Conserved gatekeeper methionine regulates the binding and access of kinase inhibitors to ATP sites of MAP2K1, 4, and 7: Clues for developing selective inhibitors.

Mitogen-activated protein kinase kinases (MAP2Ks) 1, 4, and 7 are potential targets for treating various diseases. Here, we solved the crystal structures of MAP2K1 and MAP2K4 complexed with covalent inhibitor 5Z-7-oxozeaenol (5Z7O). The elucidated structures showed that 5Z7O was non-covalently bound to the ATP binding site of MAP2K4, while it covalently attached to cysteine at the DFG-1 position of the deep ATP site of MAP2K1. In contrast, we previously showed that 5Z7O covalently binds to MAP2K7 via another cysteine on the solvent-accessible edge of the ATP site. Structural analyses and molecular dynamics calculations indicated that the configuration and mobility of conserved gatekeeper methionine located at the central ATP site regulated the binding and access of 5Z7O to the ATP site of MAP2Ks. These structural features provide clues for developing highly potent and selective inhibitors against MAP2Ks. Abbreviations: ATP, adenosine triphosphate; FDA, Food and Drug Administration; MAP2Ks, mitogen-activated protein kinase kinases; MD, molecular dynamics; NSCLC, non-small cell lung cancer; 5Z7O, 5Z-7-oxozeaenol; PDB, protein data bank; RMSD, root-mean-square deviation.

Feed contamination with zearalenone promotes growth but affects the immune system of rainbow trout.

To investigate the effects of feed contamination with zearalenone (ZEN) at the current European Commission (EC) guidance value (2 mg⋅kg-1 feed) on the growth and health of rainbow trout, we performed a long-term feeding trial under aquaculture conditions. It started with the external feeding of the fish larvae, and continued for 96 weeks, at which point the fish had reached market size. To assess the growth of fish and their feeding efficiency throughout this period, the fish were regularly weighed and measured, and their feed consumption was monitored. Additionally, to investigate potential health effects, after 72 weeks of the exposure to ZEN, the fishes' blood was analyzed for major hematological and biochemical indices, and their head kidney, spleen, and liver were examined for morphological, histopathological, cytological, and molecular changes. Finally, to gain insight into the metabolism and distribution of ZEN in fish, the content of free and glucuronidated forms of ZEN and its major metabolites was measured in the intestine, liver, and muscles of the exposed fish. The feed-borne exposure of rainbow trout to ZEN at a dose of 2 mg⋅kg-1 feed resulted in higher feeding efficiency and growth rate, most probably due to the anabolic properties of the ZEN metabolite. Importantly for the consumers of fish, despite absorption and metabolism of ZEN in the digestive system of the fish that had been exposed for 72 weeks, the residuals of ZEN were not transferred to the fishes' muscles, which rules out a potential risk to human health related to the consumption of fish meat. However, the increased growth of fish fed with the contaminated feed may come at some cost, as the exposure to ZEN was associated with modulation of key components of the adaptive and innate immune systems. Moreover, the trunk kidney of ZEN-fed fish showed massive inflammation that was likely caused by pathogen infection. These findings raise concerns about fish health under the current recommended EC guidance values.

Metabolic Profile, Bioavailability and Toxicokinetics of Zearalenone-14-Glucoside in Rats after Oral and Intravenous Administration by Liquid Chromatography High-Resolution Mass Spectrometry and Tandem Mass Spectrometry.

