Co-occurrence of Mycotoxin-Induced Hepatotoxicity in Mice Inhibited by Lycopene: Mitochondrial Impairment and Early Hepatic Fibrosis.

SCOPE: Mycotoxins co-contamination of agricultural products poses a serious threat to human and animal health, especially hepatic dysfunction. Zearalenone (ZEN), deoxynivalenol (DON), and aflatoxin B1 (AFB1) are three commonly co-occurring mycotoxins. This study is to determine whether lycopene (LYC) can alleviate hepatic toxicity induced by the co-occurrence of ZEN, DON, and AFB1 in mice. METHODS AND RESULTS: Eighty 6-week-old male ICR mice are divided into four groups: CON group (solvent control), LYC group (10 mg kg-1 LYC), Co-M group (10 mg kg-1 ZEN + 1 mg kg-1 DON + 0.5 mg kg-1 AFB1), and LYC+Co-M group (10 mg kg-1 LYC + 10 mg kg-1 ZEN + 1 mg kg-1 DON + 0.5 mg kg-1 AFB1). The results show that LYC can suppress the co-occurrence of mycotoxin-induced mitochondrial swelling and vacuolization accompanied by dysregulation of indices of mitochondrial dynamics (Mitofusin 1 (Mfn1), Mfn2, Optic atrophy 1 (Opa1), Dynamin-related protein 1 (Drp1), Fission 1 (Fis1) at the mRNA level; DRP1 and FIS1 at the protein level). LYC effectively inhibits co-occurrence of mycotoxin-induced activation of Cytochrome P450 2E1, and early fibrosis, as determined by staining with Masson's trichrome and α-SMA protein. CONCLUSION: LYC successfully attenuates early hepatic fibrosis mainly through antioxidant activities and prevented mitochondrial injury.

Lycopene alleviates multiple-mycotoxin-induced toxicity by inhibiting mitochondrial damage and ferroptosis in the mouse jejunum.

Multiple mycotoxins contamination in foods and feeds threatens human and animal health after they accumulate in the food chain, producing various toxic effects. The common mycotoxins contaimination in feeds are zearalenone (ZEN), deoxynivalenol (DON), and aflatoxin B1 (AFB1), but the effects of their co-exposure on the jejunum are not well understood. Lycopene (LYC) has been reported to have antioxidant activity that alleviates jejunal damage. In this study, we investigated the possible role of LYC as a treatment to mitigate the combined effects of ZEN, DON, and AFB1 on the jejunum of mice. Eighty male specific-pathogen-free ICR mice were randomly allocated to treatments with LYC (10 mg kg-1) and/or ZEN + DON + AFB1 (10 mg kg-1 ZEN, 1 mg kg-1 DON, and 0.5 mg kg-1 AFB1). The results indicated that LYC alleviated ZEN + DON + AFB1-induced jejunal injury by ameliorating the jejunal structural injury and increasing the villus height/crypt depth ratio and the levels of tight junction proteins (zonula occludens 1 [ZO1], occludin1 and claudin1) in the mouse jejunum. LYC also inhibited the oxidative stress induced by co-exposure to ZEN, DON, and AFB1 via reducing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and enhancing the total antioxidant capacity (T-AOC). LYC also alleviated jejunal mitochondrial damage in the ZEN + DON + AFB1-affected mice, evident as an increase in mitochondrial fission 1 (Fis1) transcription and a reduction in mitochondrial mitofusin 1 (Mfn1) and Mfn2 transcription. Co-exposure to ZEN, DON, and AFB1 also significantly increased the transcription of ferroptosis-related genes (transferrin receptor 1 (Tfr1), ferritin heavy chain 1 [Fth1], solute carrier family 3 member 2 [Slc3a2], and glutathione peroxidase 4 [Gpx4]), TFR1 and Fe2+ concentration. Notably, LYC potentially alleviated ZEN + DON + AFB1-induced jejunal ferroptosis. These results demonstrate that LYC alleviates ZEN + DON + AFB1-induced jejunal toxicity by inhibiting oxidative stress-mediated ferroptosis and mitochondrial damage in mice.

Glutamate attenuates lipopolysaccharide induced intestinal barrier injury by regulating corticotropin-releasing factor pathway in weaned pigs.

