Melatonin improves cholestatic liver disease via the gut-liver axis.

Cholestatic liver disease is characterized by disturbances in the intestinal microbiota and excessive accumulation of toxic bile acids (BA) in the liver. Melatonin (MT) can improve liver diseases. However, the underlying mechanism remains unclear. This study aimed to explore the mechanism of MT on hepatic BA synthesis, liver injury, and fibrosis in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed and Mdr2-/- mice. MT significantly improved hepatic injury and fibrosis with a significant decrease in hepatic BA accumulation in DDC-fed and Mdr2-/- mice. MT reprogramed gut microbiota and augmented fecal bile salt hydrolase activity, which was related to increasing intestinal BA deconjugation and fecal BA excretion in both DDC-fed and Mdr2-/- mice. MT significantly activated the intestinal farnesoid X receptor (FXR)/fibroblast growth factor 15 (FGF-15) axis and subsequently inhibited hepatic BA synthesis in DDC-fed and Mdr2-/- mice. MT failed to improve DDC-induced liver fibrosis and BA synthesis in antibiotic-treated mice. Furthermore, MT provided protection against DDC-induced liver injury and fibrosis in fecal microbiota transplantation mice. MT did not decrease liver injury and fibrosis in DDC-fed intestinal epithelial cell-specific FXR knockout mice, suggesting that the intestinal FXR mediated the anti-fibrosis effect of MT. In conclusion, MT ameliorates cholestatic liver diseases by remodeling gut microbiota and activating intestinal FXR/FGF-15 axis-mediated inhibition of hepatic BA synthesis and promotion of BA excretion in mice.

Mild endoplasmic reticulum stress alleviates FB1-triggered intestinal pyroptosis via the Sec62-PERK pathway.

Fumonisin B1 (FB1), a water-soluble mycotoxin released by Fusarium moniliforme Sheld, is widely present in corn and its derivative products, and seriously endangers human life and health. Recent studies have reported that FB1 can lead to pyroptosis, however, the mechanisms by which FB1-induced pyroptosis remain indistinct. In the present study, we aim to investigate the mechanisms of pyroptosis in intestinal porcine epithelial cells (IPEC-J2) and the relationship between FB1-induced endoplasmic reticulum stress (ERS) and pyroptosis. Our experimental results showed that the pyroptosis protein indicators in IPEC-J2 were significantly increased after exposure to FB1. The ERS markers, including glucose-regulated Protein 78 (GRP78), PKR-like ER kinase protein (PERK), and preprotein translocation factor (Sec62) were also significantly increased. Using small interfering RNA silencing of PERK or Sec62, the results demonstrated that upregulation of Sec62 activates the PERK pathway, and activation of the PERK signaling pathway is upstream of FB1-induced pyroptosis. After using the ERS inhibitor 4-PBA reduced the FB1-triggered intestinal injury by the Sec62-PERK pathway. In conclusion, we found that FB1 induced pyroptosis by upregulating Sec62 to activate the PERK pathway, and mild ERS alleviates FB1-triggered damage. It all boils down to one fact, the study provides a new perspective for further, and improving the toxicological mechanism of FB1.

Taurine alleviates ochratoxin A-induced pyroptosis in PK-15 cells by inhibiting oxidative stress.

Ochratoxin A (OTA) is one of the most harmful mycotoxins, which can cause multiple toxicological effects, especially nephrotoxicity in animals and humans. Taurine is an essential amino acid with various biological functions such as anti-inflammatory and anti-oxidation. However, the protective effect of taurine on OTA-induced nephrotoxicity and pyroptosis had not been reported. Our results showed that OTA exposure induced cytotoxicity and oxidative stress in PK-15 cells, including reactive oxygen species (ROS) accumulation, increased mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), and decreased mRNA levels of catalase (CAT), glutathione peroxidase 1 (GPx1), and glutathione peroxidase 4 (GPx4). In addition, OTA treatment induced pyroptosis by increasing the expressions of pyroptosis-related proteins NLRP3, GSDMD, Caspase-1 P20, ASC, Pro-caspase-1, and IL-1ß. Meanwhile, taurine could alleviate OTA-induced pyroptosis and cytotoxicity, as well as reduce ROS level, COX-2, and iNOS mRNA levels, and increase the mRNA levels of the antioxidant enzyme in PK-15 cells. Taken together, taurine alleviated OTA-induced pyroptosis in PK-15 cells by inhibiting ROS generation and altering the activity of antioxidant enzymes, thereby attenuating its nephrotoxicity.