Zearalenone-14-glucoside (ZEN-14G), a key modified mycotoxin, has attracted a great deal of attention due to the possible conversion to its free form of zearalenone (ZEN) exerting toxicity. In this study, the toxicokinetics of ZEN-14G were investigated in rats after oral and intravenous administration. The plasma concentrations of ZEN-14G and its major five metabolites were quantified using a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The data were analyzed via non-compartmental analysis using software WinNonlin 6.3. The results indicated that ZEN-14G was rapidly hydrolyzed into ZEN in vivo. In addition, the major parameters of ZEN-14G following intravenous administration were: area under the plasma concentration-time curve (AUC), 1.80 h·ng/mL; the apparent volume of distribution (VZ), 7.25 L/kg; and total body clearance (CL), 5.02 mL/h/kg, respectively. After oral administration, the typical parameters were: AUC, 0.16 h·ng/mL; VZ, 6.24 mL/kg; and CL, 4.50 mL/h/kg, respectively. The absolute oral bioavailability of ZEN-14G in rats was about 9%, since low levels of ZEN-14G were detected in plasma, which might be attributed to its extensive metabolism. Therefore, liquid chromatography high-resolution mass spectrometry (LC-HRMS) was adopted to clarify the metabolic profile of ZEN-14G in rats' plasma. As a result, eight metabolites were identified in which ZEN-14-glucuronic acid (ZEN-14GlcA) had a large yield from the first time-point and continued accumulating after oral administration, indicating that ZEN-14-glucuronic acid could serve a potential biomarker of ZEN-14G. The obtained outcomes would prompt the accurate safety evaluation of ZEN-14G.

Synergistic estrogenic effects of Fusarium and Alternaria mycotoxins in vitro.

Mycotoxins are toxic secondary metabolites formed by various fungal species that are found as natural contaminants in food. This very heterogeneous group of compounds triggers multiple toxic mechanisms, including endocrine disruptive potential. Current risk assessment of mycotoxins, as for most chemical substances, is based on the effects of single compounds. However, concern on a potential enhancement of risks by interactions of single substances in naturally occurring mixtures has greatly increased recently. In this study, the combinatory effects of three mycoestrogens were investigated in detail. This includes the endocrine disruptors zearalenone (ZEN) and α-zearalenol (α-ZEL) produced by Fusarium fungi and alternariol (AOH), a cytotoxic and estrogenic mycotoxin formed by Alternaria species. For evaluation of effects, estrogen-dependent activation of alkaline phosphatase (AlP) and cell proliferation were tested in the adenocarcinoma cell line Ishikawa. The estrogenic potential varied among the single substances. Half maximum effect concentrations (EC50) for AlP activation were evaluated for α-ZEL, ZEN and AOH as 37 pM, 562 pM and 995 nM, respectively. All three mycotoxins were found to act as partial agonists. The majority of binary combinations, even at very low concentrations in the case of α-ZEL, showed strong synergism in the AlP assay. These potentiating phenomena of mycotoxin mixtures highlight the urgent need to incorporate combinatory effects into future risk assessment, especially when endocrine disruptors are involved. To the best of our knowledge, this study presents the first investigation on synergistic effects of mycoestrogens.

Preliminary results on the interactive effects of deoxynivalenol, zearalenone and fumonisin B1 on porcine lymphocytes.

Fusarium mycotoxins, such as fumonisin B1 (FB1), deoxynivalenol (DON) and zearalenone (ZEN), frequently co-occur in feed raw materials and their presence is ubiquitous. The aims of this study were to determine the concentration that inhibits cell viability by 50% (IC50 values) for each mycotoxin (after 24, 48 and 72 h) and to investigate their combined effects in binary (DON + ZEN: DZ, DON + FB1: DF, FB1 + ZEN: FZ) and ternary (DFZ) mixtures using cyto- and genotoxicity on porcine lymphocytes as endpoints. The potency of cytotoxicity of the three toxins in an increasing order was FB1 < ZEN < DON. The range of IC values depending on the period of exposure was 0.31-0.42 µg/ml and 16.6- 22.9 µg/ml for DON and ZEN, respectively, and 101.15 µg/ml for FB1 (50% viability was reached only after 72 h). The main interaction observed was antagonism regarding cytotoxicity. Lower and higher sets of concentrations were used for the genotoxicity (comet assay) experiments. When lower concentrations were used, antagonism was again the main interaction observed. However, at higher concentrations an antagonism was confirmed only for DFZ, whereas for DZ and FZ a synergism was observed. Interactions of DF were inconsistent in different exposure periods in both series of experiments. Further studies with additional endpoints should be performed (e.g. DNA fragmentation, protein synthesis) in order to elucidate the mechanisms underlying the interactions observed.