OBJECTIVE: The purpose of this study was to evaluate the protection of glutamate (GLU) against the impairment in intestinal barrier function induced by lipopolysaccharide (LPS) stress in weaned pigs. METHODS: Twenty-four weaned pigs were divided into four treatments containing: i) non-challenged control, ii) LPS-challenged control, iii) LPS+1.0% GLU, and iv) LPS+2.0% GLU. On day 28, pigs were treated with LPS or saline. Blood samples were collected at 0, 2, and 4 h post-injection. After blood samples collection at 4 h, all pigs were slaughtered, and spleen, mesenteric lymph nodes, liver and intestinal samples were obtained. RESULTS: Dietary GLU supplementation inhibited the LPS-induced oxidative stress in pigs, as demonstrated by reduced malondialdehyde level and increased glutathione level in jejunum. Diets supplemented with GLU enhanced villus height, villus height/crypt depth and claudin-1 expression, attenuated intestinal histology and ultrastructure impairment induced by LPS. Moreover, GLU supplementation reversed intestinal intraepithelial lymphocyte number decrease and mast cell number increase induced by LPS stress. GLU reduced serum cortisol concentration at 4 h after LPS stress and downregulated the mRNA expression of intestinal corticotropin-releasing factor signal (corticotrophin-releasing factor [CRF], CRF receptor 1 [CRFR1], glucocorticoid receptor, tryptase, nerve growth factor, tyrosine kinase receptor A), and prevented mast cell activation. GLU upregulated the mRNA expression of intestinal transforming growth factor ß. CONCLUSION: These findings indicate that GLU attenuates LPS-induced intestinal mucosal barrier injury, which is associated with modulating CRF signaling pathway.

EPA and DHA confer protection against deoxynivalenol-induced endoplasmic reticulum stress and iron imbalance in IPEC-1 cells.

This study assessed the molecular mechanism of EPA or DHA protection against intestinal porcine epithelial cell line 1 (IPEC-1) cell damage induced by deoxynivalenol (DON). The cells were divided into six groups, including the CON group, the EPA group, the DHA group, the DON group, the EPA + DON group and the DHA + DON group. RNA sequencing was used to investigate the potential mechanism, and qRT-PCR was employed to verify the expression of selected genes. Changes in ultrastructure were used to estimate pathological changes and endoplasmic reticulum (ER) injury in IPEC-1 cells. Transferrin receptor 1 (TFR1) was tested by ELISA. Fe2+ and malondialdehyde (MDA) contents were estimated by spectrophotometry, and reactive oxygen species (ROS) was assayed by fluorospectrophotometry. RNA sequencing analysis showed that EPA and DHA had a significant effect on the expression of genes involved in ER stress and iron balance during DON-induced cell injury. The results showed that DON increased ER damage, the content of MDA and ROS, the ratio of X-box binding protein 1s (XBP-1s)/X-box binding protein 1u (XBP-1u), the concentration of Fe2+ and the activity of TFR1. However, the results also showed that EPA and DHA decreased the ratio of XBP-1s/XBP-1u to relieve DON-induced ER damage of IPEC-1 cells. Moreover, EPA and DHA (especially DHA) reversed the factors related to iron balance. It can be concluded that EPA and DHA reversed IPEC-1 cell damage induced by DON. DHA has the potential to protect IPEC-1 cells from DON-induced iron imbalance by inhibiting ER stress.

Xylooligosaccharide attenuates lipopolysaccharide-induced intestinal injury in piglets via suppressing inflammation and modulating cecal microbial communities.

