Zinc proteinate with moderate chelation strength enhances zinc absorption by up-regulating the expression of zinc and amino acid transporters in primary cultured duodenal epithelial cells of broiler embryos.

Recent study showed that zinc (Zn) and amino acid transporters may be involved in enhancing Zn absorption from Zn proteinate with moderate chelation strength (Zn-Prot M) in the duodenum of broilers. However, the specific mechanisms by which Zn-Prot M promotes the above Zn absorption are unknown. Therefore, in this study, three experiments were conducted to investigate specific and direct effects of Zn-Prot M and Zn sulfate (ZnS) on Zn absorption and expression of related transporters in primary duodenal epithelial cells of broiler embryos so as to preliminarily address possible mechanisms. In experiment 1, cells were treated with 100 µmol Zn/L as ZnS or Zn-Prot M for 20, 40, 60, 80, 100 or 120 min. Experiment 2 consisted of 3 sub-experiments. In experiment 2A, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 100 or 200 µmol Zn/L as ZnS or Zn-Prot M for 60 min; in experiment 2B, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 200 µmol Zn/L of as the ZnS or Zn-Prot M for 120 min; in experiment 2C, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 400 or 800 µmol Zn/L as ZnS or Zn-Prot M for 120 min. In experiment 3, cells were treated with a Zn-unsupplemented basal medium (Control) or the basal medium supplemented with 400 µmol Zn/L as ZnS or Zn-Prot M for 120 min. The results of experiment 1 indicated that the minimum incubation time for saturable Zn absorption was determined to be 50.83 min using the best fit line. The results in experiment 2 demonstrated that a Zn concentration of 400 µmol/L and an incubation time of 120 min were suitable to increase the absorption of Zn from Zn-Prot M compared to ZnS. In experiment 3, Zn absorption across cell monolayers was significantly increased by Zn addition (P < 0.05), and was significantly greater with Zn-Prot M than with ZnS (P < 0.05). Compared to the control, Zn addition significantly decreased Zn transporter 10 and peptide-transporter 1 mRNA expression levels and increased y+L-type amino transporter 2 (y+LAT2) protein abundance (P < 0.05). Moreover, protein expression levels of zrt/irt-like protein 3 (ZIP3), ZIP5 and y+LAT2 were significantly greater for Zn-Prot M than for ZnS (P < 0.05). These findings suggest that Zn-Prot M promote Zn absorption by increasing ZIP3, ZIP5 and y+LAT2 protein expression levels in primary duodenal epithelial cells.

Research progress on the biological regulatory mechanisms of selenium on skeletal muscle in broilers.

As one of the indispensable trace elements for both humans and animals, selenium widely participates in multiple physiological processes and facilitates strong anti-inflammatory, antioxidant, and immune enhancing abilities. The biological functions of selenium are primarily driven by its presence in selenoproteins as a form of selenocysteine. Broilers are highly sensitive to selenium intake. Recent reports have demonstrated that selenium deficiency can adversely affect the quality of skeletal muscles and the economic value of broilers; the regulatory roles of several key selenoproteins (e.g., GPX1, GPX4, TXNRD1, TXNRD3, SelK, SelT, and SelW) have been identified. Starting from the selenium metabolism and its biological utilization in the skeletal muscle, the effect of the selenium antioxidant function on broiler meat quality is discussed in detail. The progress of research into the prevention of skeletal muscle injury by selenium and selenoproteins is also summarized. The findings emphasize the necessity of in vivo and in vitro research, and certain mechanism problems are identified, which aids their further examination. This mini-review will be helpful to provide a theoretical basis for the further study of regulatory mechanisms of selenium nutrition in edible poultry.

Dietary Zn Deficiency Inhibits Cell Proliferation via the GPR39-Mediated Suppression of the PI3K/AKT/mTOR Signaling Pathway in the Jejunum of Broilers.