Intracerebroventricular administration of the (1→6)-ß-d-glucan (lasiodiplodan) in male rats prevents d-penicillamine-induced behavioral alterations and lipoperoxidation in the cortex.

CONTEXT: Lasiodiplodan, an exocellular (1→6)-ß-d-glucan of molecular weight >1.4 × 106 Da produced by MMPI strain of Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (Brotyosphaeriaceae) is known to exhibit anti-proliferative activity on breast cancer cells (MCF-7), anticoagulant activity when sulfonylated, and reduction in transaminase activity when administered in rats. OBJECTIVE: The effect of intracerebroventricular (I.C.V) injection of lasiodiplodan on neurotoxicity and behavioural changes induced by d-penicillamine was investigated. MATERIALS AND METHODS: Twenty-four male Wistar rats were initially separated in groups of six and treated with 0.15 µmol/µL of NaCl (Groups Ct and d-Pen) and 0.01 µg/µL of lasiodiplodan (Groups Las and Las + d-Pen). After 15 min, they received 6 µmol/µL of NaCl (Groups Ct and Las) and 2 µmol/µL of d-penicillamine (Groups d-Pen and Las + d-Pen). The animal behavior was observed in an open-field test for 60 min. Twenty-four h later, the animals were sacrificed and histopathological analysis and Thiobarbituric acid reactive substances (TBARS) production measurements were performed. RESULTS: Lasiodiplodan prevented neurotoxicity induced by d-penicillamine significantly reducing the production of TBARS (308%; p < 0.05), and behavioural signs; convulsive and pre-convulsive. No histopathological alterations in the cerebral cortex were observed. DISCUSSION AND CONCLUSION: The reduction of TBARS production and convulsive episodes suggests that the protector effect provided by lasiodiplodan passes thought an antioxidant path, possibly interfering in a cascade of neurochemical events, triggering cell death and convulsive episodes. These results demonstrated that lasiodiplodan can be effective in treating neurotoxicity, and reducing damage triggered by convulsions in neuropathies related to GABAergic system.

Interactions between plant-derived oestrogenic substances and the mycoestrogen zearalenone in a bioassay with MCF-7 cells.

Human and animal diets may contain several non-steroidal oestrogenic compounds which originate either from plants (phytoestrogens) or from fungi that infect plants (mycoestrogens such as zearalenone (ZEN)). Phytoestrogens may compete with ZEN in binding to the oestrogen receptor ß and thereby may counteract the oestrogenic activity of ZEN. Using a modified version of the E-screen assay, plant-derived oestrogenic substances were tested for their proliferative or anti-proliferative effect on oestrogen-dependent MCF-7 cells. The samples were additionally tested for their ability to influence the oestrogenic activity of ZEN (1 µM). Among the individual substances tested, 8-prenylnaringenin had the strongest effect, as cell proliferation was increased by 78% at the lowest concentration (0.23 µM), and by 167% at the highest concentration (29.4 µM). Coumestrol (5.83 µM) increased cell proliferation by 39%, and genistein (370 µM) by 61%, respectively. Xanthohumol and enterolactone did not stimulate cell proliferation significantly. In the co-incubation experiments with ZEN, none of the single substances was able to decrease the oestrogenic activity of ZEN. Only for 8-prenylnaringenin (14.7 and 29.4 µM) was a trend towards an increase in the ZEN-induced cell proliferation up to 72% observed. In conclusion, with the exception of 8-prenylnaringenin, no substantial interaction between phytoestrogens and the mycotoxin ZEN could be detected using a bioassays with MCF-7 cells.

Synergistic action of 5Z-7-oxozeaenol and bortezomib in inducing apoptosis of Burkitt lymphoma cell line Daudi.

