Golgi apparatus regulated pyroptosis through the miR-32-5p/Golga7/NLRP3 axis in chicken splenic lymphocytes exposure to ammonia.

Ammonia, a common toxic gas, posed a hazard to both human and chickens. The Golgi apparatus, an essential organelle, helped maintain the internal environment of the organism and supported the protein foundation for the endoplasmic reticulum to be involved in pyroptosis. Thus, the Golgi apparatus has garnered significant attention. The purpose of our research was to explore the mechanisms of Golgin A7 (Golga7) involved in pyroptosis after chicken exposure to ammonia. To reach our goal, we first created an in vitro ammonia model to study the effect of ammonia on chicken splenic lymphocyte pyroptosis. Then, leveraging this model, we established Golga7 and miR-32-5p knockdown and overexpression models to investigate their roles in ammonia-induced pyroptosis. We found the ultrastructural changes in the nucleus, Golgi apparatus, and mitochondria of chicken splenic lymphocytes exposure to ammonia. The damage of mitochondria increased the level of Reactive Oxygen Species (ROS), which caused the down-regulation of miR-32-5p. The miR-32-5p inhibitor increased the expression of Golga7 and pyroptosis-related genes (NOD-like receptor protein 3 (NLRP3), Cysteine aspartase-1 (Caspase-1), Golgin A3 (Golga3), Nuclear Factor-kappa B (NF-κB), and Tumor Necrosis Factor-alpha (TNF-α)), which induced the pyroptosis, but when miR-32-5p mimic/si-Golga7 (Golga7 inhibitor) was utilized, these effects were reduced. Our research demonstrated that miR-32-5p/Golga7 regulated NLRP3 involving in the pyroptosis of chicken splenic cells exposed to ammonia. Our study provided a valuable foundation for the prevention and treatment chickens ammonia poisoning in the livestock production.

Analysis revealed the molecular mechanism of oxidative stress-autophagy-induced liver injury caused by high alkalinity: integrated whole hepatic transcriptome and metabolome.

Introduction: High-alkalinity water is a serious health hazard for fish and can cause oxidative stress and metabolic dysregulation in fish livers. However, the molecular mechanism of liver damage caused by high alkalinity in fish is unclear. Methods: In this study, 180 carp were randomly divided into a control (C) group and a high-alkalinity (A25) group and were cultured for 56 days. High-alkalinity-induced liver injury was analysed using histopathological, whole-transcriptome, and metabolomic analyses. Results: Many autophagic bodies and abundant mitochondrial membrane damage were observed in the A25 group. High alkalinity decreased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activity and the total antioxidant capacity (T-AOC) and increased the malondialdehyde (MDA) content in liver tissues, causing oxidative stress in the liver. Transcriptome analysis revealed 61 differentially expressed microRNAs (miRNAs) and 4008 differentially expressed mRNAs. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that mammalian target of rapamycin (mTOR), forkhead box O (FoxO), mitogen-activated protein kinase (MAPK), and the autophagy signalling pathway were the molecular mechanisms involved. High alkalinity causes oxidative stress and autophagy and results in autophagic damage in the liver. Bioinformatic predictions indicated that Unc-51 Like Kinase 2 (ULK2) was a potential target gene for miR-140-5p, demonstrating that high alkalinity triggered autophagy through the miR-140-5p-ULK2 axis. Metabolomic analysis revealed that the concentrations of cortisol 21-sulfate and beta-aminopropionitrile were significantly increased, while those of creatine and uracil were significantly decreased. Discussion: The effects of high alkalinity on oxidative stress and autophagy injury in the liver were analysed using whole-transcriptome miRNA-mRNA networks and metabolomics approaches. Our study provides new insights into liver injury caused by highly alkaline water.

Environmental endocrine disrupting chemical 4-tert-butylphenol induced calcium overload and subsequent autophagy impairment via miRNA-363/CACNA1D Axis in epithelioma papulosum cyprini cells.

Environmental endocrine disrupting chemical 4-tert-butylphenol (4-tBP), a widely-utilized surfactant in various industries, poses potential risks to aquatic organisms. Our previous sequencing results suggested that 4-tBP-induced common carp liver injury might be associated with Ca2+ signaling and autophagy. However, the intricate involvement of these pathways in 4-tBP-induced cytotoxic mechanisms remained unexplored. To bridge these knowledge gaps, this study focused on epithelioma papulosum cyprini (EPC) cells, a significant cell type in fish biology. Initial observations showed that 4-tBP induced a dose-dependent perturbation in Ca2+ levels. Further investigations, with siRNA and L-type Ca2+ channel agonist (BAYK8644), identified L-type calcium channel gene CACNA1D as a critical regulator of 4-tBP-induced Ca2+ overload. Predictive analysis using miRanda platform suggested a potential interaction between miR-363 and CACNA1D, which was subsequently verified through dual-luciferase reporter gene assays. We then established miR-363 mimic/inhibitor models, along with miR-363 and CACNA1D co-suppression models in EPC cells. Through TEM observation, immunofluorescence assay, Ca2+ staining, and qRT-PCR analysis, we evaluated the role of miR-363/CACNA1D axis in modulating the effects of 4-tBP on Ca2+ signaling and autophagy. Results showed that miR-363 inhibitor exacerbated 4-tBP-induced increase in CALM2, CAMKII, Calpain2, and p62 expression and also led to decrease in ATG5, ATG7, and LC3b expression. In contrast, miR-363 mimic notably alleviated these changes. Notably, siRNA CACNA1D effectively modulating miR-363 inhibitor's effect. Our study revealed that 4-tBP induced Ca2+ overload and subsequent autophagy impairment via miR-363/CACNA1D axis. These findings illuminated a profound understanding of molecular mechanisms underlying 4-tBP-induced cytotoxicity and spotlighted a potential therapeutic target.