A prior investigation revealed that a lack of Zinc (Zn) could hinder intestinal cell proliferation in broiler chickens; however, the mechanisms responsible for this effect remain unclear. We aimed to investigate the possible mechanisms of dietary Zn deficiency in inhibiting the jejunal cell proliferation of broilers. For this study, a total of 112 chickens (21 days old) were randomly divided into two treatments (seven replicate cages per treatment, eight chickens per replicate cage): the control group (CON) and the Zn deficiency group. The duration of feeding was 21 d. Chickens in the control group were provided with a basal diet containing an extra addition of 40 mg Zn/kg in the form of Zn sulfate, whereas chickens in the Zn deficiency group were given the basal diet with no Zn supplementation. The results indicated that, in comparison to the CON, Zn deficiency increased (p < 0.05) the duodenal and jejunal crypt depth (CD) of broilers on d 28 and jejunal and ileal CD on d 35, and decreased (p < 0.05) the duodenal, jejunal, and ileal villus height/crypt depth (VH/CD) on d 28 and the jejunal VH, jejunal and ileal villus surface area, and VH/CD on d 35. Furthermore, Zn deficiency decreased (p < 0.0001) the number of proliferating cell nuclear antigen-positive cells and downregulated (p < 0.01) the mRNA or protein expression levels of phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K, phosphorylated serine-threonine kinase (AKT), phosphorylated mechanistic target of rapamycin (mTOR), G protein-coupled receptor 39 (GPR39), and extracellular-regulated protein kinase, but upregulated (p < 0.05) the mRNA or protein expression levels of P38 mitogen-activated protein kinase, c-jun N-terminal kinase (JNK) 1 and JNK2, and phosphorylated protein kinase C in the jejunum of the broilers on d 42. It was concluded that dietary Zn deficiency inhibited cell proliferation possibly via the GPR39-mediated suppression of the PI3K/AKT/mTOR signaling pathway in the jejunum of broilers.

Dietary copper requirement of broilers fed a corn-soybean meal diet during 22-42 d of age.

This research was to assess the dietary copper (Cu) requirement of broiler chickens fed a practical corn-soybean meal diet during 22-42 d of age. A total of 288 numbered Arbor Acres male broilers at 22 d of age were randomly allotted 6 treatments with 8 replicate cages (6 broilers per cage) per treatment. Broilers were fed a Cu-unsupplemented corn-soybean meal basal diet (control, containing 7.36 mg Cu/kg) or the basal diet added with 3, 6, 9, 12, or 15 mg Cu/kg from CuSO4·5H2O for 21 d. Quadratic, asymptotic and broken-line models were fitted and the best fitted models were selected to determine dietary Cu requirements. The results revealed that the contents of Cu in serum and liver, mRNA expression levels of Cu- and zinc-containing superoxide dismutase (CuZnSOD) in liver and monoamine oxidase b (MAO B) in heart, as well as protein expression level of CuZnSOD in liver were affected (P < 0.05) by supplemental Cu levels, and the above indices varied linearly and quadratically (P < 0.05) with increasing Cu levels. Dietary Cu requirements assessed according to the best fitted broken-line models (P < 0.05) of the above indexes were 10.45-13.81 mg/kg. It was concluded that mRNA expression levels of CuZnSOD in liver and MAO B in heart, as well as liver CuZnSOD protein expression level were new specific sensitive biomarkers for estimating dietary Cu requirements, and the dietary Cu requirement was recommended to be 14 mg/kg to support Cu metabolic needs related to key Cu-containing enzymes in broilers fed the corn-soybean meal diet during 22-42 d of age, which was higher than the dietary Cu requirement (8 mg/kg) for broilers at the corresponding stage suggested by the Chinese Feeding Standard of Chicken.

The chemical characteristics of different sodium iron ethylenediaminetetraacetate sources and their relative bioavailabilities for broilers fed with a conventional corn-soybean meal diet.