Treatment failure in cancer chemotherapy is largely due to the toxic effects of chemotherapeutic agents on normal cells/tissues. The proteasome inhibitor bortezomib has been successfully applied to treat multiple myeloma (MM), but there are some common adverse reactions in the clinic including peripheral neuropathy (PN). The TAK1 selective inhibitor 5Z-7-oxozeaenol has been widely studied in cancer therapy. Here, we investigated the potential synergy of bortezomib and 5Z-7-oxozeaenol in Burkitt's lymphoma (BL) cell lines. Cell viability assay showed that co-treatment of bortezomib at 8 nM, representing a one-eighth concentration for growth arrest, and 5Z-7-oxozeaenol at 2 µM, a dose that exhibited insignificant cytotoxic effects, synergistically induced apoptosis in the cell line Daudi. In parallel with the increasing dose of the bortezomib, and 5Z-7-oxozeaenol at 0.5 µM, lower colony formation efficiencies were seen in the cell line Daudi. Western blotting analysis verified that TAK1 inhibition by 5Z-7-oxozeaenol completely blocked JNK, p38, Erk, IKK, and IκB phosphorylation, which was almost instantly activated by TAK1 both directly or indirectly. Both agents synergistically prevented nuclear translocation of NF-κB, a characteristic of NF-κB inactivation. Moreover, a synergistic effect of bortezomib and 5Z-7-oxozeaenol on Western blotting analysis and flow cytometry was disclosed. Collectively, our results indicated that the proteasome inhibitor bortezomib and the TAK1 inhibitor 5Z-7-oxozeaenol displayed synergy on inhibiting BL cell apoptosis by inhibiting NF-κB activity.

Immunohistochemical evaluation of apoptosis and proliferation in the mucous membrane of selected uterine regions in pre-pubertal bitches exposed to low doses of zearalenone.

Mycotoxins are toxic secondary metabolites produced by fungi. Those biologically active compounds occur naturally and they include zearalenone (ZEN), a mycotoxin that contaminates plant material, including the ingredients used in the production of commercial dog food. The influence of monotonic, low-dose and long-term exposure to ZEN on pre-pubertal bitches has not been fully explored to date. This paper describes a 42-day experiment performed on clinically healthy female dogs aged approximately 70 days, with estimated body weight of 8 kg. The animals were randomly divided into two experimental groups (EI and EII) and a control group (C) of 10 animals each. Group EI received 50 µg ZEN/kg (of body weight) per os, group EII received 75 µg ZEN/kg BW per os, and the control group was administered placebo. The bitches were ovariohysterectomized at the end of the experiment (at around 112 days of age), and selected sections of the uterine wall were subjected to immunohistochemical analyses (TUNEL and PCNA). A shift towards higher apoptotic (AI) and proliferative index (PI) was observed, in particular in group EI. Higher AI and PI values were noted in the epithelium of all uterine regions analysed and in the uterine glands in the uterine horn proximal to the body of the uterus.

Intra-amniotic pharmacological blockade of inflammatory signalling pathways in an ovine chorioamnionitis model.