Se Alleviated Pb-Caused Neurotoxicity in Chickens: SPS2-GPx1-GSH-IL-2/IL-17-NO Pathway, Selenoprotein Suppression, Oxidative Stress, and Inflammatory Injury.

Lead (Pb), a heavy metal environmental pollutant, poses a threat to the health of humans and birds. Inflammation is one of the most common pathological phenomena in the case of illness and poisoning. However, the underlying mechanisms of inflammation remain unclear. The cerebellum and the thalamus are important parts of the nervous system. To date, there have been no reports of Pb inducing inflammation in animal cerebellums or thalami. Selenium (Se) can relieve Pb poisoning. Therefore, we aimed to explore the mechanism by which Se alleviates Pb toxicity to the cerebellums and thalami of chickens by establishing a chicken Pb or/and Se treatment model. Our results demonstrated that exposure to Pb caused inflammatory damage in cerebellums and thalami, evidenced by the characteristics of inflammation, the decrease in anti-inflammatory factors (interleukin (IL)-2 and interferon-γ (INF-γ)), and the increase in pro-inflammatory factors (IL-4, IL-6, IL-12ß, IL-17, and nitric oxide (NO)). Moreover, we found that the IL-2/IL-17-NO pathway took part in Pb-caused inflammatory injury. The above findings were reversed by the supplementation of dietary Se, meaning that Se relieved inflammatory damage caused by Pb via the IL-2/IL-17-NO pathway. In addition, an up-regulated oxidative index malondialdehyde (MDA) and two down-regulated antioxidant indices (glutathione (GSH) and total antioxidant capacity (TAC)) were recorded after the chickens received Pb stimulation, indicating that excess Pb caused an oxidant/antioxidant imbalance and oxidative stress, and the oxidative stress mediated inflammatory damage via the GSH-IL-2 axis. Interestingly, exposure to Pb inhibited four glutathione peroxidase (GPx) family members (GPx1, GPx2, GPx3, and GPx4), three deiodinase (Dio) family members (Dio1, Dio2, and Dio3), and fifteen other selenoproteins (selenophosphate synthetase 2 (SPS2), selenoprotein (Sel)H, SelI, SelK, SelM, SelO, SelP1, SelPb, SelS, SelT, SelU, and selenoprotein (Sep)n1, Sepw1, Sepx1, and Sep15), suggesting that Pb reduced antioxidant capacity and resulted in oxidative stress involving the SPS2-GPx1-GSH pathway. Se supplementation, as expected, reversed the changes mentioned above, indicating that Se supplementation improved antioxidant capacity and mitigated oxidative stress in chickens. For the first time, we discovered that the SPS2-GPx1-GSH-IL-2/IL-17-NO pathway is involved in the complex inflammatory damage mechanism caused by Pb in chickens. In conclusion, this study demonstrated that Se relieved Pb-induced oxidative stress and inflammatory damage via the SPS2-GPx1-GSH-IL-2/IL-17-NO pathway in the chicken nervous system. This study offers novel insights into environmental pollutant-caused animal poisoning and provides a novel theoretical basis for the detoxification effect of Se against oxidative stress and inflammation caused by toxic pollutants.

Molecular mechanism of selenium against lead-induced apoptosis in chicken brainstem relating to heat shock protein, selenoproteins, and inflammatory cytokines.