BACKGROUND: Our previous studies demonstrated that divalent organic iron (Fe) proteinate sources with higher complexation or chelation strengths as expressed by the greater quotient of formation (Qf) values displayed higher Fe bioavailabilities for broilers. Sodium iron ethylenediaminetetraacetate (NaFeEDTA) is a trivalent organic Fe source with the strongest chelating ligand EDTA. However, the bioavailability of Fe when administered as NaFeEDTA in broilers and other agricultural animals remains untested. Herein, the chemical characteristics of 12 NaFeEDTA products were determined. Of these, one feed grade NaFeEDTA (Qf = 2.07 × 108), one food grade NaFeEDTA (Qf = 3.31 × 108), and one Fe proteinate with an extremely strong chelation strength (Fe-Prot ES, Qf value = 8,590) were selected. Their bioavailabilities relative to Fe sulfate (FeSO4·7H2O) for broilers fed with a conventional corn-soybean meal diet were evaluated during d 1 to 21 by investigating the effects of the above Fe sources and added Fe levels on the growth performance, hematological indices, Fe contents, activities and gene expressions of Fe-containing enzymes in various tissues of broilers. RESULTS: NaFeEDTA sources varied greatly in their chemical characteristics. Plasma Fe concentration (PI), transferrin saturation (TS), liver Fe content, succinate dehydrogenase (SDH) activities in liver, heart, and kidney, catalase (CAT) activity in liver, and SDH mRNA expressions in liver and kidney increased linearly (P < 0.05) with increasing levels of Fe supplementation. However, differences among Fe sources were detected (P < 0.05) only for PI, liver Fe content, CAT activity in liver, SDH activities in heart and kidney, and SDH mRNA expressions in liver and kidney. Based on slope ratios from multiple linear regressions of the above indices on daily dietary analyzed Fe intake, the average bioavailabilities of Fe-Prot ES, feed grade NaFeEDTA, and food grade NaFeEDTA relative to the inorganic FeSO4·7H2O (100%) for broilers were 139%, 155%, and 166%, respectively. CONCLUSIONS: The bioavailabilities of organic Fe sources relative to FeSO4·7H2O were closely related to their Qf values, and NaFeEDTA sources with higher Qf values showed higher Fe bioavailabilities for broilers fed with a conventional corn-soybean meal diet.

Polystyrene microplastics with different sizes induce the apoptosis and necroptosis in liver through the PTEN/PI3K/AKT/autophagy axis.

The production of plastics worldwide has been instrumental in the progress of modern society, while the increasing accumulation of plastics castoff in oceans, soils and anywhere else has become a major pressure source on environmental sustainability and animal health. Meanwhile, from a biological perspective, our understanding of the toxicological fingerprints of plastics, especially microplastics (MPs), is still poor. Here, we reported a phenomenon of hepatotoxicity dominated by MPs in the form of polystyrene (PS), was observed in mice model systems and cellular assays. Apoptosis and necroptosis related to the size of particles were seen upon PS-MPs introduction, as revealed by transmission electron microscopy, fluorescence microscopy, flow cytometry, and quantitative analysis of signaling pathways in vivo and vitro. Collectively, the current study demonstrated that the levels of liver cell injury caused by PS-MPs were negatively correlated with the particle diameters. Small-sized particles (1-10 µm) induced cell death primarily as necroptosis whereas the large-sized particles (50-100 µm) mainly induced apoptosis, which was directly accomplished by PTEN/PI3K/AKT signaling axis and its targeted autophagy flux. More interestingly, inhibition of autophagy not only alleviated PS-MPs-triggered cell death, but also changed the form of death injury to a certain extent. This uncovered crosstalk relationship opens up a new avenue for investigating the biological and toxicological effects of MPs, and may provide important insights for preventing and limiting of health hazards from MPs.

Polystyrene microplastics-induced macrophage extracellular traps contributes to liver fibrotic injury by activating ROS/TGF-ß/Smad2/3 signaling axis.

