Selenium deficiency exacerbates ROS/ER stress mediated pyroptosis and ferroptosis induced by bisphenol A in chickens thymus.

Bisphenol A (BPA) is an industrial pollutant that can cause immune impairment. Selenium acts as an antioxidant, as selenium deficiency often accompanies oxidative stress, resulting in organ damage. This study is the first to demonstrate that BPA and/or selenium deficiency induce pyroptosis and ferroptosis-mediated thymic injury in chicken and chicken lymphoma cell (MDCC-MSB-1) via oxidative stress-induced endoplasmic reticulum (ER) stress. We established a broiler chicken model of BPA and/or selenium deficiency exposure and collected thymus samples as research subjects after 42 days. The results demonstrated that BPA or selenium deficiency led to a decrease in antioxidant enzyme activities (T-AOC, CAT, and GSH-Px), accumulation of peroxides (H2O2 and MDA), significant upregulation of ER stress-related markers (GRP78, IER 1, PERK, EIF-2α, ATF4, and CHOP), a significant increase in iron ion levels, significant upregulation of pyroptosis-related gene (NLRP3, ASC, Caspase1, GSDMD, IL-18 and IL-1ß), significantly increase ferroptosis-related genes (TFRC, COX2) and downregulate GPX4, HO-1, FTH, NADPH. In vitro experiments conducted in MDCC-MSB-1 cells confirmed the results, demonstrating that the addition of antioxidant (NAC), ER stress inhibitor (TUDCA) and pyroptosis inhibitor (Vx765) alleviated oxidative stress, endoplasmic reticulum stress, pyroptosis, and ferroptosis. Overall, this study concludes that the combined effects of oxidative stress and ER stress mediate pyroptosis and ferroptosis in chicken thymus induced by BPA exposure and selenium deficiency.

Co-exposure of bisphenol A and selenium deficiency induces pyroptosis via ROS/NLRP3 pathway in chicken spleen.

Bisphenol A (BPA) is widely applied in plastic products, which will produce immunotoxicity to organisms after spilling in the environment, and become a kind of endocrine disruptor. Selenium (Se) is an essential trace element and plays an important role in maintaining redox homeostasis and immune function. BPA exposure and Se deficiency often occur together in livestock and poultry farming, however, studies on the effects of joint exposure on chicken immunotoxins have not been reported. Therefore, this study established a chicken spleen and MDCC-MSB1 cell model under the combined effects of BPA exposure or/and Se deficiency. Transcriptomic analysis showed that BPA exposure and/or Se deficiency induced differential enrichment of positive regulatory pathways such as NLRP3 inflammatory complex assembly, inflammatory response and cellular oxidative stress response. In the -Se+BPA group, pathological damage was significantly increased, Se content decreased, BPA accumulation, oxidative stress and pyroptosis. Meanwhile, the roles and mechanisms of oxidative stress and pyroptosis in BPA exposure or/and Se deficiency-induced splenic tissue injury were investigated by using IF and qRT-PCR methods. The results showed that joint BPA exposure with Se deficiency resulted in more significant changes in the above outcomes than 1 of them. The oxidative stress inhibitor NAC effectually reduced Se deficiency and BPA-induced oxidative stress and pyroptosis, further suggests that oxidative stress mediated Se deficiency or/and BPA-induced pyroptosis. This study revealed that BPA exposure and Se deficiency induced spleen pyroptosis in chickens via the ROS/NLRP3 pathway. These results provide the theoretical basis for the toxicity of BPA in poultry and enrich the toxicological mechanism of combined exposure of Se deficiency and environmental toxins.

Quercetin antagonizes apoptosis, autophagy and immune dysfunction induced by di(2-ethylhexyl) phthalate via ROS/ASK1/JNK pathway.

Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer that can damage various organizations and physiques through oxidative stress. Quercetin (Que) is a rich polyphenol flavonoid with good anti-inflammatory and antioxidant effects. However, the protection mechanism of Que against DEHP exposure-induced IPEC-J2 cell injury and the implication of autophagy, apoptosis and immunity are still unclear. In this experiment, we looked into the toxicity regime of DEHP exposure on IPEC-J2 cells and the antagonistic function of Que on DEHP. In the experiment, 135 µM DEHP and/or 80 µM Que were used to treat the IPEC-J2 cells for 24h. Experiments indicated that DEHP exposure can cause increased reactive oxygen species (ROS) levels leading to oxidative stress, decreased CAT, T-AOC and GSH-Px activities, increased MDA and H2O2 accumulation, activated the ASK1/JNK signalling pathway, and further increases in the levels of apoptosis markers Bax, Caspase3, Caspase9, and Cyt-c, while reduced the Bcl-2 expression. DEHP also increased the expression of genes linked to autophagy (ATG5, Beclin1, LC3), while decreasing the expression of P62. Additionally, DEHP exposure led to elevated levels of IL1-ß, IL-6, MCP-1, and TNF expression. When exposed to Que alone, there were no significant changes in cellular oxidative stress level, ASK1/JNK signalling pathway expression level, apoptosis, autophagy and cellular immune function. The combination of DEHP and Que treatment remarkably decreased the proportion of autophagy and apoptosis, and recovered cellular immunity. In summary, Que can attenuate DEHP-induced apoptosis and autophagy in IPEC-J2 cells by regulating the ROS/ASK1/JNK signalling pathway and improving the immune dysfunction of IPEC-J2 cells.

Emamectin benzoate exposure induced carp kidney injury by triggering mitochondrial oxidative stress to accelerate ferroptosis and autophagy.

Emamectin benzoate (EMB), commonly used as an insecticide in fishery production, inevitably leaves residual chemicals in aquatic environments. High-level EMB exposure can cause severe damage to multiple systems of marine animals, potentially through mechanisms involving severe mitochondrial damage and oxidative stress. However, it is not clear yet how EMB exposure at a certain level can cause damage to fish kidney tissue. In this study, we exposed carps to an aquatic environment containing 2.4 µg/L of EMB and cultured carp kidney cells in vitro, established a cell model exposed to EMB. Our findings revealed that EMB exposure resulted in severe kidney tissue damage in carp and compromised the viability of grass carp kidney cells (CIK cells). By RNA-seq analysis, EMB exposure led to significant differences in mitochondrial homeostasis, response to ROS, ferroptosis, and autophagy signals in carp kidney tissue. Mechanistically, EMB exposure induced mitochondrial oxidative stress by promoting the generation of mitochondrial superoxide and reducing the activity of antioxidant enzymes. Additionally, EMB exposure triggered loss of mitochondrial membrane potential, an imbalance in mitochondrial fusion/division homeostasis, and dysfunction in oxidative phosphorylation, ultimately impairing ATP synthesis. Notably, EMB exposure also accelerated excessive autophagy and ferroptosis of cells by contributing to the formation of lipid peroxides and autophagosomes, and the deposition of Fe2+. However, N-acetyl-L-cysteine (NAC) treatment alleviated the damage and death of CIK cells by inhibiting oxidative stress. Overall, our study demonstrated that EMB exposure induced mitochondrial oxidative stress, impaired mitochondrial homeostasis, and function, promoted autophagy and ferroptosis of kidney cells, and ultimately led to kidney tissue damage in carp. Our research enhanced the toxicological understanding on EMB exposure and provides a model reference for comparative medicine.

Effects of atrazine and curcumin exposure on TCMK-1 cells: Oxidative damage, pyroptosis and cell cycle arrest.

Atrazine (ATR), a commonly used herbicide, is highly bioaccumulative and toxic, posing a threat to a wide range of organisms. Curcumin has strong antioxidant properties. However, it is unclear whether curcumin counteracts cellular pyroptosis as well as cell cycle arrest induced by ATR exposure. Therefore, we conducted a study using TCMK-1 cells and established cell models by adding 139 µmol/L ATR and 20 µmol/L curcumin. The results showed that ATR exposure produced excessive reactive oxygen species (ROS), reduced activities of enzymes such as GSH-PX, SOD and Total Antioxidant Capacity, markedly increased the content of H2O2, disrupted the antioxidant system, activated Caspase-1, and the expression levels of the pyroptosis-related genes NLRP3, GSDMD, ASC, Caspase-1, IL-1ß and IL-18 were increased. The simultaneous excess of ROS led to DNA damage, activation of P53 led to elevated expression levels of P53 and P21, as a consequence, the expression levels of cyclinE, CDK2 and CDK4 were reduced. These results suggest that Cur can modulate ATR exposure-induced pyroptosis as well as cell cycle arrest in TCMK-1 cells by governing oxidative stress.

Insight into the mechanism of melatonin in attenuating PCB126-induced liver injury: Resistance to ROS-dependent NETs formation to alleviate inflammation and lipid metabolism dysfunction.