Xylooligosaccharide (XOS) has been considered to be an effective prebiotic, but its exact mechanisms remain unknown. This research was conducted to evaluate the effects of XOS on pig intestinal bacterial community and mucosal barrier using a lipopolysaccharide (LPS)-caused gut damage model. Twenty-four weaned pigs were assigned to 4 treatments in a 2 × 2 factorial design involving diet (with or without XOS) and immunological challenge (saline or LPS). After 21 d of feeding 0% or 0.02% commercial XOS product, piglets were treated with saline or LPS. After that, blood, small intestinal mucosa and cecal digesta were obtained. Dietary XOS enhanced intestinal mucosal integrity demonstrated by higher villus height, villus height-to-crypt depth ratio, disaccharidase activities and claudin-1 protein expression and lower crypt depth. XOS also caused down-regulation of the gene expression of toll-like receptor 4 and nucleotide-binding oligomerization domain protein signaling, accompanied with decreased pro-inflammatory cytokines and cyclooxygenase 2 contents or mRNA expression and increased heat shock protein 70 mRNA and protein expression. Additionally, increased Bacteroidetes and decreased Firmicutes relative abundance were observed in the piglets fed with XOS. At the genus level, XOS enriched the relative abundance of beneficial bacteria, e.g., Faecalibacterium, Lactobacillus, and Prevotella. Moreover, XOS enhanced short chain fatty acids contents and inhibited histone deacetylases. The correlation analysis of the combined datasets implied some potential connections between the intestinal microbiota and pro-inflammatory cytokines or cecal metabolites. These results suggest that XOS inhibits inflammatory response and beneficially modifies microbes and metabolites of the hindgut to protect the intestine from inflammation-related injury.

Necroptosis is active and contributes to intestinal injury in a piglet model with lipopolysaccharide challenge.

Necroptosis, a newly discovered form of programmed cell death that combines the features of apoptosis and necrosis, is important in various physiological and pathological disorders. However, the role of necroptosis on intestinal injury during sepsis has been rarely evaluated. This study aimed to investigate the presence of necroptosis in intestinal injury, and its contribution to intestinal injury in a piglet model challenged with Escherichia coli lipopolysaccharide (LPS). Firstly, a typical cell necrotic phenomenon was observed in jejunum of LPS-challenged pigs by transmission electron microscope. Protein expression of necroptosis signals including receptor-interacting protein kinase (RIP) 1, RIP3, and phosphorylated mixed-lineage kinase domain-like protein (MLKL), mitochondrial proteins including phosphoglycerate mutase family member 5 (PGAM5) and dynamin-related protein 1 (DRP1), and cytoplasmic high-mobility group box 1 (HMGB1) were time-independently increased in jejunum of LPS-challenged piglets, which was accompanied by the impairment of jejunal morphology, and digestive and barrier function indicated by lower activities of jejunal disaccharidases and protein expression of jejunal tight junction proteins claudin-1 and occludin. Pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6 were also dynamically induced in serum and jejunum of piglets after LPS challenge. Moreover, pretreatment with necrostatin-1 (Nec-1), an specific inhibitor of necroptosis, inhibited necroptosis indicated by decreased necrotic ultrastructural changes and decreased protein expression of RIP1, RIP3, and phosphorylated MLKL as well as PGAM5, DRP1, and cytoplasmic HMGB1. Nec-1 pretreatment reduced jejunal morphological injury, and improved digestive and barrier function. Nec-1 pretreatment also decreased the levels of serum and jejunal pro-inflammatory cytokines and the numbers of jejunal macrophages and monocytes. These findings indicate for the first time that necroptosis is present and contributes to LPS-induced intestinal injury. Nec-1 may have a preventive effect on intestinal injury during sepsis.

Kinetics of changes in gene and microRNA expression related with muscle inflammation and protein degradation following LPS-challenge in weaned piglets.

To test the dynamic changes of the expression of genes and microRNA in the gastrocnemius muscle after LPS challenge, 36 piglets were assigned to a control group (slaughtered 0 h after saline injection) and LPS groups (slaughtered at 1 h, 2 h, 4 h, 8 h, and 12 h after LPS treatment, respectively). After LPS treatment, the mRNA expression of IL-1ß, IL-6, and TNF-α reached maximal levels at 1 h, 2 h, and 1 h, respectively (P < 0.05), and mRNA expression of TLR4, NODs, muscle-specific ring finger 1, and muscle atrophy F-box peaked at 12 h (P < 0.05). Moreover, the expression of miR-122, miR-135a, and miR-370 reduced at 1 h, 1 h, and 2 h, respectively (P < 0.05), and miR-34a, miR-224, miR-132, and miR-145 reached maximum expression levels at 1 h, 1 h, 2 h, and 4 h, respectively (P < 0.05). These results suggested that mRNA expression of pro-inflammatory cytokines was elevated in the early stage, mRNA expression of genes related to TLR4 and NODs signaling pathways and protein degradation increased in the later phase, and the expression of microRNA related to muscle inflammation and protein degradation changed in the early stage after LPS injection.