Microplastics (MPs) are a type of emerging pollutant, posing a great threat to human and animal health. While recent studies have revealed the link between MPs exposure and liver injury of organisms, the effect of particle size on the level of MPs-induced hepatotoxicity and the intrinsic mechanism remain to be explored. Here, we established a mouse model exposed to two-diameter polystyrene MPs (PS-MPs, 1-10 µm or 50-100 µm) for 30 days. The in vivo results revealed that PS-MPs caused liver fibrotic injury in mice, accompanied with macrophages recruitment and macrophage extracellular traps (METs) formation, which were negatively correlated with particle size. The data in vitro showed that PS-MPs treatment could induce macrophages to release METs in a reactive oxygen species (ROS)-independent manner, and the METs formation level caused by large-size particles was higher than small-size particles. Further mechanistic analysis of a cell co-culture system revealed that PS-MPs-induced METs release led to a hepatocellular inflammatory response and epithelial-mesenchymal transition (EMT) via activating the ROS/TGF-ß/Smad2/3 signaling axis, and this biological crosstalk could be relieved by DNase I. Overall, this findings demonstrates the key role of the action mechanism of METs in aggravating MPs-caused liver injury.

TBBPA induces inflammation, apoptosis, and necrosis of skeletal muscle in mice through the ROS/Nrf2/TNF-α signaling pathway.

Tetrabromobisphenol A (TBBPA) is present in large quantities in the environment due to its widespread use. And TBBPA is capable of accumulating in animals, entering the ecological chain and causing widespread damage to organisms. TBBPA is capable of causing the onset of oxidative stress, which induces tissue damage and cell death, which in turn affects the physiological function of tissues. Skeletal muscle is a critical tissue for maintaining growth, movement, and health in the body. However, the mechanism of TBBPA-induced skeletal muscle injury remains unclear. In this study, we constructed mouse skeletal muscle models (10, 20, and 40 mg/kg TBBPA) and mouse myoblasts (C2C12) cell models (2,4, and 8 µg/L TBBPA) at different concentrations. The results of this experiment showed that under TBBPA treatment, the levels of reactive oxygen species (ROS) and Malondialdehyde (MDA) in mouse skeletal and C2C12 cells were increased significantly, but the activities of some antioxidant enzymes decreased. TBBPA can inhibit Nuclear factor E2-related factor 2 (Nrf2) entry into the nucleus, thus affecting the expression of the Nrf2 downstream factors. With the increase of TBBPA concentration, the expression levels of inflammatory factors were significantly increased, while the anti-apoptotic factors were significantly decreased. The expression of pro-apoptotic factors increased in a dose-dependent manner. Programmed necrosis-related factors were also significantly elevated. Our results suggest that TBBPA induces oxidative stress and inflammation, apoptosis, and necrosis in the skeletal muscle of mice by regulating Nrf2/ROS/TNF-α signaling pathway.

Profiling Substrate Specificity of the SUMO Protease Ulp1 by the YESS-PSSC System to Advance the Conserved Mechanism for Substrate Cleavage.

SUMO modification is a vital post-translational regulation process in eukaryotes, in which the SUMO protease is responsible for the maturation of the SUMO precursor and the deconjugation of the SUMO protein from modified proteins by accurately cleaving behind the C-terminal Gly-Gly motif. To promote the understanding of the high specificity of the SUMO protease against the SUMO protein as well as to clarify whether the conserved Gly-Gly motif is strictly required for the processing of the SUMO precursor, we systematically profiled the specificity of the S. cerevisiae SUMO protease (Ulp1) on Smt3 at the P2-P1↓P1' (Gly-Gly↓Ala) position using the YESS-PSSC system. Our results demonstrated that Ulp1 was able to cleave Gly-Gly↓ motif-mutated substrates, indicating that the diglycine motif is not strictly required for Ulp1 cleavage. A structural-modeling analysis indicated that it is the special tapered active pocket of Ulp1 conferred the selectivity of small residues at the P1-P2 position of Smt3, such as Gly, Ala, Ser and Cys, and only which can smoothly deliver the scissile bond into the active site for cleavage. Meanwhile, the P1' position Ala of Smt3 was found to play a vital role in maintaining Ulp1's precise cleavage after the Gly-Gly motif and replacing Ala with Gly in this position could expand Ulp1 inclusivity against the P1 and P2 position residues of Smt3. All in all, our studies advanced the traditional knowledge of the SUMO protein, which may provide potential directions for the drug discovery of abnormal SUMOylation-related diseases.

