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Zearalenone (ZEN), a non-steroidal Fusarium graminearum with an estrogen effect, can cause damage to the gastrointestinal tract, immune organs, liver, and reproductive system. Further analysis of the mechanism of ZEN has become an important scientific issue. We have established in vivo and in vitro models of ZEN intervention, used AMPK/mTOR as a targeted pathway for ZEN reproductive toxicity, and explored the molecular mechanism by which ZEN may induce uterine hypertrophy in weaned piglets. Our study strongly suggested that ZEN can activate the phosphorylation of AMPK in uterine endometrial epithelium cells, affect the phosphorylation level of mTOR through TSC2 and Rheb, induce autophagy, upregulate the expression of proliferative genes PCNA and BCL2, downregulate the expression of apoptotic gene BAX, promote uterine endometrial epithelium cells proliferation, and ultimately lead to thickening of the endometrial and myometrium, increased density of uterine glands, and induce uterine hypertrophy.

This study aims to examine the impact of zearalenone (ZEA) on glucose nutrient absorption and the role of the Kelch-like erythroid cell-derived protein with CNC homology-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in zearalenone-induced oxidative stress of porcine jejunal epithelial cells (IPEC-J2). For 24 and 36 h, the IPEC-J2 cells were exposed to ZEA at concentrations of 0, 10, 20, and 40 (Control, ZEA10, ZEA20, ZEA40) mol/L. With the increase of ZEA concentration and prolongation of the action time, the apoptosis rate and malondialdehyde level and relative expression of sodium-dependent glucose co-transporter 1 (Sglt1), glucose transporter 2 (Glut2), Nrf2, quinone oxidoreductase 1 (Nqo1), and hemeoxygenase 1 (Ho1) at mRNA and protein level, fluorescence intensity of Nrf2 and reactive oxygen species increased significantly ( p < 0.05), total superoxide dismutase and glutathione peroxidase activities and relative expression of Keap1 at mRNA and protein level, fluorescence intensity of Sglt1 around the cytoplasm and the cell membrane of IPEC-J2 reduced significantly ( p < 0.05). In conclusion, ZEA can impact glucose absorption by affecting the expression of Sglt1 and Glut2, and ZEA can activate the Keap1-Nrf2 signaling pathway by enhancing Nrf2, Nqo1, and Ho1 expression of IPEC-J2.

In this study, we aimed to assess the effect of diet ZEA on serum hormones, the location and expression of estrogen receptor ERα/β and progesterone receptor (PR) of the uterus in weaned piglets and to reveal the mechanism underneath. A total of 40 healthy weaned gilts were randomly allocated to basal diet supplemented with 0 (Control), 0.5 (ZEA0.5), 1.0 (ZEA1.0) and 1.5 (ZEA1.5) mg ZEA/kg and fed individually for 35 days. Meanwhile, the porcine endometrial epithelial cells (PECs) were incubated for 24 h with ZEA at 0 (Control), 5 (ZEA5), 20 (ZEA20) and 80 (ZEA80) μmol/L, respectively. The results showed that nutrient apparent digestibility (CP and GE), nutrient apparent availability (ME/GE, BV and NPU), the uterine immunoreactive integrated optic density (IOD), relative mRNA and protein expression of ER-α, ER-β and PR and the relative mRNA and protein expression of ER-α and ER-β in PECs all increased linearly ( p < 0.05) with ZEA. Collectively, ZEA can interfere with the secretion of some reproductive hormones in the serum and promote the expression of estrogen/progesterone receptors in the uterus and PECs. All these indicate that ZEA may promote the development of the uterus in weaned gilts through estrogen receptor pathway.

Zearalenone (ZEN), also known as the F-2 toxin, is a common contaminant in cereal crops and livestock products. This experiment aimed to reveal the changes in the proteomics of ZEN-induced intestinal damage in weaned piglets by tandem mass spectrometry tags. Sixteen weaned piglets either received a basal diet or a basal diet supplemented with 3.0 mg/kg ZEN in a 32 d study. The results showed that the serum levels of ZEN, α-zearalenol, and β-zearalenol were increased in weaned piglets exposed to ZEN (p < 0.05). Zearalenone exposure reduced apparent nutrient digestibility, increased intestinal permeability, and caused intestinal damage in weaned piglets. Meanwhile, a total of 174 differential proteins (DEPs) were identified between control and ZEN groups, with 60 up-regulated DEPs and 114 down-regulated DEPs (FC > 1.20 or <0.83, p < 0.05). Gene ontology analysis revealed that DEPs were mainly involved in substance transport and metabolism, gene expression, inflammatory, and oxidative stress. The Kyoto Encyclopedia of Genes and Genomes analysis revealed that DEPs were significantly enriched in 25 signaling pathways (p < 0.05), most of which were related to inflammation and amino acid metabolism. Our study provides valuable clues to elucidate the possible mechanism of ZEN-induced intestinal injury.