Extensive application of lead (Pb) brought about environmental pollution and toxic reactions of organisms. Selenium (Se) has the effect of antagonizing Pb poisoning in humans and animals. However, it is still unclear how Pb causes brainstem toxicity. In the present study, we wanted to investigate whether Se can alleviate Pb toxicity in chicken brainstems by reducing apoptosis. One hundred and eighty chickens were randomly divided into four groups, namely the control group, the Se group, the Pb group, and the Se/Pb group. Morphological examination, ultrastructural observation, relative mRNA expressions of genes on heat shock proteins (HSPs); selenoproteins; inflammatory cytokines; and apoptosis-related factors were investigated. The results showed that Pb exposure led to tissue damage and apoptosis in chicken brainstems. Furthermore, an atypical expression of HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90); selenoprotein family glutathione peroxidase (GPx) 1, GPx2, GPx3, and GPx4), thioredoxin reductases (Txnrd) (Txnrd1, Txnrd2, and Txnrd3), dio selenoprotein famliy (diodothyronine deiodinases (Dio)1, Dio2, and Dio3), as well as other selenoproteins (selenoprotein (Sel)T, SelK, SelS, SelH, SelM, SelU, SelI, SelO, Selpb, selenoprotein n1 (Sepn1), Sepp1, Sepx1, Sepw1, 15-kDa selenoprotein (Sep15), and selenophosphate synthetases 2 (SPS2)); inflammatory cytokines (Interleukin 2 (IL-2), IL-4, IL-6, IL-12ß, IL-17, and Interferon-γ (IFN-γ)); and apoptosis-related genes (B-cell lymphoma-2 (Bcl-2), tumor protein 53 (p53), Bcl-2 Associated X (Bax), Cytochrome c (Cyt c), and Caspase-3) were identified. An inflammatory reaction and apoptosis were induced in chicken brainstems after exposure to Pb. Se alleviated the abnormal expression of HSPs, selenoproteins, inflammatory cytokines, and apoptosis in brainstem tissues of chickens treated with Pb. The results indicated that HSPs, selenoproteins, inflammatory, and apoptosis were involved in Se-resisted Pb poisoning. Overall, Se had resistance effect against Pb poisoning, and can be act as an antidote for Pb poisoning in animals.

Luteolin enhanced antioxidant capability and induced pyroptosis through NF-κB/NLRP3/Caspase-1 in splenic lymphocytes exposure to ammonia.

During feeding process in intensive chicken farms, the prolonged exposure of chickens to elevated level of ammonia leads to substantial economic losses within poultry farming industry. Luteolin (Lut), known as its anti-inflammatory and antioxidant properties, possesses the ability to eliminate free radicals and enhance the activities of antioxidant enzymes, thus rendering it highly esteemed in production. The objective of this study was to examine the effects of Lut on antioxidant and anti-inflammatory responses of chicken splenic lymphocytes exposed to ammonia. In order to achieve this, we have replicated a protective model involving Lut against ammonia exposure in chicken splenic lymphocytes. The findings of the study indicated that Lut mitigated the elevation of lactate dehydrogenase (LDH), malondialdehyde (MDA), and reactive oxygen species (ROS) induced by ammonia poisoning. Additionally, Lut demonstrated an increase in the expression of antioxidant enzymes, namely superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Furthermore, Lut exhibited a protective effect on cell morphology and ultrastructure following exposure to ammonia. Moreover, Lut exhibited a reduction in the expression of heat shock proteins (HSPs) and inflammatory cytokines, which were found to be highly expressed in splenic lymphocytes after ammonia exposure. Additionally, Lut demonstrated the ability to inhibit the overexpression of pyroptosis-related genes and proteins (NLRP3 and Caspase-1) in splenic lymphocytes following ammonia exposure. Lut exerted an antioxidant effect on lymphocytes, counteracting elevated levels of oxidative stress following exposure to ammonia. Additionally, Lut had the potential to modulate the expression of HSPs, suppressed the inflammatory response subsequent to ammonia exposure, and influenced the expression of NLRP3 and Caspase-1, thereby mitigating pyroptosis induced by ammonia exposure. The exploration of this subject matter can elucidate the protective properties of Lut against NH4Cl-induced damage in chicken splenic lymphocytes, while also offer insights and experimental groundwork for the utilization of natural therapeutics in animal husbandry to prevent and treat ammonia-related conditions.

The protective effect of Luteolin on chicken spleen lymphocytes from ammonia poisoning through mitochondria and balancing energy metabolism disorders.

Ammonia poses a significant challenge in the contemporary intensive breeding industry, resulting in substantial economic losses. Despite this, there is a dearth of research investigating efficacious strategies to prevent ammonia poisoning in poultry. Consequently, the objective of this study was to investigate the molecular mechanisms through which Luteolin (Lut) safeguards mitochondria and restores equilibrium to energy metabolism disorders, thereby shielding chicken spleen lymphocytes from the detrimental effects of ammonia poisoning. Chicken spleen lymphocytes were categorized into 3 distinct groups: the control group, the ammonia group (with the addition of 1 mmol/L of ammonium chloride), and the Lut group (with the treatment of 0.5 µg/mL of Lut for 12 h followed by the addition of 1 mmol/L of ammonium chloride). These groups were then cultured for a duration of 24 h. To investigate the potential protective effect of Lut on lymphocytes exposed to ammonia, various techniques were employed, including CCK-8 analysis, ultrastructural observation, reagent kit methodology, fluorescence microscopy, and quantitative real-time PCR (qRT-PCR). The findings indicate that Lut has the potential to mitigate the morphological damage of mitochondria caused by ammonia poisoning. Additionally, it can counteract the decline in mitochondrial membrane potential, ATP content, and ATPase activities (specifically Na+/K+-ATPase, Ca2+-ATPase, Mg2+-ATPase, and Ca/Mg2+-ATPase) following exposure to ammonia in lymphocytes. Lut also has the ability to regulate the expression of genes involved in mitochondrial fusion (Opa1, Mfn1, and Mfn2) and division (Drp1 and Mff) in spleen lymphocytes after ammonia exposure. This regulation leads to a balanced energy metabolism (HK1, HK2, LDHA, LDHB, PFK, PK, SDHB, and ACO2) and provides protection against ammonia poisoning.