Polystyrene microplastics induce apoptosis and necroptosis in swine testis cells via ROS/MAPK/HIF1α pathway.

Microplastics (MPs) generally refer to the plastic fragments or particles smaller than 5 mm in diameter, which are closely concerned due to their widespread presence in the environment. Recent studies have shown that MPs have a serious threat on the reproductive health of organisms. Pigs are often selected as the model animals because of their high similarity to human tissues and organs. However, there are no reports on the effects and mechanisms of MPs exposure on swine germ cells. In the present study, we established swine testis (ST) cell models exposed to 250, 500, and 1000 µg/ml polystyrene microplastics (PS-MPs, 1-10 µm), respectively. The findings revealed that PS-MPs reduced cell viability dose-dependently. Acridine orange/ethidium bromide staining and flow cytometry results indicated the occurrence of apoptosis and necrosis in ST cells under PS-MPs exposure, and the expression changes of relevant marker genes (B-cell lymphoma-2, Bcl-2 Associated X, Caspase-3, Caspase-9, Receptor-interacting protein kinase 1, Receptor-interacting protein kinase 3, Mixed lineage kinase domain-like, and Caspase-8) were clarified via quantitative real-time PCR and western blot. Further mechanistic studies found that PS-MPs treatment induced excessive intracellular reactive oxygen species (ROS) production, which promoted the phosphorylation of mitogen-activated protein kinase (MAPK) pathway-related genes (P38, c-Jun N-terminal kinase, extracellular regulated protein kinases) and activated the downstream gene hypoxia-inducible factor (HIF1α). In conclusion, our study suggests that PS-MPs treatment causes apoptosis and necroptosis in ST cells via ROS/MAPK/HIF1α signaling pathway.

Cadmium induces apoptosis by miR-9-5p targeting PTEN and regulates the PI3K/AKT pathway in the piglet adrenal gland.

Cadmium (Cd) is an environmental pollutant that can cause endocrine organ damage. To explore the effect of subacute CdCl2 exposure on piglet adrenal gland tissue and its mechanism based on the establishment of this model, bioinformatics, TUNEL assay, western blot (WB), and qRT-PCR methods were used to detect related indicators. The results showed that after Cd exposure, antioxidant enzymes decreased, heat shock protein increased, and miR-9-5p-gene of phosphatase and tensin homolog (PTEN) upregulates the phosphatidylinositol-3-kinase (PI3K/AKT) pathway. After this pathway was activated, the expression of the apoptosis-related factors cysteinyl aspartate-specific proteinase 3 and 9 (caspase 3 and 9), B-cell lymphoma-2-associated X (BAX) was increased sharply, and the expression of B-cell lymphoma-2 (BCL2) was significantly decreased. The changes in these indicators indicate that Cd exposure induces apoptosis and causes tissue damage in the adrenal gland of piglets. This study aims to reveal the toxic effects of CdCl2 in animals and will provide new ideas for the toxicology of Cd.

BPA exposure aggravates necroptosis of myocardial tissue in selenium deficient broilers through NO-dependent endoplasmic reticulum stress.

The environmental problem of BPA pollution has seriously endangered the health of humans and animals. As an essential trace element, the health problems caused by insufficient intake of selenium have always been widespread. Under toxic and other harmful stimuli, severe endoplasmic reticulum (ER) stress is one of multitude factors leading to death such as apoptosis and necrotic apoptosis. For investigating the damage of BPA and selenium deficiency combined effect to the myocardial tissue of broilers and the role of ER stress, 1-day-old broilers were fed with toxic feed and selenium deficiency feed for 35 days. Histopathological results showed that the combined exposure of BPA and low selenium resulted in more severe necrosis of cardiomyocytes in broilers than the single exposure, but there was no significant change in apoptosis compared with single exposure. Molecular biology studies showed that NO-dependent ER stress induced by BPA increased the expression of necroptosis related genes in myocardium of selenium deficient broilers, but had no effect on apoptosis pathway. In conclusion, our records state that the NO-dependent ER stress caused by combined exposure of BPA and low selenium can cause serious damage to the myocardial tissue of broilers by promoting the activation of the necroptosis pathway.