3,3',4',4',5-Polychlorinated biphenyls (PCB126) is classified as a persistent organic environmental pollutant that can cause liver damage by producing excessive reactive oxygen species (ROS). ROS also can stimulate neutrophil extracellular traps (NETs) formation, which cause damage to organism if NETs are produced in excess. Melatonin is generally considered to possess strong antioxidant and anti-inflammation prosperities, but it is unclear whether it can alleviate PCB126-induced injury. To explore whether PCB126-induced liver injury is related to the formation of NETs and whether melatonin has a potent protective effect, we established PCB126 exposure/ PCB126 and melatonin co-treatment mouse models by gavage. To further clarify the specific mechanism, we also cultured neutrophils and AML12 cells to replicate in vivo model. Here, we found PCB126 exposure resulted in an elevation in the activities of MDA, LPO, PCO, and 8-OHdG, and a reduction in the activities of CAT, GSH-PX and SOD. We found that PCB126 exposure led to an elevation in the expression levels of chemokines (CCL2, CCL3, CCL4, CXCL12, and CXCL8) and marker factors for NETs formation (MPO, NE, NOX2, PKCα, and PKCζ) in the PCB126 group. IF, SYTOX staining, and SEM results also revealed that PCB126 could stimulate NETs formation. In addition, results of a co-culture system of PBNs and AML12 cells revealed that the expression levels of inflammatory cytokines (IL-1ß, IL-6, and TNF-α) significantly decreased and the expression levels of metabolism factors (Fas, Acc, and Srebp) slightly decreased for scavenging NETs, indicating NETs formation aggravated PCB126-induced hepatic damages. Noteworthy, treatment with melatonin reversed these results. In summary, our findings revealed that melatonin alleviated hepatic damage aggravated by PCB126-induced ROS-dependent NETs formation through suppressing excessive ROS production. This finding not only enriches toxicological mechanism of PCB126, but more importantly extends biological effects of melatonin and its potential application values.

Selenomethionine modulates the JAK2 / STAT3 / A20 pathway through oxidative stress to alleviate LPS-induced pyroptosis and inflammation in chicken hearts.

Selenium (Se) is involved in many physiopathologic processes in humans and animals and is strongly associated with the development of heart disease. Lipopolysaccharides (LPS) are cell wall components of gram-negative bacteria that are present in large quantities during environmental pollution. To investigate the mechanism of LPS-induced cardiac injury and the efficacy of the therapeutic effect of SeMet on LPS, a chicken model supplemented with selenomethionine (SeMet) and/or LPS treatment, as well as a primary chicken embryo cardiomyocyte model with the combined effect of SeMet / JAK2 inhibitor (INCB018424) and/or LPS were established in this experiment. CCK8 kit, Trypan blue staining, DCFH-DA staining, oxidative stress kits, immunofluorescence staining, LDH kit, real-time fluorescence quantitative PCR, and western blot were used. The results proved that LPS exposure led to ROS explosion, hindered the antioxidant system, promoted the expression of the JAK2 pathway, and increased the expression of genes involved in the pyroptosis pathway, inflammatory factors, and heat shock proteins (HSPs). Upon co-treatment with SeMet and LPS, SeMet reduced LPS-induced pyroptosis and inflammation and restored the expression of HSPs by inhibiting the ROS burst and modulating the antioxidant capacity. Co-treatment with INCB018424 and LPS resulted in inhibited of the JAK2 pathway, attenuating pyroptosis, inflammation, and high expression of HSPs. Thus, LPS induced pyroptosis, inflammation, and changes in HSPs activity by activating of the JAK2 / STAT3 / A20 signaling axis in chicken hearts. Moreover, SeMet has a positive effect on LPS-induced injury. This work further provides a theoretical basis for treating cardiac injury by SeMet.

Resveratrol alleviates imidacloprid-induced mitochondrial apoptosis, necroptosis, and immune dysfunction in chicken lymphocyte lines by inhibiting the ROS/MAPK signaling pathway.