Cadmium exposure caused cardiotoxicity in common carps (Cyprinus carpio L.): miR-9-5p, oxidative stress, energetic impairment, mitochondrial division/fusion imbalance, inflammation, and autophagy.

Cadmium (Cd), a toxic heavy metal pollutant, is a threat to human and eatable fish health. Common carps are widely cultivated and eaten by humans. However, there are no reports about Cd-damaged common carp hearts. Our experiment attempted to investigate the cardiotoxicity of Cd to common carps by establishing a common carp Cd exposure model. Our results showed that Cd injured hearts. Moreover, Cd treatment induced autophagy via miR-9-5p/Sirt1/mTOR/ULK1 pathway. Cd exposure caused oxidant/antioxidant imbalance and oxidative stress; and led to energetic impairment. Energetic impairment partook in oxidative stress-mediated autophagy through AMPK/mTOR/ULK1 pathway. Furthermore, Cd caused mitochondrial division/fusion imbalance and resulted in inflammatory injury via NF-κB-COX-2-PTGEs and NF-κB-COX-2-TNF-α pathways. Oxidative stress mediated mitochondrial division/fusion imbalance, further induced inflammation and autophagy via OPA1/NF-κB-COX-2-TNF-α-Beclin1 and OPA1/NF-κB-COX-2-TNF-α/P62 pathways under Cd treatment. Taken together, miR-9-5p, oxidative stress, energetic impairment, mitochondrial division/fusion imbalance, inflammation, and autophagy participated in the mechanism of Cd-cardiotoxicity to common carps. Our study revealed harmful effect of Cd on hearts, and provided new information for researches of environmental pollutant toxicity.

Effect of arsenic stress on the intestinal structural integrity and intestinal flora abundance of Cyprinus carpio.

Aquatic organisms such as fish can accumulate high concentrations of arsenic (As), which has toxic effects on fish. However, whether the intestinal flora are involved in As damage to fish intestinal tissues and the underlying process are unclear. Common carp (Cyprinus carpio) were exposed to As (2.83 mg/L) in water for 30 days, and blood, muscle, intestine, and intestine samples were collected. Intestinal pathological sections were observed, and the lipopolysaccharide (LPS) levels in serum and the levels of As accumulation and tight junction-related factors in intestinal tissues were measured. The gut microbiota was analysed by 16S rRNA sequencing. The results showed that As treatment decreased the abundance of microbiota, increased the number of harmful bacteria, and decreased the number of beneficial bacteria in the intestine. In our experiment, the top 30 harmful and beneficial bacteria with the highest relative abundance were identified. Among the top 30 harmful and beneficial bacteria, As treatment resulted in a significant (P < 0.05) increase in harmful bacteria (such as Fusobacteriota, Bacteroidota (LPS-producing bacteria), Verrucomicrobiota, Bacteroides, Aeromonas, and Stenotrophomonas) and a significant (P < 0.05) decrease in beneficial bacteria (such as Actinobacteriota, Planctomycetota, Firmicutes, Reyranella, Akkermansia, and Pseudorhodobacter), which further demonstrated that As affects the abundance of intestinal flora. In addition, As exposure increased the LPS level in serum and the abundance of Bacteroidota (LPS-producing bacteria) in the intestine. Bacteroidota exhibits the six highest relative abundance at the phylum level, which indicates that LPS produced by Bacteroidota can increase the LPS level in serum. Additionally, the protein and gene levels of the tight junction markers ZO-1 and occludin in the intestine were reduced by As treatment, which further indicated that As exposure impaired the structural integrity of the intestine. In conclusion, the results obtained in our study indicate that the intestinal flora, LPS, and tight junctions participate in the impairment of the structural integrity of the common carp intestine resulting from As exposure.

Cadmium induced time-dependent kidney injury in common carp via mitochondrial pathway: Impaired mitochondrial energy metabolism and mitochondrion-dependent apoptosis.

