane potential (MMP), apoptosis rate, mRNA expression of pyroptosis-related and apoptosis-related genes, and Caspase-1, Caspase-3 proteins expression were determined. These results indicated that Cu markedly induced the mRNA expression of pyroptosis-related genes (Caspase-1, IL-1ß, IL-18, and NLRP3) and Caspase-1 protein expression. Furthermore, contents of Caspase-1, IL-1ß, and IL-18 in the supernatant fluid of culture hepatocytes were significantly increased in hepatocytes. NAC relieved excess Cu-caused the changes of above genes and proteins. Additionally, Z-YVAD-FMK, caspase-1 inhibitor, which attenuated Cu-induced the increased lactic dehydrogenase (LDH), aspartate amino transferase (AST), alanine aminotransferase (ALT) activities. Furthermore, treatment with Cu and Z-YVAD-FMK could down-regulate the mRNA levels of Caspase-3, Bak1, Bax, and CytC and Caspase-3 protein expression, up-regulate the mRNA expression of Bcl2, increase the MMP and reduce cell apoptosis compared to treatment with Cu in hepatocytes. Collectively, these finding evidenced that excess Cu induced pyroptosis by generating ROS in hepatocytes, and the inhibition of Caspase-1-dependent pyroptosis might attenuate Cu-induced apoptosis.

Mitochondrial Protein Profile in Mice with Low or Excessive Selenium Diets.

Dietary selenium putatively prevents oxidative damage, whereas excessive selenium may lead to animal disorder. In this study, we investigated the effects of low and excessive levels of dietary selenium on oxidative stress and mitochondrial proteins in mouse liver. Six to eight week old mice were fed a diet with low, excessive, or moderate (control) levels of selenium (sodium selenite). The selenium concentration and oxidative stress-related parameters in hepatic mitochondria were evaluated. Two-dimensional electrophoresis and mass spectrometry were applied to identify the differentially-expressed proteins associated with dietary selenium. The selenium content of the livers in mice with the low selenium diet was significantly lower than that of the control, while that of mice fed excessive levels was significantly higher. In both groups oxidative stress in hepatic mitochondria was found; accompanied by lower superoxide dismutase (SOD) and glutathione peroxidase (GPX) levels and higher malondialdehyde (MDA) content, compared with the control group. Furthermore, ten proteins in the hepatic mitochondria of the selenium-low or -excessive groups with more than two-fold differences in abundance compared with the control group were identified. The differentially-expressed proteins in hepatic mitochondria may be associated with dietary (low or excessive) selenium-induced oxidative stress.

Multi-omics reveals the protective effects of curcumin against AFB1-induced oxidative stress and inflammatory damage in duckling intestines.

Aflatoxin B1 (AFB1) is the most prevalent and toxic class of aflatoxins, which is considered a significant risk factor for food safety. Curcumin, a phytoconstituent with anti-inflammatory and antioxidant properties, has potential therapeutic value for intestinal inflammatory diseases. In this study, the duckling model susceptible to AFB1 was selected for toxicity testing, aiming to explore the effect of curcumin on AFB1 enterotoxicity and its possible mechanism of action. The results showed that curcumin promoted the growth and development of ducklings and mitigated the changes in morphology and permeability serological index (DAO and D-LA) after AFB1 exposure. Curcumin also mitigated AFB1-induced oxidative stress by activating the Nrf2 pathway, and ameliorated intestinal inflammation by inhibiting the NF-κB/IκB signaling pathway and boosting intestinal autophagy. In terms of gut flora and their metabolites, we found that curcumin supplementation significantly increased the intestinal flora's abundance index and diversity index compared to the AFB1 group, mitigating the decline in the abundance of Actinobacteria and the rise in that of harmful bacteria Clostridia. Furthermore, untargeted metabolomic analysis revealed that the protective effect of curcumin on the intestine was mainly through the regulation of AFB1-induced disorders of lipid metabolism, involving linoleic acid metabolism, α-linolenic acid metabolism, and glycerolipid metabolism. Overall, the enteroprotective effects of curcumin may be of significant value in the future for treating chronic AFB1 poisoning and also provide new therapeutic ideas for other mycotoxicosis.

5-Hydroxytryptamine levels in the pulmonary arterioles of broilers with induced pulmonary hypertension and its relationship to pulmonary vascular remodelling.

This experiment was performed to explore the relationship between 5-hydroxytryptamine (5-HT) levels in pulmonary arterioles and in pulmonary vascular remodelling in broilers. Pulmonary arterial hypertension was induced by injecting cellulose microparticles intravenously. Pulmonary hypertension syndrome (PHS) morbidity, right ventricle/total ventricle weight ratio (RV/TV), packed cell volume (PCV), haemoglobin concentration (HB), vessel wall area to vessel total area ratio (WA/TA) and mean tunica media thickness in pulmonary arterioles (mMTPA) were measured. Proliferating cell nuclear antigen (PCNA), argyrophilic nucleolar organizer region proteins (Ag-NORs) and 5-HT content in pulmonary arterioles were determined. The results showed that injecting cellulose microparticles intravenously in broilers could successfully increase the PHS morbidity, significantly elevate RV/TV, PCV and HB, significantly increase mMTPA and WA/TA, and significantly increase the argyrophilic particles in smooth muscle cell nucleoli, PCNA-positive cells in the medial layer, and the 5-HT content in pulmonary arterioles. Correlation analysis showed that the level of 5-HT was strongly positively correlated with PCNA and Ag-NORs. The results indicated that the increase of 5-HT in the tunica media could possibly promote the proliferation of smooth muscle cells in pulmonary arterioles and thus the occurrence of pulmonary vascular remodelling.