Imidacloprid (IMI) is a neonicotinoid insecticide with the highest global market share, and IMI exposure in the environment can negatively affect many nontarget organisms (a general term for organisms affected by drugs other than target organisms). Resveratrol (RSV), a non-flavonoid polyphenolic organic compound derived from peanuts, grapes, and other plants, has anti-inflammatory and antioxidant effects. It is currently unclear how RSV protects against cell damage caused by IMI. Therefore, we established an experimental model of chicken lymphocyte lines exposed to 110 µg/mL IMI and/or 0.5 µM RSV for 24 h. According to the experimental results, IMI markedly raised intracellular reactive oxygen species levels and diminished the activity of the cellular antioxidant enzymes (CAT, SOD, and GPx), leading to MDA accumulation and decreased T-AOC. JNK, ERK, and P38, the essential components of the mitogen-activated protein kinase (MAPK) signaling pathway, were also expressed more when IMI was present. Additionally, IMI resulted in upregulation of mitochondrial apoptosis (Caspase 3, Caspase 9, Bax, and Cyt-c) and necroptosis (Caspase 8, RIPK1, RIPK3, and MLKL) related factors expression, downregulation of Bcl-2 expression, induction of upregulation of cytokine IL-6 and TNF-α expression, and downregulation of IFN-γ expression. The combined treatment of RSV and IMI significantly reduced cellular oxidative stress levels, inhibited the MAPK signaling pathway, and alleviated IMI-induced mitochondrial apoptosis, necroptosis, and immune dysfunction. To summarize, RSV antagonized IMI-induced mitochondrial apoptosis, necroptosis, and immune dysfunction in chicken lymphocyte lines by inhibiting the ROS/MAPK signaling pathway.

Bisphenol A Regulates the TNFR1 Pathway and Excessive ROS Mediated by miR-26a-5p/ADAM17 Axis to Aggravate Selenium Deficiency-Induced Necroptosis in Broiler Veins.

Selenium (Se) deficiency can affect the expression of microRNA (miRNA) and induce necroptosis, apoptosis, etc., resulting in damage to various tissues and organs. Bisphenol A (BPA) exposure can cause adverse consequences such as oxidative stress, endothelial dysfunction, and atherosclerosis. The toxic effects of combined treatment with Se-deficiency and BPA exposure may have a synergistic effect. We replicated the BPA exposure and Se-deficiency model in broiler to investigate whether the combined treatment of Se-deficiency and BPA exposure induced necroptosis and inflammation of chicken vascular tissue via the miR-26A-5p/ADAM17 axis. We found that Se deficiency and BPA exposure significantly inhibited the expression of miR-26a-5p and increased the expression of ADAM17, thereby increasing reactive oxygen species (ROS) production. Subsequently, we discovered that the tumor necrosis factor receptor (TNFR1), which was highly expressed, activated the necroptosis pathway through receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), and mixed-lineage kinase domain-like (MLKL), and regulated the heat shock proteins-related genes expressions and inflammation-related genes expressions after exposure to BPA and selenium deficiency. In vitro, we found that miR-26a-5p knockdown and increased ADAM17 can induce necroptosis by activating the TNFR1 pathway. Similarly, both N-Acetyl-L-cysteine (NAC), Necrostatin-1 (Nec-1), and miR-26a-5p mimic prevented necroptosis and inflammation caused by BPA exposure and Se deficiency. These results suggest that BPA exposure activates the miR-26a-5p/ADAM17 axis and exacerbates Se deficient-induced necroptosis and inflammation through the TNFR1 pathway and excess ROS. This study lays a data foundation for future ecological and health risk assessments of nutrient deficiencies and environmental toxic pollution.

Selenium deficiency exacerbated Bisphenol A-induced intestinal toxicity in chickens: Apoptosis and cell cycle arrest mediated by ROS/P53.

Bisphenol A (BPA) is a phenolic organic synthetic compound that is used as the raw material of polycarbonate plastics, and its safety issues have recently attracted wide attention. Selenium (Se) deficiency has gradually developed into a global disease affecting intestinal function via oxidative stress and apoptosis. However, the toxic effects and potential mechanisms of BPA exposure and Se deficiency in the chicken intestines have not been studied. In this study, BPA exposure and/or Se deficiency models were established in vivo and in vitro to investigate the effects of Se deficiency and BPA on chicken jejunum. The results showed that BPA exposure and/or Se deficiency increased jejunum oxidative stress and DNA damage, activated P53 pathway, led to mitochondrial dysfunction, and induced apoptosis and cell cycle arrest. Using protein-protein molecular docking, we found a strong binding ability between P53 and peroxisome proliferator-activated receptor γ coactivator-1, thereby regulating mitochondrial dysfunctional apoptosis. In addition, we used N-acetyl-L-cysteine and pifithrin-α for in vitro intervention and found that N-acetyl-L-cysteine and pifithrin-α intervention reversed the aforementioned adverse effects. This study clarified the potential mechanism by which Se deficiency exacerbates BPA induced intestinal injury in chickens through reactive oxygen species/P53, which provides a new idea for the study of environmental combined toxicity of Se deficiency, and insights into animal intestinal health from a new perspective.