The aim of this study was to explore the effect of zearalenone (ZEA) exposure on uterine development in weaned gilts by quantitative proteome analysis with tandem mass spectrometry tags (TMT). A total of 16 healthy weaned gilts were randomly divided into control (basal diet) and ZEA3.0 treatments groups (basal diet supplemented with 3.0 mg/kg ZEA). Results showed that vulva size and uterine development index were increased (p < 0.05), whereas serum follicle stimulation hormone, luteinizing hormone and gonadotropin-releasing hormone were decreased in gilts fed the ZEA diet (p < 0.05). ZEA, α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) were detected in the uteri of gilts fed a 3.0 mg/kg ZEA diet (p < 0.05). The relative protein expression levels of creatine kinase M-type (CKM), atriopeptidase (MME) and myeloperoxidase (MPO) were up-regulated (p < 0.05), whereas aldehyde dehydrogenase 1 family member (ALDH1A2), secretogranin-1 (CHGB) and SURP and G-patch domain containing 1 (SUGP1) were down-regulated (p < 0.05) in the ZEA3.0 group by western blot, which indicated that the proteomics data were dependable. In addition, the functions of differentially expressed proteins (DEPs) mainly involved the cellular process, biological regulation and metabolic process in the biological process category. Some important signaling pathways were changed in the ZEA3.0 group, such as extracellular matrix (ECM)-receptor interaction, focal adhesion and the phosphoinositide 3-kinase−protein kinase B (PI3K-AKT) signaling pathway (p < 0.01). This study sheds new light on the molecular mechanism of ZEA in the uterine development of gilts.

This study aimed to investigate the effect of zearalenone (ZEA) exposure on follicular development in weaned gilts, and its mechanism based on the silent information regulator 1 (SIRT1)/peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α) signaling pathway. A total of 32 healthy female weaned piglets (Landrace × Yorkshire × Duroc) with an average body weight of 12.39 ± 0.24 kg were randomly allotted to a basal diet supplemented with 0, 0.15, 1.5, or 3.0 mg/kg ZEA for a 32-d feeding test. Blood and ovarian samples were obtained at the end of the experiment to determine serum toxin concentrations, ovarian histology, and the expressions of proliferating cell nuclear antigen (PCNA) and SIRT1/PGC-1α signaling pathway-related genes. Results showed that the vulva area, serum concentrations of ZEA, α-zearalenol and β-zearalenol, the thickness of the growing follicular layer, and the diameter of the largest growing follicles, as well as the expressions of SIRT1, PGC-1α, estrogen-related receptor α (ERRα), ATP synthase subunit beta (ATP5B), and PCNA, increased linearly (P < 0.05) with increasing dietary ZEA, whereas the thickness of the primordial follicle layer decreased linearly (P < 0.05). Immunohistochemical analysis showed that the immunoreactive substances of SIRT1 and PGC-1α in the ovaries enhanced with the increasing dietary ZEA (P < 0.05). In addition, the thickness of the growing follicular layer and the diameter of the largest growing follicle were positively correlated with relative mRNA and protein expressions of SIRT1, PGC-1α, ERRα, ATP5B, and PCNA (P < 0.05). However, the thickness of the primordial follicle layer was negatively correlated with the mRNA and protein expression of SIRT1, PGC-1α, ERRα, ATP5B, and PCNA (P < 0.05). Interestingly, the 1.5 mg/kg ZEA treatment had highly hyperplastic follicles, whereas 3.0 mg/kg ZEA resulted in a large number of follicular atresia, which indicated that low-dose ZEA exposure accelerated follicular proliferation, while high-dose ZEA promoted follicular atresia, although the critical value interval needs further confirmation. Results provide a theoretical basis for finding the therapeutic target of ZEA-induced reproductive disorders in weaned gilts., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. 
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This study aims to investigate the effects of exposure to different dosages of zearalenone (ZEA) on cecal physical barrier functions and its mechanisms based on the TGF-β1/Smads signaling pathway in weaned piglets. Thirty-two weaned piglets were allotted to four groups and fed a basal diet supplemented with ZEA at 0, 0.15, 1.5, and 3.0 mg/kg, respectively. The results showed that 1.5 and 3.0 mg/kg ZEA damaged cecum morphology and microvilli, and changed distribution and shape of M cells. Moreover, 1.5 and 3.0 mg/kg ZEA decreased numbers of goblet cells, the expressions of TFF3 and tight junction proteins, and inhibited the TGF-β1/Smads signaling pathway. Interestingly, the 0.15 mg/kg ZEA had no significant effect on cecal physical barrier functions but decreased the expressions of Smad3, p-Smad3 and Smad7. Our study suggests that high-dose ZEA exposure impairs cecal physical barrier functions through inhibiting the TGF-β1/Smads signaling pathway, but low-dose ZEA had no significant effect on cecum morphology and integrity through inhibiting the expression of smad7. These findings provide a scientific basis for helping people explore how to reduce the toxicity of ZEA in feeds.