Intrauterine inflammation (IUI) associated with infection is the major cause of preterm birth (PTB) at <32 weeks' gestation and accounts for ∼40% of all spontaneous PTBs. Pharmacological strategies to prevent PTB and improve fetal outcomes will likely require both antimicrobial and anti-inflammatory therapies. Here we investigated the effects of two cytokine-suppressive anti-inflammatory drugs (CSAIDs), compounds that specifically target inflammatory signalling pathways, in an ovine model of lipopolysaccharide (LPS)-induced chorioamnionitis. Chronically catheterized ewes at 116 days gestation (n = 7/group) received an intra-amniotic (IA) bolus of LPS (10 mg) plus vehicle or CSAIDS: TPCA-1 (1.2 mg/kg fetal weight) or 5z-7-oxozeaenol (OxZnl; 0.4 mg/kg fetal weight); controls received vehicle (dimethylsulphoxide). Amniotic fluid (AF), fetal and maternal blood samples were taken 0, 2, 6, 12, 24 and 48 h later; tissues were taken at autopsy (48 h). Administration of TPCA-1 or OxZnl abrogated the stimulatory effects of LPS (P < 0.01 versus vehicle control) on production of PGE2 in AF, with lesser (non-significant) effects on IL-6 production. Fetal membrane polymorphonuclear cell infiltration score was significantly higher in LPS versus vehicle control animals (P < 0.01), and this difference was absent with TPCA-1 and OxZnl treatment. LPS-induced systemic fetal inflammation was highly variable, with no significant effects of CSAIDs observed. Lung inflammation was evident with LPS exposure, but unaffected by CSAID treatment. We have shown in a large animal model that IA administration of a single dose of CSAIDs can suppress LPS-induced IA inflammatory responses, while fetal effects were minimal. Further development and investigation of these compounds in infectious models is warranted.

The Effect of Low Monotonic Doses of Zearalenone on Selected Reproductive Tissues in Pre-Pubertal Female Dogs--A Review.

The growing interest in toxic substances combined with advancements in biological sciences has shed a new light on the problem of mycotoxins contaminating feeds and foods. An interdisciplinary approach was developed by identifying dose-response relationships in key research concepts, including the low dose theory of estrogen-like compounds, hormesis, NOAEL dose, compensatory response and/or food tolerance, and effects of exposure to undesirable substances. The above considerations increased the researchers' interest in risk evaluation, namely: (i) clinical symptoms associated with long-term, daily exposure to low doses of a toxic compound; and (ii) dysfunctions at cellular or tissue level that do not produce clinical symptoms. Research advancements facilitate the extrapolation of results and promote the use of novel tools for evaluating the risk of exposure, for example exposure to zearalenone in pre-pubertal female dogs. The arguments presented in this paper suggest that low doses of zearalenone in commercial feeds stimulate metabolic processes and increase weight gains. Those processes are accompanied by lower proliferation rates in the ovaries, neoangiogenesis and vasodilation in the ovaries and the uterus, changes in the steroid hormone profile, and changes in the activity of hydroxysteroid dehydrogenases. All of the above changes result from exogenous hyperestrogenizm.

Evaluation of selected serum biochemical and haematological parameters in gilts exposed per os to 100 ppb of zearalenone.

Zearalenone (ZEN) widely contaminates animal feed of plant origin. The recommended safe concentrations of ZEN in feeds for various animal species are set mainly based on the mycotoxin's hormonal properties (NOEL). Our growing knowledge about biologically active concentrations of ZEN, molecular mechanisms and cells/tissues targeted by ZEN indicates that the harmful effects exerted by this mycotoxin on animals may be far greater than previously believed. This experiment was performed on pre-pubertal gilts divided into a control group (n=9) and an experimental group (ZEN, n=9). The control group received placebo, whereas the experimental group was administered ZEN at a dose of 0.1 mg/kg feed (equivalent to 5 µg/kg BW/day) for 42 days. On days 14, 28 and 42 blood samples were collected from the animals to determine the concentrations of selected zearalenols, serum biochemical and haematological parameters. Conjugated ZEN was found in the blood serum of the experimental gilts. Changes in the analysed biochemical parameters included a transient increase in albumin and cholesterol levels. A statistically significant increase in the concentrations of neutrophilic and acidophilic granulocytes was observed in the white blood cell system. The results indicate that long-term per os exposure of pre-pubertal gilts to low doses of ZEN (below NOEL) has a modulatory effect on liver function and white blood cells.

Cecal enzyme activity in gilts following experimentally induced Fusarium mycotoxicosis.