TBBPA and lead co-exposure induces grass carp liver cells apoptosis via ROS/JAK2/STAT3 signaling axis.

Tetrabromobisphenol A (TBBPA) and lead (Pb) are widely used in industrial field, which poses a serious threat to human and animal health. In particular, a large volume of wastewater containing TBBPA and Pb was discharged into the aquatic environment, causing a seriously negative impact on fish. Currently, whether TBBPA and Pb have a synergistic toxicity on fish remains unclear. In this study, we used the grass carp hepatocytes (L8824 cell line) exposed to either TBBPA or Pb, or both to determine their potential impacts on fish. The results showed that Pb or TBBPA induced oxidative stress and the loss of mitochondrial membrane potential in grass carp hepatocytes. In contrast to the control cells, the levels of JAK2, p-JAK2, STAT3 and p-STAT3 were significantly upregulated after exposure to TBBPA and Pb. Furthermore, the levels of Caspase3, Caspase9 and Bax were all increased while the level of Bcl2 was decreased in hepatocytes exposed to TBBPA or Pb. Results of flow cytometry and AO/EB staining reveled significant increases in the number of apoptotic cells in the TBBPA and Pb group compared to the controls. Notably, cells exposed to both TBBPA and Pb exhibited more severe damage than the single exposure, manifested by a higher number of apoptotic cells in the co-exposure group than the single exposure groups. Nevertheless, N-acetyl-l-cysteine (NAC) treatment could remarkably alleviate oxidative damage and loss of membrane potential in grass carp hepatocytes induced by TBBPA and Pb. Altogether, our study showed that combined exposure of TBBPA and Pb has a synergistic toxicity due to, inducing oxidative stress to activate JAK2/STAT3 signaling pathway, resulting in apoptosis of carp hepatocytes. This study shed a new light on the toxicological mechanism of exposure of TBBPA and Pb and provided a potential treatment of toxicity induced by TBBPA and Pb.

Bisphenol A exposure exacerbates tracheal inflammatory injury in selenium-deficient chickens by regulating the miR-155/TRAF3/ROS pathway.

Bisphenol A (BPA) is an endocrine disruptor. Excessive BPA intake can damage the structure and function of the respiratory tract. Dietary selenium (Se) deficiency may also cause immune tissue damage. To investigate the potential mechanism of BPA on tracheal damage in selenium-deficient chickens and the role of microRNAs (miRNAs) in this process, we established in vitro and in vivo Se deficiency and BPA exposure models and screened out miR-155 for follow-up experiments. We further predicted and confirmed the targeting relationship between miR-155 and TRAF3 using TargetScan and dual luciferase assays and found that miR-155 was highly expressed and caused inflammatory damage. Further studies showed that BPA exposure increased airway oxidative stress, activated the NF-κB pathway, and caused inflammation and immune damage in selenium-deficient chickens, but down-regulating miR-155 and NAC treatment could reverse this phenomenon. This suggested that these pathways are regulated by the miR-155/TRAF3/ROS axis. In conclusion, BPA exposure aggravates airway inflammation in selenium-deficient chickens by regulating miR-155/TRAF3/ROS. This study revealed the mechanism of BPA exposure combined with Se deficiency in tracheal inflammatory injury in chickens and enriched the theoretical basis of BPA injury in poultry.

New insights into brain injury in chickens induced by bisphenol A and selenium deficiency-Mitochondrial reactive oxygen species and mitophagy-apoptosis crosstalk homeostasis.