Selenoprotein K protects skeletal muscle from damage and is required for satellite cells-mediated myogenic differentiation.

The regeneration of adult skeletal muscle after injury is primarily initiated by satellite cells (SCs), but the regulatory mechanisms of cells committed to myogenic differentiation remain poorly explored. Small molecular selenoprotein K (SelK) plays crucial roles in the modulation of endoplasmic reticulum (ER) stress and against oxidative stress. Here, we first showed that SelK expression is activated in myogenic cells during differentiation both in vivo and in vitro. Meanwhile, loss of SelK delayed skeletal muscle regeneration, inhibited the development of myoblasts into myotubes, and was accompanied by reduced expression of myogenic regulatory factors (MRFs). Moreover, ER stress, intracellular reactive oxygen species (ROS), autophagy and apoptosis under myogenesis induction were more severe in SelK-deficient mice and cells than in the corresponding control groups. Supplementation with specific inhibitors to alleviate excessive ER stress or oxidative stress partly rescued the differentiation potential and formation of myotubes. Notably, we demonstrated that Self-mediated regulation of cellular redox status was primarily derived from its subsequent effects on ER stress. Together, our results suggest that SelK protects skeletal muscle from damage and is a crucial regulator of myogenesis.

A new discovery of polystyrene microplastics toxicity: The injury difference on bladder epithelium of mice is correlated with the size of exposed particles.

Microplastics (MPs), as widespread hazardous substances in the environment, can cause potential adverse effects on biological health. However, reports on the toxic effects of different diameters MPs on urinary system are limited. Here, we investigated the types and mechanisms of damage to mice bladder epithelial cells treated with diameter (1-10 µm and 50-100 µm) polystyrene microplastics (PS-MPs). The results showed that exposure to PS-MPs of both diameters resulted in necroptosis and inflammation to bladder epithelium. However, 1-10 µm PS-MPs posed more severe necroptosis and 50-100 µm PS-MPs led to a higher degree of inflammatory injury at the same exposure concentration. Mechanistically, PS-MPs were found to induce necroptosis as well as p-NFκB-mediated inflammation by triggering oxidative stress and excessive release of reactive oxygen species (ROS). Furthermore, N-Acetyl-l-cysteine (NAC) attenuated the toxic effects of PS-MPs on bladder epithelial cells. In conclusion, our study demonstrated for the first time that PS-MPs caused necroptosis and inflammation in mice bladders tissues, and the difference of injury correlates with the size of PS-MPs particles.

CircRNA-IGLL1/miR-15a/RNF43 axis mediates ammonia-induced autophagy in broilers jejunum via Wnt/ß-catenin pathway.

With the continued increase of global ammonia emission, the damage to human or animal caused by ammonia pollution has attracted wide attention. The noncoding RNAs have been reported to regulate a variety of biological processes under different environmental stimulation via ceRNA (competing endogenous RNA) networks. Autophagy is a hallmark of tissue damage from air pollution. However, the specific role of circular RNAs (circRNAs) in the injury of intestinal tissue caused by autophagy remains unclear. Here, we established 42-days old ammonia-exposed broiler models and observed that autophagy flux in broiler jejunum was activated under ammonia exposure. Meanwhile, a total of eight significantly dysregulated expressed circRNAs were obtained and a circRNAs-miRNAs-genes interaction networks were constructed by bioinformatics analysis. Furthermore, an axis named circRNA-IGLL1/miR-15a/RNF43 was predicted to participate in the excessive autophagy by targeting RNF43. The target relationship was proved by dual-luciferase reporter assay in vitro. Mechanistically, downregulated circRNA-IGLL1 could suppress the expression of RNF43 in ammonia-exposed jejunum and the Wnt/ß-catenin pathway was activated. Inhibition of miR-15a reversed autophagy caused by downregulated circRNA-IGLL1. CircRNA-IGLL1 could competitively bind miR-15a to regulate RNF43 expression, thus modulating the occurrence of autophagy. Taken together, our results showed that circRNA-IGLL1/miR-15a/RNF43 axis is involved in ammonia-induced intestinal autophagy in broilers.