The objective of the presented study was to examine the influence of Fusarium mycotoxins (zearalenone--ZEN and deoxynivalenol--DON), administered separately and in combination, on the activity of cecal enzymes (ß-glucosidase and ß-glucuronidase) in gilts which were fed fodder con- taminated with these mycotoxins. The activity of ß-glucosidase and ß-glucuronidase varied in the range of 0.170-1.236 µmol · h(-1) · mg(-1) and 8.701-96.704 µmol · h(-1) · mg(-1), respectively. In the first two weeks, the toxins had no significant effect on the activity of ß-glucosidase and ß-glucuronidase in the ascending and descending colon. After week 3 and later on, ZEN and DON administered as a mix- ture led to the highest increase in the activity of both enzymes. Administered separately, DON affected the activity of enzymes more than ZEN. From the third week of the experiment, an increase in the activity of CW ß-glucosidase and ß-glucuronidase was observed.

5Z-7-Oxozaenol attenuates cuprizone-induced demyelination in mice through microglia polarization regulation.

INTRODUCTION: Demyelination is a key factor in axonal degeneration and neural loss, leading to disability in multiple sclerosis (MS) patients. Transforming growth factor beta activated kinase 1 (TAK1) is a critical molecule involved in immune and inflammatory signaling pathways. Knockout of microglia TAK1 can inhibit autoimmune inflammation of the brain and spinal cord and improve the outcome of MS. However, it is unclear whether inhibiting TAK1 can alleviate demyelination. METHODS: Eight-week-old male c57bl/6j mice were randomly divided into five groups: (a) the control group, (b) the group treated with cuprizone (CPZ) only, (c) the group treated with 5Z-7-Oxozaenol (OZ) only, and (d) the group treated with both cuprizone and 15 µg/30 µg OZ. Demyelination in the mice of this study was induced by administration of CPZ (ig) at a daily dose of 400 mg/kg for consecutive 5 weeks. OZ was intraperitoneally administered at mentioned doses twice a week, starting from week 3 after beginning cuprizone treatment. Histology, rotarod test, grasping test, pole test, Western blot, RT-PCR, and ELISA were used to evaluate corpus callosum demyelination, behavioral impairment, oligodendrocyte differentiation, TAK1 signaling pathway expression, microglia, and related cytokines. RESULTS: Our results demonstrated that OZ protected against myelin loss and behavior impairment caused by CPZ. Additionally, OZ rescued the loss of oligodendrocytes in CPZ-induced mice. OZ inhibited the activation of JNK, p65, and p38 pathways, transformed M1 polarized microglia into M2 phenotype, and increased brain-derived neurotrophic factor (BDNF) expression to attenuate demyelination in CPZ-treated mice. Furthermore, OZ reduced the expression of proinflammatory cytokines and increases anti-inflammatory cytokines in CPZ-treated mice. CONCLUSION: These findings suggest that inhibiting TAK1 may be an effective approach for treating demyelinating diseases.

Oxomollugin, an oxidized substance in mollugin, inhibited LPS-induced NF-κB activation via the suppressive effects on essential activation factors of TLR4 signaling.

Oxomollugin is a degraded product of mollugin and was found to be an active compound that inhibits LPS-induced NF-κB activation. In this study, we investigated the inhibitory activity of oxomollugin, focusing on TLR4 signaling pathway, resulting in NF-κB activation. Oxomollugin inhibited the LPS-induced association of essential factors for initial activation of TLR4 signaling, MyD88, IRAK4 and TRAF6. Furthermore, oxomollugin showed suppressive effects on LPS-induced modification of IRAK1, IRAK2 and TRAF6, LPS-induced association of TRAF6-TAK1/TAB2, and followed by IKKα/ß phosphorylation, which critical in signal transduction leading to LPS-induced NF-κB activation. The consistent results suggested that oxomollugin inhibits LPS-induced NF-κB activation via the suppression against signal transduction in TLR4 signaling pathway.The activities of oxomollugin reported in this study provides a deeper understanding on biological activity of mollugin derivatives as anti-inflammatory compounds.