Bisphenol A (BPA), a component of plastic products, can penetrate the blood-brain barrier and pose a threat to the nervous system. Selenium (Se) deficiency can also cause nervous system damage. Resulting from the rapid industrial development, BPA pollution and Se deficiency often coexist. However, it is unclear whether brain damage in chickens caused by BPA exposure and Se deficiency is related to the crosstalk disorder between mitophagy and apoptosis. In this study, 60 chickens (1 day old) were fed with a diet that contained 20 mg/kg BPA but was insufficient in Se (only 0.039 mg/kg) for 42 days to establish a chicken brain injury model. In vitro, the primary chicken embryo brain neurons were treated for 24 h with Se-deficient medium containing 75 µM BPA. The results showed that BPA exposure and Se deficiency inhibited the expression of the mitochondrial respiratory chain complex in brain neurons, and a large number of mitochondrial reactive oxygen species were released. Furthermore, the expression levels of mitochondrial fusion proteins (OPA1, Mfn1, and Mfn2) decreased, while the expression levels of mitochondrial fission proteins (Drp1, Mff, and Fis1) increased, thus exacerbating mitochondrial division. In addition, the results of immunofluorescence and flow cytometry analysis, as well as the elevated expressions of mitophagy related genes (PINK1, Parkin, ATG5, and LC3II/I) and pro-apoptotic markers (Bax, Cytc, Caspase3, and Caspase9) indicated that BPA exposure and Se deficiency disrupted the crosstalk homeostasis between mitophagy and apoptosis. However, this crosstalk homeostasis was restored after Mito-Tempo and Rapamycin treatment. In contrast, 3-methyladenine treatment exacerbated this crosstalk disorder. In conclusion, BPA exposure and Se deficiency can induce mitochondrial reactive oxygen species bursts and disorders of mitochondrial dynamics by destroying the mitochondrial respiratory chain complex. The result is indicative of an imbalance in mitochondrial autophagy and apoptosis crosstalk homeostasis, which damages the chicken brain.

Selenium alleviates lead-induced CIK cells pyroptosis and inflammation through IRAK1/TAK1/IKK pathway.

The toxic heavy metal lead is widely found in rivers and soils as an environmental pollutant, posing a threat to the health of aquatic organisms. Selenium is an essential trace element and a powerful antioxidant that has been shown to have anti-inflammatory and antioxidant properties as well as alleviating heavy metal poisoning. Many studies have shown that lead poisoning produces inflammatory responses and damage to the kidneys of a wide range of animals, but the effects on cellular pyroptosis and immune function and selenium antagonism in CIK cells are not clear. In this study, 500 µM Pb and 20 nM Se were applied to grass carp kidney cells, and the results showed that Pb exposure to CIK cells resulted in oxidative stress, activation of the IRAK1/TAK1/IKK pathway, up-regulation of the expression of cellular pyroptosis markers GSDMD and NLRP3, and cellular pyroptosis of CIK cells, as well as up-regulation of IL-1ß and IL-18, and the generation of cellular inflammatory response. In contrast, Se treatment significantly reduced the ROS level, the expression of cellular pyroptosis markers GSDMD, NLRP3 and inflammatory element IL-1ß and IL-18. Taken together, Se alleviated cellular pyroptosis and immune dysfunction caused by Pb exposure through oxidative stress and activation of the IRAK1/TAK1/IKK pathway. This study complements the harmful effects of the heavy metal Pb on fish and the real-life application of selenium in the healthy culture of fish as a reference will be provided.

Evodiamine alleviates DEHP-induced hepatocyte pyroptosis, necroptosis and immunosuppression in grass carp through ROS-regulated TLR4 / MyD88 / NF-κB pathway.

Di (2-ethylhexyl) phthalate (DEHP) is a neuroendocrine disruptor that can cause multi-tissue organ damage by inducing oxidative stress. Evodiamine (EVO) is an indole alkaloid with anti-inflammatory, antitumor, and antioxidant pharmacological activity. In this manuscript, the effects of DEHP and EVO on the pyroptosis, necroptosis and immunology of grass carp hepatocytes (L8824) were investigated using DCFH-DA staining, PI staining, IF staining, AO/EB staining, LDH kit, qRT-PCR and protein Western blot. The results showed that DEHP exposure upregulated reactive oxygen species (ROS) levels, promoted the expression of TLR4/MyD88/NF-κB pathway, increased the expression of genes involved in cell pyroptosis pathway (LDH, NLRP3, ASC, caspase1, IL-1ß, IL-18 and GSDMD) and necroptosis-related genes (RIPK1, RIPK3 and MLKL). The expression of DEHP can also affect immune function, which can be demonstrated by variationsin the activation of antimicrobial peptides (LEAP2, HEPC, and ß-defensin) and inflammatory cytokines (TNF-α, IL-2, IL-6 and IL-10). EVO regulates cellular antioxidant capacity by inhibiting ROS burst, reduces DEHP-induced cell pyroptosis and necroptosis to some extent, and restores cellular immune function, after co-exposure with EVO. The TLR4 pathway was inhibited by the co-treatment of TLR4 inhibitor TLR-IN-C34 and DEHP, which attenuated the expression of cell pyroptosis, necroptosis, and immunosuppression. Thus, DEHP induced pyroptosis, necroptosis and abnormal immune function in L8824 cells by activating TLR4/MyD88/NF-κB pathway. In addition, EVO has a therapeutic effect on DEHP-induced toxic injury. This study further provides a theoretical basis for the risk assessment of plasticizer DEHP on aquatic organisms.