Toxic effect of heavy metal cadmium (Cd) on fish kidneys had been reported. Mitochondrion is an important organelle for maintaining kidney function, while its role in Cd-induced kidney injury in common carp remained unclarified. In this experiment, we established a poisoning model of common carp with Cd exposure (0.26 mg/L) for 15, 30, and 45 days. Serum biochemistry determination, histological observation, TUNEL assay, qRT-PCR, Western blot, and integrated biomarker response (IBR) were applied to assess the nephrotoxicity of Cd to common carp. Our results displayed that Cd exposure increased the levels of serum biochemical indexes (UREA, CRE, and UA), indicating kidney injury. We further revealed via histological observation that Cd damaged structural integrity of kidneys, as evidenced by renal glomerulus and renal tubular injury, hallmark phenotypes of apoptosis, and mitochondrial damage, suggesting that mitochondria damage and apoptosis were involved in Cd-induced kidney injury. Moreover, Cd exposure decreased ATPase (Na+/K+-ATPase, Ca2+-ATPase, Mg2+-ATPase, and Ca2+Mg2+-ATPase) activities as well as PGC-1a and Mfn2 levels, while increased Drp1 and PINK1 levels as well as LC3-II/LC3-I ratio, which indicated that Cd-impaired renal energy metabolism was related to mitochondrial dysfunction. Additionally, we found that Cd induced oxidative stress (abnormal levels of SOD, CAT, GPX, MDA, and H2O2) in kidneys, which was involved in triggering mitochondrial dysfunction and further impairing mitochondrial energy metabolism. Moreover, the occurrence of mitochondria-dependent apoptosis was found after Cd-exposure in common carp kidneys, as indicated by enhanced levels of Bax, CytC, APAF1, Caspase-9, and Caspase-3, while declined level of Bcl-2. Subsequently, we confirmed a time-dependent nephrotoxicity of Cd to common carp via IBR assessment. In conclusion, Cd induced time-dependent nephrotoxicity in common carp via mitochondrial pathway. This mitochondria-oriented study shed light on underlying mechanisms of Cd-induced renal pathologies and provided a theoretical basis for evaluating Cd toxicity to aquatic organisms.

Chlorpyrifos induced autophagy and mitophagy in common carp livers through AMPK pathway activated by energy metabolism disorder.

Water pollution caused by widely used agricultural pesticide chlorpyrifos (CPF) has aroused extensive public concern. While previous studies have reported on toxic effect of CPF on aquatic animal, little is known about its effect on common carp (Cyprinus carpio L.) livers. In this experiment, we exposed common carp to CPF (11.6 µg/L) for 15, 30, and 45 days to establish a poisoning model. Histological observation, biochemical assay, quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and integrated biomarker response (IBR) were applied to assess the hepatotoxicity of CPF in common carp. Our results displayed that CPF exposure damaged histostructural integrity and induced liver injury in common carp. Furthermore, we found that CPF-induced liver injury may be associated with mitochondrial dysfunction and autophagy, as evidenced by swollen mitochondria, broken mitochondrial ridges, and increased the number of autophagosomes. Moreover, CPF exposure decreased the activities of ATPase (Na+/K+-ATPase, Ca2+-ATPase, Mg2+-ATPase, and Ca2+Mg2+-ATPase), altered glucose metabolism-related genes (GCK, PCK2, PHKB, GYS2, PGM1, and DLAT), and activated energy-sensing AMPK, indicating that CPF caused energy metabolism disorder. The activation of AMPK further induced mitophagy via AMPK/Drp1 pathway, and induced autophagy via AMPK/mTOR pathway. Additionally, we found that CPF induced oxidative stress (abnormal levels of SOD, GSH, MDA, and H2O2) in common carp livers, which further contributed to the induction of mitophagy and autophagy. Subsequently, we confirmed a time-dependent hepatotoxicity caused by CPF in common carp via IBR assessment. Our findings presented a new insight into molecular mechanism of CPF induced-hepatotoxicity in common carp, and provided a theoretical basis for evaluating CPF toxicity to aquatic organisms.

Transcriptome analysis revealed the mechanism of Luciobarbus capito (L. capito) adapting high salinity: Antioxidant capacity, heat shock proteins, immunity.

Salinity has a significant influence on the physiology of freshwater aquatic organisms. However, there are few studies on the hematology and immunology of freshwater fish under high salinity. In the current study, we aimed to analyze the adaptive effect of salt stress on L. capito spleen immune function and hematology using transcriptomic analysis. We replicated a L. capito acute salinity stress model, and collected blood and spleens from freshwater and saltwater fish. It was found that salinity affected significantly the numbers of leukocytes, lymphocytes, neutrophils, and red blood cells, as well as the content of haemoglobin. Salt treatment resulted in a significant increase in the expression of HSP70, HSP90, CAT, SOD, and GPX1 genes in L. capito spleens. Transcriptomic analysis revealed a total of 546 differentially expressed genes (DEGs) in spleens, including 224 up-regulated DEGs and 322 down-regulated DEGs. In addition, GO enrichment analysis revealed immune system process, multicellular organismal process, and biological regulation of genes with the most differences in biological processes. KEGG enrichment analysis showed that the regulation of lipolysis in adipocyte, FoxO signaling pathway, Hematopoietic cell lineage signaling pathway, and HIF-1 signaling pathway were significantly enriched. L. capito adapted oxidative to high salinity through FoxO signaling pathway and immune to high salinity through Hematopoietic cell lineage signaling pathway. At the same time, we selected 10 DEGs for qRT-PCR detection, and the results showed that the qRT-PCR results were consistent with our RNA-Seq results, indicating that transcriptome sequencing was accurate and reliable. In conclusion, our results demonstrated that the improvement of antioxidant capacity, heat shock protein and immunity are involved in the molecular mechanism of L. capito adapting to high salinity. Our findings provided a rationale for further study on high salinity adaptation and related enrichment pathways.