Scutellarin inhibits inflammatory PANoptosis by diminishing mitochondrial ROS generation and blocking PANoptosome formation.

PANoptosis is manifested with simultaneous activation of biomarkers for both pyroptotic, apoptotic and necroptotic signaling via the molecular platform PANoptosome and it is involved in pathologies of various inflammatory diseases including hemophagocytic lymphohistiocytosis (HLH). Scutellarin is a flavonoid isolated from herbal Erigeron breviscapus (Vant.) Hand.-Mazz. and has been shown to possess multiple pharmacological effects, but it is unknown whether scutellarin has any effects on PANoptosis and related inflammatory diseases. In this study, we found that scutellarin inhibited cell death in bone marrow-derived macrophages (BMDMs) and J774A.1 cells treated with TGF-ß-activated kinase 1 (TAK1) inhibitor 5Z-7-oxozeaenol (OXO) plus lipopolysaccharide (LPS), which has been commonly used to induce PANoptosis. Western blotting showed that scutellarin dose-dependently inhibited the activation biomarkers for pyroptotic (Caspase-1p10 and GSDMD-NT), apoptotic (cleaved Casp3/8/9 and GSDME-NT), and necroptotic (phosphorylated MLKL) signaling. The inhibitory effect of scutellarin was unaffected by NLRP3 or Caspase-1 deletion. Interestingly, scutellarin blocked the assembly of PANoptosome that encompasses ASC, RIPK3, Caspase-8 and ZBP1, suggesting its action on upstream signaling. Consistent with this, scutellarin inhibited mitochondrial damage and mitochondrial reactive oxygen species (mtROS) generation in cells treated with OXO+LPS. Further, mito-TEMPO that can scavenge mtROS significantly inhibited OXO+LPS-induced PANoptotic cell death. In line with the in vitro results, scutellarin markedly alleviated systemic inflammation, multiple organ injury, and activation of PANoptotic biomarkers in mice with HLH. Collectively, our data suggest that scutellarin can inhibit PANoptosis by suppressing mitochondrial damage and mtROS generation and thereby mitigating multiple organ injury in mice with inflammatory disorders.

TAK1 inhibition mitigates intracerebral hemorrhage-induced brain injury through reduction of oxidative stress and neuronal pyroptosis via the NRF2 signaling pathway.

Introduction: Intracerebral hemorrhage (ICH) often triggers oxidative stress through reactive oxygen species (ROS). Transforming growth factor-ß-activated kinase 1 (TAK1) plays a pivotal role in regulating oxidative stress and inflammation across various diseases. 5Z-7-Oxozeaenol (OZ), a specific inhibitor of TAK1, has exhibited therapeutic effects in various conditions. However, the impact of OZ following ICH and its underlying molecular mechanisms remain elusive. This study aimed to explore the possible role of OZ in ICH and its underlying mechanisms by inhibiting oxidative stress-mediated pyroptosis. Methods: Adult male Sprague-Dawley rats were subjected to an ICH model, followed by treatment with OZ. Neurobehavioral function, blood-brain barrier integrity, neuronal pyroptosis, and oxidative stress markers were assessed using various techniques including behavioral tests, immunofluorescence staining, western blotting, transmission electron microscopy, and biochemical assays. Results: Our study revealed that OZ administration significantly inhibited phosphorylated TAK1 expression post-ICH. Furthermore, TAK1 blockade by OZ attenuated blood-brain barrier (BBB) disruption, neuroinflammation, and oxidative damage while enhancing neurobehavioral function. Mechanistically, OZ administration markedly reduced ROS production and oxidative stress by facilitating nuclear factor-erythroid 2-related factor 2 (NRF2) nuclear translocation. This was accompanied by a subsequent suppression of the NOD-like receptor protein 3 (NLRP3) activation-mediated inflammatory cascade and neuronal pyroptosis. Discussion: Our findings highlight that OZ alleviates brain injury and oxidative stress-mediated pyroptosis via the NRF2 pathway. Inhibition of TAK1 emerges as a promising approach for managing ICH.