Mechanism of evodiamine blocking Nrf2/MAPK pathway to inhibit apoptosis of grass carp hepatocytes induced by DEHP.

Di (2-ethylhexyl) phthalate (DEHP) is often used as a plasticizer for plastic products, and its excessive use can cause irreversible damage to aquatic animals and humans. Evodiamine (EVO) is an alkaloid component in the fruit of Evodia rutaecarpa, which has antioxidant and detoxification functions. To investigate the toxic mechanism of DEHP on grass carp (Ctenopharyngodon idellus) hepatocyte cell line (L8824) and the therapeutic effect of evodiamine, an experimental model of L8824 cells exposed to 800 µM DEHP and/or 10 µM EVO for 24 h was established. Flow cytometry, AO/EB fluorescence staining, real-time quantitative PCR, and western blot were used to detect the degree of cell injury, oxidative stress level, MAPK signaling pathway relative genes, and the expression of apoptosis-related molecules. The results showed that DEHP exposure could significantly increase the level of reactive oxygen species (ROS), inhibit the activities of antioxidant enzymes (CAT, SOD, GSH-Px), and cause the accumulation of MDA. DEHP also activated MAPK signaling pathway-related molecules (JNK, ERK, P38 MAPK), and then up-regulated the expression of pro-apoptotic factors Bcl-2-Associated X (Bax) and caspase 3, while inhibiting the anti-apoptotic factor B-cell lymphoma-2 (Bcl-2). In addition, EVO can also promote the dissociation of nuclear factor-E2-related factor 2 (Nrf2) into the nucleus, reduce the level of ROS and the occurrence of oxidative stress in grass carp hepatocytes, down-regulate the MAPK pathway, alleviate DEHP-induced apoptosis, and restore the expression of antioxidant genes. These results indicated that evodiamine could block Nrf2/MAPK pathway to inhibit DEHP-induced apoptosis of grass carp hepatocytes.

Bisphenol A exacerbates selenium deficiency-induced pyroptosis via the NF-κB/NLRP3/Caspase-1 pathway in chicken trachea.

Selenium deficiency can lead to multiple tissue and organ damage in the body and could coexist with chronic toxic exposures. Contamination from Bisphenol A (BPA) exposure can induce the occurrence of various injuries including pyroptosis. However, it is not clear whether selenium deficiency and BPA exposure affect tracheal tissue pyroptosis in chickens. To investigate whether selenium deficiency and BPA exposure induce chicken tracheal tissue pyroptosis via the NF-κB/NLRP3/Caspase-1 pathway and the effect of their combined exposure on tissue injury, we developed a model of relevant chicken tracheal injury. Sixty broilers were divided into four groups: the control group (C group), selenium-deficient group (SeD group), BPA-exposed group (BPA group) and combined exposure group (SeD + BPA group). The study examined the expression indicators of markers of pyroptosis (NLRP3&GSDMD), NF-κB pathway-related inflammatory factors (NF-κB, iNOS, TNF-α, COX-2), pyroptosis-related factors (ASC, Caspase-1, IL-1ß, IL-18), and some heat shock proteins and interleukins (HSP60, HSP90, IL-6, IL-17) in the samples. The results showed that the expression of the above indicators was significantly upregulated in the different treatment groups (P < 0.05). In addition, the expression levels of the above related indicators were more significantly up-regulated in the combined selenium-deficient and BPA-exposed group compared to the group in which they were individually exposed. It was concluded that selenium deficiency and BPA exposure induced tracheal tissue pyroptosis in chickens through NF-κB/NLRP3/Caspase-1 pathway, and BPA exposure exacerbated selenium deficiency-induced tracheal pyroptosis. The present study provides new ideas into studies related to the co-exposure of organismal micronutrient deficiency and chronic toxicants.

Roles of selenoprotein K in oxidative stress and endoplasmic reticulum stress under selenium deficiency in chicken liver.