The effect of acute toxicity from tributyltin on Liza haematocheila liver: Energy metabolic disturbance, oxidative stress, and apoptosis.

Tributyltin (TBT), a highly toxic and persistent organic pollutant, is widely distributed in coastal waters. Liza haematocheila (L. haematocheila) is one of bony fish distributing coincident with TBT, and exposure risk of TBT to this fish is unknown. In this study, L. haematocheila was exposed to TBT of 0, 3.4, 6.8, and 17.2 µg/L for 48 h to explore hepatic response mechanism. Our results showed that Sn content in livers increased after 48 h of exposure. HSI and histological changes indicated that TBT suppressed liver development of L. haematocheila. TBT reduced ATPase activities. The increased RB in blood and the reduced TBC were measured after exposure to TBT. T-AOC and antioxidant enzymes SOD, CAT, and GPx activities were inhibited while MDA content was increased. Liver cells showed apoptosis characteristics after TBT exposure. Furthermore, transcriptome analysis of livers was performed and the results showed energy metabolism-related GO term (such as ATPase complex and ATPase dependent transmembrance transport complex), oxidative stress-related GO term (such as Celllular response to oxidative stress and Antioxidant activity), and apoptosis-related GO term (such as Regulation of cysteine-type endopeptidase activity involved in apoptosic signaling pathway). Moreover, we found six energy metabolism-related differentially expressed genes (DEGs) including three up-regulated DEGs (atnb233, cftr, and prkag2) and three down-regulated DEGs (acss1, abcd2, and smarcb1); five oxidative stress-related DEGs including one up-regulated DEG (mmp9) and four down-regulated DEG (prdx5, hsp90, hsp98, and gstf9); as well as six apoptosis-related DEGs including five up-regulated DEGs (casp8, cyc, apaf1, hccs, and dapk3) and one down-regulated DEG (bcl2l1). Our transcriptome data above further confirmed that acute stress of TBT led energy metabolic disturbance, oxidative stress, and apoptosis in L. haematocheila livers.

Nano-selenium protects grass carp hepatocytes against 4-tert-butylphenol-induced mitochondrial apoptosis and necroptosis via suppressing ROS-PARP1 axis.

4-tert-butylphenol (4-tBP) is a monomer widely used in the synthesis of industrial chemicals, and posed a high risk to aquatic animals. Our study focused on toxic phenotype and mechanism of detoxification in grass carp hepatocytes (L8824) after 4-tBP-treatment. In this experiment, L8824 displayed hallmark phenotypes of apoptosis and necroptosis after 4-tBP exposure, as evidenced by changes in cell morphology, increased rates of apoptosis and necrosis, the loss of MMP, the accumulation of ROS, and changes in associated factors (PARP1, JNK, Bid, Bcl-2, Bax, AIFM1, CytC, Caspase 9, APAF1, Caspase 3, TNF-α, TNFR1, RIPK1, RIPK3, and MLKL). Furthermore, we found that 4-tBP-induced apoptosis and necroptosis were reversed by pretreating with N-Acetylcysteine (a ROS scavenger) and 3-Aminobenzamide (a PARP1 inhibitor), indicating that 4-tBP induced the onset of mitochondrial apoptosis and necroptosis in L8824 via activating ROS-PARP1 axis. Nano-selenium (Nano-Se) is a novel form of Se with a noteworthy antioxidant capacity. Here, Nano-Se was found to have preventive, therapeutic, and resistance effects on 4-tBP-induced L8824 apoptosis and necroptosis. Nano-Se co-treatment with 4-tBP was an optimal way to alleviate 4-tBP-induced apoptosis and necroptosis. We demonstrated for the first time that Nano-Se protected L8824 against 4-tBP-induced mitochondrial apoptosis and necroptosis through ROS-PARP1 pathway. This study will provide a new theoretical basis for 4-tBP toxicology researches and aquatic animal protection.

EGCG alleviated Mn exposure-caused carp kidney damage via trpm2-NLRP3-TNF-α-JNK pathway: Oxidative stress, inflammation, and tight junction dysfunction.