This study explored and investigated how zearalenone (ZEA) affects the morphology of small intestine and the distribution and expression of ghrelin and proliferating cell nuclear antigen (PCNA) in the small intestine of weaned gilts. A total of 20 weaned gilts (42-day-old, D × L × Y, weighing 12.84 ± 0.26 kg) were divided into the control and ZEA groups (ZEA at 1.04 mg/kg in diet) in a 35-d study. Histological observations of the small intestines revealed that villus injuries of the duodenum, jejunum and ileum, such as atrophy, retardation and branching dysfunction, were observed in the ZEA treatment. The villi branch of the ileum in the ZEA group was obviously decreased compared to that of the ileum, jejunum and duodenum, and the number of lymphoid nodules of the ileum was increased. Additionally, the effect of ZEA (1.04 mg/kg) was decreased by the immunoreactivity and distribution of ghrelin and PCNA in the duodenal and jejunal mucosal epithelial cells. Interestingly, ZEA increased the immunoreactivity of ghrelin in the ileal mucosal epithelial cells and decreased the immunoreactivity expression of PCNA in the gland epithelium of the small intestine. In conclusion, ZEA (1.04 mg/kg) had adverse effects on the development and the absorptive capacity of the villi of the intestines; yet, the small intestine could resist or ameliorate the adverse effects of ZEA by changing the autocrine of ghrelin in intestinal epithelial cells.

This study aims to investigate the effects of zearalenone (ZEA) on the localizations and expressions of follicle stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), gonadotropin releasing hormone (GnRH) and gonadotropin releasing hormone receptor (GnRHR) in the ovaries of weaned gilts. Twenty 42-day-old weaned gilts were randomly allocated into two groups, and treated with a control diet and a ZEA-contaminated diet (ZEA 1.04 mg/kg), respectively. After 7-day adjustment, gilts were fed individually for 35 days and euthanized for blood and ovarian samples collection before morning feeding on the 36th day. Serum hormones of E 2 , PRG, FSH, LH and GnRH were determined using radioimmunoassay kits. The ovaries were collected for relative mRNA and protein expression, and immunohistochemical analysis of FSHR, LHR, GnRH and GnRHR. The results revealed that ZEA exposure significantly increased the final vulva area ( p < 0.05), significantly elevated the serum concentrations of estradiol, follicle stimulating hormone and GnRH ( p < 0.05), and markedly up-regulated the mRNA and protein expressions of FSHR, LHR, GnRH and GnRHR ( p < 0.05). Besides, the results of immunohistochemistry showed that the immunoreactive substances of ovarian FSHR, LHR, GnRH and GnRHR in the gilts fed the ZEA-contaminated diet were stronger than the gilts fed the control diet. Our findings indicated that dietary ZEA (1.04 mg/kg) could cause follicular proliferation by interfering with the localization and expression of FSHR, LHR, GnRH and GnRHR, and then affect the follicular development of weaned gilts.