Exposure to imidacloprid induce oxidative stress, mitochondrial dysfunction, inflammation, apoptosis and mitophagy via NF-kappaB/JNK pathway in grass carp hepatocytes.

Imidacloprid (IMI) is a neonicotinoid compound widely used in agriculture production, causing surface water pollution and threatening non-target organisms. The aim of this study was to analyze the effects of IMI on grass carp (Ctenopharyngodon idellus) liver cell (L8824) injury. The L8824 cells were exposed to different doses of IMI (65 mg/L, 130 mg/L and 260 mg/L) for 24 h. Our results demonstrated that exposure IMI significantly suppressed the activity of anti-oxidant enzymes (SOD, CAT and T-AOC) and accumulated oxidase (MDA) levels, and promoting reactive oxygen species (ROS) generation in L8824 cells. Additionally, mitochondrial membrane potential (ΔΨ m), mitochondria-derived ROS and ATP content and the MitoTracker Green indicated that IMI aggravated mitochondrial dysfunction, thereby inducing inflammation and enhancing pro-inflammatory genes (NF-kappaB, TNFα, IL-1ß and IL-6) expressions. However, the addition of 2 mM N-acetyl-l-cysteine (NAC) can reverse these adverse effects of high-dose IMI- induced. Hence, ROS is the main factor of IMI-induced mitochondrial dysfunction and inflammation. We further found that exposure to IMI induced apoptosis, which is characterized by promoting release of cytochrome c (Cyt-C), and increasing the expression of Bcl-2-Associated X (BAX), cysteinyl aspartate specific proteinases (Caspase 9 and 3), decreasing Bcl-2 level. Immunofluorescent staining, qRT-PCR and Western Blot results indicated that IMI exposure also activated mitophagy, which was demonstrated by the expression of mitophagy-related genes (BNIP3, LC3B and P62). Conversely, scavenging JNK by SP600125(10 µM) alleviated the expression of mitochondrial apoptosis and mitophagy-related gene induced by high-dose IMI. Therefore, these results of study demonstrated that IMI-induced oxidative stress to regulate mitochondrial dysfunction, thus causing inflammation, mitochondrial apoptosis and mitophagy in grass carp hepatocytes through NF-kappaB/JNK pathway.

Quercetin antagonizes imidacloprid-induced mitochondrial apoptosis through PTEN/PI3K/AKT in grass carp hepatocytes.

Imidacloprid (IMI) is widely used in agriculture, and is toxic to non-target aquatic species. Quercetin (Que) is a flavonoid abundant in fruits and vegetables that exhibits anti-oxidant activity. In the present study, we treated grass carp hepatocytes (L8824) with 0.1 µM Que and/or 1 mM IMI for 24 h to explore the effect of Que on IMI-induced mitochondrial apoptosis. We found that IMI exposure enhanced reactive oxygen species (ROS) generation, inhibiting the activities of SOD, CAT and T-AOC, exacerbating the accumulation of MDA, aggravating the expression of mitochondrial apoptosis pathway (Cyt-C, BAX, Caspase9 and Caspase3) related genes and decreased the expression of anti-apoptosis gene B-cell lymphoma-2 (Bcl-2). In addition, Que and IMI co-treatment significantly restored the activity of anti-oxidant enzymes, downregulated ROS level and apoptosis rate, thereby alleviating the depletion of mitochondrial membrane potential (ΔΨm) and the expression of cytochrome c (Cyt-C), Bcl-2-associated X (BAX), and cysteinyl aspartate specific proteinases (Caspase9 and 3), increasing the Bcl-2 level. Furthermore, we elucidated that Que could inhibit the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), thus activating phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway to attenuate IMI-induced apoptosis. Molecular docking provides assertive evidence for the interaction between Que ligand and PTEN receptor. Consequently, these results indicate that Que effectively antagonizes IMI-induced mitochondrial apoptosis in grass carp hepatocytes via regulating the PTEN/PI3K/AKT pathway.