Manganese (Mn), an essential trace metal element in organisms. However, with extensive use of Mn in industry and agriculture, Mn becomes a heavy metal pollutant in water. (-)-epigallocatechin gallate (EGCG), an tea polyphenols, can alleviate metal toxicity. Kidney is an important detoxifying organ, but toxic mechanism of Mn to kidneys is unclear, which needs further research. Carp is an Asian important economical species for fisheries and a biological model for studying environmental toxicology. Thus, we established excess Mn and EGCG-supplemented carp model to explore molecular mechanism of EGCG alleviating Mn-caused carp kidney damage. In this experiment, we set a control group (the Con group), a Mn treatment group (the Mn group, 90 mg/L Mn), a EGCG supplement group (the EG group, 75 mg/kg EGCG), and a combined group (the Mn + EG group, 90 mg/L Mn and 75 mg/kg EGCG). Transcriptome, qRT-PCR, kit, and morphology method results indicated that excess Mn caused oxidative stress, inflammatory damage, and tight junction dysfunction in carp kidneys. Excess Mn-triggered oxidative stress caused tight junction dysfunction via trpm2-NLRP3-TNF-α-JNK pathway and inflammation. EGCG reversed the harm of Mn to fish through the above mechanism. The findings of this study provided the evidence of EGCG-alleviated Mn poisoning and offered new ideas for reducing heavy metal environmental pollution risk.

Mitochondrion Participated in Effect Mechanism of Manganese Poisoning on Heat Shock Protein and Ultrastructure of Testes in Chickens.

Manganese (Mn) poisoning can happen in the case of environmental pollution and occupational exposure. However, the underlying mechanisms of Mn-induced teste toxicity and whether mitochondrion and heat shock proteins (HSPs) are involved in toxic effect of Mn on chicken testes remain poorly understood. To investigate this, MnCl2·4H2O was administered in the diet (600, 900, and 1800 mg/kg Mn) of chickens for 30, 60, and 90 days. Electron microscopy and qPCR were performed. Results showed that Mn exposure suppressed dose- and time-dependently HSP40 and HSP60 mRNA levels, meanwhile increased does-dependently HSP27, HSP70, and HSP90 mRNA levels at all three time points under three Mn exposure concentrations. Furthermore, Mn treatment damaged myoid cells, spermatocytes, and Sertoli cells through electron microscopic observation, indicating that Mn treatment damaged chicken testes. In addition, abnormal shapes of mitochondria were found, and mitochondria displayed extensive vacuolation. The increase of HSP90 and HSP70 induced by Mn exposure inhibited HSP40 and stimulated HSP27, respectively, in chicken testes, which needs further to be explored. Taken together, our study suggested that there was toxic effect in excess Mn on chickens, and HSPs and mitochondria were involved in the mechanism of dose-dependent injury caused by Mn in chicken testes. This study provided new insights for Mn toxicity identification in animal husbandry production practice.

Melatonin alleviates lead-induced fatty liver in the common carps (Cyprinus carpio) via gut-liver axis.

As a widespread aquatic environmental contaminant, Lead (Pb) can provoke hepatic injury in various animals. Melatonin (MT) plays a crucial role in the regulation of inflammatory response. Accumulating evidence elucidates exogenous toxins can elicit hepatic lipid metabolic disorders by influencing the gut microbiome. Nevertheless, the effects of Pb on gut microbiota and hepatic lipid metabolism of the common carps, and whether MT can prevent and cure Pb-induced toxicity via regulating microbiome remains unknown. Here, metagenomic and transcriptomic analysis were subsequently implemented to identify the Pb exposure-triggered prominent alternation of gut-liver signal. In the present study the severe intestinal injury and fatty liver formation caused by Pb in common carp were preliminarily determined. Metagenomic analysis confirmed that the gut microbiome dominant phyla, family and genus of the common carps were Fusobacteria, Fusobacteriaceae and Cetobacterium. Meanwhile, lipopolysaccharide (LPS) biosynthesis pathway was regarded as one of the main responsible for Pb exposure. Subsequently, LPS was demonstrated as the Pb-triggered microbial-derived signal of the common carps by ELISA analysis, and involves in the hepatic metabolic disorders via deteriorating the intestinal barrier. Additionally, it confirmed that hepatocytes ferroptosis associated with Pb-evoked fatty liver of the common carps, and the aggravation of lysosomal dyshomeostasis as well as inhibition of AMPK phosphorylation were referred to lipid metabolic disorders. The results of the present study demonstrated microbial-derived signal induced by aquatic Pb contaminant cause fatty liver formation in the common carps, and the protective effects of MT on Pb toxicity were performed by receding LPS over-synthesis, restraining microbiota-sourced LPS transport, along with attenuation of hepatocytes ferroptosis.

HSP27-HSP40-HSP70-HSP90 pathway participated in molecular mechanism of selenium alleviating lead-caused oxidative damage and proteotoxicity in chicken Bursa of Fabricius.