Disruption of TIGAR-TAK1 alleviates immunopathology in a murine model of sepsis.

Macrophage-orchestrated inflammation contributes to multiple diseases including sepsis. However, the underlying mechanisms remain to be defined clearly. Here, we show that macrophage TP53-induced glycolysis and apoptosis regulator (TIGAR) is up-regulated in murine sepsis models. When myeloid Tigar is ablated, sepsis induced by either lipopolysaccharide treatment or cecal ligation puncture in male mice is attenuated via inflammation inhibition. Mechanistic characterizations indicate that TIGAR directly binds to transforming growth factor ß-activated kinase (TAK1) and promotes tumor necrosis factor receptor-associated factor 6-mediated ubiquitination and auto-phosphorylation of TAK1, in which residues 152-161 of TIGAR constitute crucial motif independent of its phosphatase activity. Interference with the binding of TIGAR to TAK1 by 5Z-7-oxozeaenol exhibits therapeutic effects in male murine model of sepsis. These findings demonstrate a non-canonical function of macrophage TIGAR in promoting inflammation, and confer a potential therapeutic target for sepsis by disruption of TIGAR-TAK1 interaction.

The effects of experimental administration of low doses of zearalenone on the histology of ovaries in pre-pubertal bitches.

The experiment involved 30 clinically healthy Beagle bitches aged approximately 70 days with an initial body weight of approximately 8 kg. The animals were randomly divided into two experimental groups (EI and EII) and a control group of 10 animals each. Group EI was administered 50 microg of body weight zearalenone/kg per os for 42 days, group EII received 75 microg of body weight zearalenone/kg per os for 42 days, and the control group was administered placeboper os for 42 days. The bitches were ovariectomized at the end of the treatment period for anatomopathological examination. At the same time, peripheral blood samples were collected for endocrinological analyses (17beta-estradiol and progesterone). Administartion of zearalenone particularly higher doses, resulted in the hyperestrogenism degeneration and atrophy of ovarian cells and tissues with accompanying edema and blood extravasation, leading to increased 17beta-estradiol concentrations and an insignificant decrease in progesterone levels.

The effect of experimental low zearalenone intoxication on ovarian follicles in pre-pubertal bitches.

Companion animals, including bitches, may be exposed to zearalenone (ZEN) toxins that are often present in feed, and ZEN intoxication may lead to ovarian dysfunction. This study involved evaluation of the degree of ZEN-induced hypo stimulation of ovary by determination of proliferative and apoptotic indices and description of the ultra-structural organization of ovarian follicles in pre-pubertal bitches subjected to experimental, long-term exposure to low-dose ZEN mycotoxicosis. The experiment involved 30 clinically healthy, immature Beagle bitches aged approximately 70 days with initial average body weight of 8 kg, randomly divided into three groups of 10 animals each: two experimental groups (EI and EII) and a control group (C). Over a period of 42 days, ZEN was administered per os to EI animals at a dose of 50 microg/kg BW, and to EII bitches at a dose of 75 microg/kg BW. Control group animals were fed placebo containing no ZEN for 42 days. Analytical samples of the mycotoxin were administered daily in gelatin capsules before morning feeding. All the bitches were subjected to ovario-hysterectomy at the end of the experiment. Proliferation index (PCNA method) and apoptotic index (TUNEL test) values were determined by immune-histological analyses. The median for apoptotic index was determined at 13.45 for group EI, 17.84 for group EII, and 8.59 for group C. The median for proliferation index was determined at 35.25 for group EII, 42.44 for group EI, and 70.60 for group C. The results of ultra-structural examinations of the ovaries revealed that experimental, ZEN-induced hyperestrogenism enhanced apoptosis and lowered the proliferative ability of follicular cells what contributed to organelle destruction in pre-pubertal bitches. The changes observed were particularly advanced in EII animals, which were administered a higher dose of ZEN.