Subacute cadmium exposure promotes M1 macrophage polarization through oxidative stress-evoked inflammatory response and induces porcine adrenal fibrosis.

Cadmium (Cd) is a widely distributed environmental pollutant with immunotoxicity and endocrine toxicity. M1/M2 macrophages participate in the immune response and exert an essential influence on fibrosis. Nevertheless, whether Cd can induce porcineadrenal fibrosis by affecting the polarization of M1/M2 macrophages and its potential regulatory mechanism have not been explored. We added 20 mg/kg CdCl2 to the pig diet for 40 days to investigate the fibrogenic effect of subacute Cd exposure on the adrenal gland. The results indicated that the ACTH and CORT in serum were decreased by 15.26 % and 21.99 %, respectively. The contents of adrenal mineral elements Cd, Cr, Mn were increased up to 34, 1.93, 1.42 folds and Co, Zn, Sn were reduced by 21.57 %, 20.52 %, 15.75 %. Concurrently, the pro-oxidative indicators (LPO, MDA and H2O2) were increased by 1.85, 2.20, 2.77 folds and 3.60, 11.15, 4.11 folds upregulated mRNA levels of TLR4, NF-κB, NLRP3 were observed. Subsequently, the expression of M1 macrophages polarization markers (IL-6, iNOS, TNF-α, CCL2 and CXCL9) were raised by 2.03, 2.30, 2.35, 1.58, 1.56 folds, while M2 macrophages (IL-4, CCL24, Arg1, IL-10, MRC1) showed a 62.34 %, 31.88 %, 50.26 %, 74.00 %, 69.34 % downregulation. The expression levels of AMPK subunits and genes related to glycolysis, oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) were also markedly increased. Additionally, the expression level of TGF-ß1, Smad2/3 and downstream pro-fibrotic markers was obviously upregulated. Taken together, we conclude that Cd activates the oxidative stress-mediated TLR4/NF-κB/NLRP3 inflammatory signal transduction, leading to porcine adrenal fibrosis by promoting macrophage polarization toward M1.

miR-130-CYLD Axis Is Involved in the Necroptosis and Inflammation Induced by Selenium Deficiency in Pig Cerebellum.

Selenium (Se) is an essential trace element in creatures which deficiency can cause necroptosis and inflammation of multiple tissues. MicroRNAs (miRNAs) have been identified to participate multiple biological processes by regulating the expression of target genes. In the present study, the Se-deficient pig cerebellar model was established and conducted by light microscopy, qRT-PCR, and Western blot. Morphological observation exhibited necrosis-like lesions and inflammatory infiltration in the cerebellum of the Se-deficient group. Quantitative analysis result showed that Se deficiency significantly suppressed miR-130 expression, which in turn disinhibited the expression of CYLD. Meanwhile, in comparison to the control group, the expression levels of TNF-α pathway genes (TNF-α, TNFR1, and NF-κB p65) and necroptosis-related genes (RIPK1, RIPK3, and MLKL) in Se deficiency group were obviously increased (P < 0.05). Moreover, Se deficiency induced the occurrence of inflammation by upregulating the expression of inflammatory cytokines (IL-1ß, IL-2, IL-8, IL-18, IFN-γ, COX-2, PTGEs, and NLRP3). In conclusion, we proved Se deficiency could induce the deregulation of miR-130-CYLD axis to cause RIPK3-dependent necroptosis and inflammation in pig cerebellum.