Lead (Pb), a toxic environmental pollutant, is hazardous to the health of humans and birds. Bursa of Fabricius (BF) is a unique organ of birds. Toxic substances can attack BF and induce proteotoxicity. Increased heat shock proteins (HSPs) can induce oxidative damage. Selenium (Se) can alleviate harmful substance-caused oxidative damage. This study aimed to investigate whether Pb can cause oxidative damage and proteotoxicity, as well as Se reverse Pb-caused chicken BF toxicity. A model of chickens treated with Se and Pb alone and in combination was established. BFs were collected on days 30, 60, and 90. H&E and qRT-PCR were performed to observe the microstructure and to detect HSP27, HSP40, HSP60, HSP70, and HSP90 mRNA levels, respectively, in BFs. Multivariate correlation analysis and principal component analysis were conducted to explore the correlation among the five HSPs. In our results, Pb caused BF damage and up-regulated the five HSPs at three time points, causing oxidative damage and proteotoxicity via HSP27-HSP40-HSP70-HSP90 pathway. Furthermore, Pb caused time-dependent stress on HSP27, HSP40, HSP60, and HSP70. In addition, Se relieved Pb-caused damage and up-regulation of HSPs. Taken together, we concluded that Se alleviated Pb-caused oxidative injury and proteotoxicity in chicken BFs via the HSP27-HSP40-HSP70-HSP90 pathway.

Melatonin alleviates lead-induced intestinal epithelial cell pyroptosis in the common carps (Cyprinus carpio) via miR-17-5p/TXNIP axis.

Lead (Pb) has been concerned as one of the most severe hazardous contaminants, because it can cause pyroptosis in multiple tissues of mammals and birds. Melatonin (Mel) has attracted much interest for its role in governing intestinal injury via microRNAs (miRNAs). To explore the effect of Mel on Pb exposure-induced intestinal epithelial cell pyroptosis in common carps by regulating miR-17-5p/TXNIP axis, the Pb exposure and Pb-Mel treated models were constructed in vivo. The results elucidated that the suppressed expression of miR-17-5p and intensified level of TXNIP were primarily detected in Pb-exposed gut tissues, and both abolished with Mel addition, along with downregulated Pb-mediated elevated expression of NLRP3, CASP1, IL1ß and GSDMD. Additionally, the targeting relationship between miR-17-5p and TXNIP were demonstrated by dual-luciferase reporter assay, and on this basis, miR-17-5p NC, mimic and inhibitor cell models were established. Thereby, Thereby, the expression of TXNIP in the miR-17-5p mimic groups was significant lower in the Pb-exposure but still elevated than the Control group, and the expression of NLRP3 and NLRP3-dependent pyrotposis-related genes performed consistent alterations. Noticeably, the expression of TXNIP suppressed with Mel addition even in the miR-17-5p inhibitor cell model, resulting in the inactivation of NLRP3 inflammasome-dependent pyroptosis. Overall, we draw the conclusion as Mel attenuates Pb-induced intestinal epithelial cell pyroptosis via miR-17-5p/TXNIP axis. The present study provides a novel perspective for toxicological mechanism of Pb, and new insights for the detoxification mechanism of Mel.

4-tert-butylphenol triggers common carp hepatocytes ferroptosis via oxidative stress, iron overload, SLC7A11/GSH/GPX4 axis, and ATF4/HSPA5/GPX4 axis.

4-tert-butylphenol (4-tBP) is a toxic environmental pollutant with moderate bioaccumulation, environmental persistence, and long-term toxicity. Its toxicity to aquatic organisms has become an issue of concern. However, the molecular mechanism of 4-tBP toxicity to aquatic organisms remained unclear. Liver is a target organ for environmental pollutants. Here, we established 4-tBP-exposed toxicity model in vivo and primary hepatocyte model in vitro in common carp (Cyprinus carpio L.). We found increased hepatic-somatic index (HSI) and abnormal serum biochemical indexes (ALT, AST, and LDH) after 4-tBP exposure, indicating liver damage. We further revealed that 4-tBP damaged the structural integrity of the livers with typical features of ferroptosis. Based on toxicogenomics analysis, we found ferroptosis is likely to be involved in the mechanism of 4-tBP-induced liver damage. Moreover, our in vivo and in vitro experiment provided evidences that 4-tBP-exposure led to excess oxidative stress, iron overload, decreased MMP, and abnormal expression of ferroptosis-related factors. Interestingly, ferrostatin-1 (Fer-1, a ferroptosis inhibitor) pretreatment alleviated above changes. In summary, we demonstrated that 4-tBP triggered hepatocytes ferroptosis via oxidative stress, iron overload, SLC7A11/GSH/GPX4 axis, and ATF4/HSPA5/GPX4 axis. For the first time, we discovered that Fer-1 can ameliorate the toxicity of 4-tBP, which needs more investigations. Our results provided a scientific basis of molecular mechanism of 4-tBP-induced fish poisoning.