Zearalenone (ZEA) contamination of feed affects animal husbandry and the human health. Currently, the molecular mechanism underlying small intestine-related diseases caused by ZEA-induced oxidative stress is not well understood. In this study, we aimed to identify the mechanisms involved in ZEA (0.5-1.5 mg/kg)-induced oxidative stress in the ileum and mesenteric lymph nodes (MLNs) and the role of the Kelch-like erythroid cell-derived protein with CNC homology-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in post-weaning gilts. Forty post-weaning gilts (Landrace × Yorkshire × Duroc) with an average body weight of 14.01 ± 0.86 kg were randomly allocated to four groups and fed a corn-soybean meal basal diet supplemented with < 0.1, 0.5, 1.0, or 1.5 mg/kg ZEA. The results showed that the activity of total superoxide dismutase and glutathione peroxidase decreased (p < 0.05) linearly and quadratically and that the content of malondialdehyde increased (p < 0.05) quadratically in the ileum and MLNs with increasing ZEA in the diet. Immunohistochemical analysis showed that the expression of Nrf2 and glutathione peroxidase 1 (Gpx1) immunoreactive proteins in the ileum and MLNs were significantly enhanced with increasing ZEA. The relative mRNA and protein expression of Nrf2, Gpx1, quinone oxidoreductase 1 (Nqo1), hemeoxygenase 1 (Ho1), modifier subunit of glutamate-cysteine ligase (Gclm), and catalytic subunit of glutamate-cysteine ligase (Gclc) increased (p < 0.05) linearly and quadratically, and the relative mRNA and protein expression of Keap1 decreased (p < 0.05) linearly and quadratically in the ileum with increasing ZEA concentrations in the diet. Further, the relative mRNA and protein expression of Nrf2 and Gpx1 increased (p < 0.05) linearly and quadratically, and the relative mRNA and protein expression of Nqo1, Ho1, and Gclm decreased (p < 0.05) quadratically in the MLNs as ZEA concentrations increased in the diet. Our results provide valuable genetic information on ZEA-induced oxidative stress in the ileum and MLNs of post-weaning gilts and have elucidated the key regulatory genes involved in the Keap1-Nrf2 signaling pathway. Results indicated that the Keap1-Nrf2 signaling pathway might be a key target to further prevent and treat ZEA-induced injury to the ileum in post-weaning gilts., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Researches have shown that dietary zearalenone (ZEA) caused oxidative stress in the liver and reproductive organs of postweaning gilts. However, information on the effects of ZEA on oxidative stress of the small intestine in the piglets is limited. The objective of this study was to determine the effects of ZEA exposure on oxidative stress, the Kelch-like erythroid cell-derived protein with CNC homology (ECH)-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and on immunohistochemistry of the jejunum in postweaning gilts. A 35-d feeding experiment using 40 postweaning gilts (Landrace × Yorkshire × Duroc) with an average BW of 14.01 ± 0.86 kg in 4 groups fed corn-soybean meal-based diets containing 0, 0.5, 1.0, and 1.5 mg ZEA/kg was conducted. The jejunum was obtained at the end of the experiment and used for analyses. The results showed that the activities of total superoxide dismutase and glutathione peroxidase and the relative expressions of Keap1 mRNA and protein in the jejunum linearly and quadratically decreased (P < 0.05) with increasing concentrations of ZEA in the diets. The malondialdehyde content, the integrated optical density of Nrf2 and glutathione peroxidase 1 (GPX1), and the relative expressions of Nrf2, GPX1, quinone oxidoreductase 1 (NQO1), and modifier subunit of glutamate-cysteine ligase (GCLM) mRNA and proteins linearly and quadratically increased (P < 0.05) with increasing levels of ZEA. Immunohistochemical analysis showed that Nrf2 and GPX1 immunoreactivity was enhanced by the ZEA treatments, and block localization of yellow and brown immunoreactive substances in the jejunum was observed with increasing levels of ZEA. The results suggest that ingested ZEA induced oxidative stress in the jejunum in postweaning gilts through upregulation of the Keap1-Nrf2 signaling pathway and downstream target genes NQO1, HO1, and GCLM, indicating the important role of the Keap1-Nrf2 signaling pathway in oxidative stress induced by ZEA in the jejunum of the postweaning piglets., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Zearalenone (ZEA) is an estrogenic toxin produced by Fusarium species, which is widely distributed and posed a great health risk to both humans and farm animals. Reproductive disorders associated with ZEA such as premature puberty, infertility and abortion have plagued the animal husbandry, but the molecular mechanism is unclear. Because transforming growth factor-β1 (TGF-β1) signaling pathway is involved in the proliferation and apoptosis of cells, proliferating cell nuclear antigen (PCNA), B-cell lymphoma/leukemia-2 (BCL-2) and BCL-2 associated X protein (BAX) that all play indispensable roles in the normal development of the uterus, it is hypothesized that ZEA induces reproductive disorders is closely related to the expression of these genes. The objective of this study was to assess the effects of dietary ZEA at the concentrations of 0.5 to 1.5 mg/kg on the mRNA and protein expression of these genes in the uteri of post-weaning gilts and to explore the possible molecular mechanism. Forty healthy post-weaning female piglets (Duroc × Landrace × Large White) aged 38 d were randomly allocated to basal diet supplemented with 0 (Control), 0.5 (ZEA0.5), 1.0 (ZEA1.0), or 1.5 (ZEA1.5) mg/kg purified ZEA, and fed for 35 d. Piglets were euthanized at the end of the experiment and samples were taken and subjected to immunohistochemistry, qRT-PCR and Western blot analyses. The relative mRNA expressions of PCNA, BCL-2 and Smad3 in the uteri of post-weaning gilts increased linearly ( p < 0.05) and quadratically ( p < 0.05) as ZEA concentration increased in the diet. The relative protein expressions of PCNA, BAX, BCL-2, TGF-β1, Smad3, and phosphorylated Smad3 (p-Smad3) in the uteri of post-weaning gilts increased linearly ( p < 0.05) and quadratically ( p < 0.001) with an increasing level of ZEA. The results showed that uterine cells in the ZEA (0.5-1.5 mg/kg) treatments were in a high proliferation state, indicating that ZEA could accelerate the proliferation of uteri and promote the development of the uteri. At the same time, the results suggested that ZEA activates the TGF-β1/Smad3 signaling pathway, suggesting it plays an important role in accelerating the development of the uterus., Competing Interests: We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