Selenoprotein K (SELENOK) is a major part of selenoprotein family. Selenoproteins have been proven playing vital roles in a variety of physiological processes. However, as a necessary supplement to the body of trace elements, how SELENOK regulates necroptosis in chicken liver has none clear claim. The purpose of this study was to cover the mechanism of SELENOK act in necroptosis of chicken liver. By feeding Se-deficiency diet for 1-day-old hyline chickens, we successfully built SELENOK-deficiency and discussed the regulation SELENOK have done. The test of liver function showed there has dysfunction appeared in the -Se groups. Results of TEM showed necroptosis occurred in the 35-Se group. After that western blot and qRT-PCR results prompted us SELENOK-deficiency caused large accumulation of ROS, enhanced endoplasmic reticulum stress, abnormally elevated HSPs family expression, and activated RIPK1-RIPK3 complex. In order to show the regulation of SELENOK in chicken liver, we artificially knocked off SELENOK gene in LMH cells. Through AO/EB staining we also found necroptosis in the siRNA-Se group. Furthermore, the results in LMH cells were coincided with those in chicken (Gallus gallus) liver. Our experiment clarified the molecular mechanism of SELENOK in the regulation and liver necroptosis, and provided reference for the healthy feeding mode of broilers.

Subacute Cadmium Exposure Induces Necroptosis in Swine Lung via Influencing Th1/Th2 Balance.

Cadmium (Cd) is a type of toxic substance, which widely exists in nature. However, the effect of Cd exposure on the toxicity of swine lungs and its underlying mechanism involved have not yet been reported. In our study, we divided swine into two groups, including a control group (C group) and Cd-exposed group. Swine in the C group were fed a basic diet, whereas swine in the Cd group were fed a 20 mg Cd/kg diet. Immunofluorescence, qRT-PCR, western blot analysis, and H&E staining were performed to detect necroptosis-related indicators. Our results found that after Cd exposure, Th1/Th2 imbalance occurred, miR-181-5p was down-regulated, TNF-α expression was increased, and the NF-κB/NLRP3 and JAK/STAT pathways and RIPK1/RIPK3/MLKL axis were activated. Furthermore, histopathological examination showed necrosis in swine lung after Cd exposure. Together, the above-mentioned results indicate that subacute Cd exposure is closely linked with necroptosis in swine lung. Our study provided evidence that Cd may act through miR-181-5p/TNF-α to induce necroptosis in swine lung. The findings of this study supplement the toxicological study of Cd and provide a reference for comparative medicine.

Tannic acid antagonizes atrazine exposure-induced autophagy and DNA damage crosstalk in grass carp hepatocytes via NO/iNOS/NF-κB signaling pathway to maintain stable immune function.

Atrazine (ATR) is a herbicide widely used in grass crops. The pollution of the soil and water environment is extremely harmful to aquatic animals and their offspring. iNOS/NO upregulation, DNA damage and cellular autophagy affect the immune function of fish liver cells. The effects of ATR at exposure doses on grass carp hepatocytes in terms of autophagy and DNA damage effects in genotoxicity, as well as the antagonistic effects of TAN on the above phenotypes and the internal mechanisms are not known. Therefore, we constructed control (Con group), ATR exposure (ATR group), TAN exposure (TAN group) and mixed group (ATR + TAN group) models on grass carp hepatocytes. Validation was performed by comet assay, MDC staining, qRT-PCR and protein blotting assay as well as iNOS/NO indicator levels and expression of immune factors as these experimental methods. Our data indicate that iNOS/NO assay kit measured that ATR treatment resulted in a significant increase in iNOS/NO activity and levels in grass carp hepatocytes (p < 0.05). We also found that NO/iNOS/NF-κB pathway genes were significantly activated (p < 0.05) at the exposure dose of ATR (3 µg mL-1). In addition, the proportion of cells that died due to DNA damage, autophagy, and immunotoxic effects was significantly increased at the exposure dose of ATR. Comet assay protein blotting detected increased DNA damage in cells at the ATR exposure dose (p < 0.05). MDC staining and qRT-PCR and protein blotting to detect the proportion of autophagic cells and autophagy-related genes also appeared upregulated at the exposed dose of ATR (p < 0.05). In brief, this study showed that ATR exposure caused cellular DNA damage and autophagy via the NO/iNOS/NF-κB axis, which led to immunotoxic effects and eventual death of grass carp hepatocytes. The present study facilitates the demonstration of the molecular mechanism of TAN alleviation of ATR cytotoxicity from the perspective of NO-mediated iNOS/NF-κB axis. It provides insights into the protection of farmed fish from agricultural contaminants and opens up new horizons in the use of natural plant-derived monomers for the clinical treatment of antagonistic triazine pesticide poisoning.