Numerous reports have provided evidence that zearalenone (ZEN) can increase the weight of genital organs. These findings have been confirmed by many studies in which the ghrelin gene was expressed in the ovary and was implicated in the control of cells in reproductive tissues. The proliferating cell nuclear antigen (PCNA) is an important marker of cell proliferation. The present study investigates the effects of a ZEN-treated diet on the development of ovaries in post-weaning piglets by the detection of ghrelin and PCNA protein and relative abundance of mRNA using optical microscopy, immunohistochemistry and quantitative real-time (qRT-PCR). A total of 20 piglets (Duroc × Landrace × Yorkshire) weaned at 28 d, with an average body weight of 8.74 ± 0.26 kg (P=0.919) were used in this study. Piglets in the control group (n=10) were fed a normal basal diet, and those in the treatment group (n=10) were fed a diet containing ZEN (1.04 mg/kg), for 35 d. The proportion of growing follicles and diameter of the largest growing follicle in ovaries were greater in piglets fed the diet with ZEN. The total integrated optical densities of protein and mRNA of ghrelin and PCNA were greater with the feeding of the ZEN-treatment diet. The results suggested that 1.04 mg/kg ZEN could promote the autocrine action or expression of the ghrelin gene in piglet ovary, and further accelerate the development of ovaries (follicles)., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Simple Summary: This study was conducted to assess the effects of Zearalenone (ZEA) exposure on the growth performance, small intestine disaccharidase, and antioxidant activities of weaned gilts. Twenty weaned gilts were randomly divided into control and ZEA treatment (1.04 mg/kg) groups. The data showed that 1.04 mg/kg ZEA in gilt's diet could reduce the activity of disaccharidase enzymes and induce oxidative stress in the small intestine. Therefore, ZEA may induce intestinal injury by oxidative stress, or induce oxidative stress through intestinal injury, thus reducing the effect of animals on nutrient absorption. Zearalenone (ZEA) is a secondary metabolite with estrogenic effects produced by Fusarium fungi and mainly occurs as a contaminant of grains such as corn and wheat. ZEA, to which weaned gilts are extremely sensitive, is the main Fusarium toxin detected in corn–soybean meal diets. Our aim was to examine the effects of ZEA on the growth performance, intestinal disaccharidase activity, and anti-stress capacity of weaned gilts. Twenty 42-day-old healthy Duroc × Landrace × Large White weaned gilts (12.84 ± 0.26 kg) were randomly divided into control and treatment (diet containing 1.04 mg/kg ZEA) groups. The experiment included a 7-day pre-trial period followed by a 35-day test period, all gilts were euthanized and small intestinal samples were collected and subjected to immunohistochemical and western blot analyses. The results revealed that inclusion of 1.04 mg/kg ZEA in the diet significantly reduced the activities of lactase, sucrase, and maltase in the duodenum, jejunum, and ileum of gilts. Similarly, the activities of superoxide dismutase and glutathione peroxidase in the duodenum, jejunum, and ileum, and activities of catalase in the jejunum and ileum were reduced (p < 0.05). Conversely, the content of malondialdehyde in the duodenum, jejunum, and ileum, and the integrated optical density (IOD), IOD in single villi, and the mRNA and protein expression of heat shock protein 70 (Hsp70) were significantly increased (p < 0.05). The results of immunohistochemical analyses revealed that the positive reaction of Hsp70 in the duodenum, jejunum, and ileum of weaned gilts was enhanced in the ZEA treatment, compared with the control. The findings of this study indicate the inclusion of ZEA (1.04 mg/kg) in the diet of gilts reduced the activity of disaccharidase enzymes and induced oxidative stress in the small intestine, thereby indicating that ZEA would have the effect of reducing nutrient absorption in these animals. [ABSTRACT FROM AUTHOR]