Combined protective effects of icariin and selenomethionine on novel chronic tubulointerstitial nephropathy models in vivo and in vitro.

Chronic tubulointerstitial nephropathy (CTIN) is one of the most common kidney diseases. However, treatment for CTIN has multiple limits. Adjuvant therapy through nutritional regulation has become a hot research topic at present. Icariin (ICA), an extraction of Chinese herbal medicine epimedium, has many pharmacological functions including anti-inflammation and tonifying kidney. Selenomethionine (SeMet) possesses the effects of antioxidant and lightening nephrotoxicity. However, little is known about the combined nephroprotection of them. This study was investigated to evaluate the joint effects of ICA and SeMet on CTIN and explore the mechanism. Based on a novel CTIN model developed in our previous study, mice were randomly divided into five groups (a: control; b: model; c: model + ICA; d: model + SeMet; e: model + ICA + SeMet). Renal tubule epithelial cells were treated with cyclosporine A and ochratoxin A without/with ICA or/and SeMet. The results showed that ICA or/and SeMet ameliorated CTIN by inhibiting the uptrends of blood urine nitrogen, serum creatinine, urine protein, urine gravity, histopathological damage degree and collagen I deposition. ICA or/and SeMet also increased cell proliferation and decreased apoptosis and the expression of transforming growth factor-beta 1 and α-smooth muscle actin. Emphatically, ICA and SeMet joint had better nephroprotection than alone in most indexes including fibrosis. Furthermore, ICA and SeMet joint decreased the activation of toll-like receptor 4 (TLR4)/NFκB pathway induced by CTIN. TLR4 overexpression counteracted the joint protection of ICA and SeMet. Therefore, ICA and SeMet in combination could protect against CTIN through blocking TLR4/NFκB pathway. The study will provide novel insights to explore an adjuvant therapeutic orientation.

Melatonin mitigates aflatoxin B1-induced liver injury via modulation of gut microbiota/intestinal FXR/liver TLR4 signaling axis in mice.

Aflatoxin B1 (AFB1) is a widespread contaminant in foods and feedstuffs, and its target organ is the liver. Melatonin (MT) has been shown to alleviate inflammation in organs and remodel gut microbiota in animals and humans. However, the underlying mechanism by which MT alleviates AFB1-induced liver injury remains unclear. In the present study, MT pretreatment markedly increased the expression of intestinal tight junction proteins (ZO-1, Occludin, and Claudin-1), decreased intestinal permeability, reduced production of gut-derived Lipopolysaccharide (LPS) and remodeled gut microbiota, ultimately alleviated AFB1-induced liver injury in mice. Interestingly, MT pretreatment failed to exert beneficial effects on the intestine and liver in antibiotic-treated mice. Meanwhile, MT pretreatment significantly increased the farnesoid X receptor (FXR) protein expression of ileum, and decreased the TLR4/NF-κB signaling pathway-related messenger RNA (mRNA) and proteins (TLR4, MyD88, p-p65, and p-IκBα) expression in livers of AFB1-exposed mice. Subsequently, pretreatment by Gly-ß-MCA, an intestine-selective FXR inhibitor, blocked the alleviating effect of MT on liver injury through increasing the liver-specific expression of TLR4/NF-κB signaling pathway-related mRNA and proteins (TLR4, MyD88, p-p65, and p-IκBα). In conclusion, MT pretreatment ameliorated AFB1-induced liver injury and the potential mechanism may be related to regulate gut microbiota/intestinal FXR/liver TLR4 signaling axis, which provides a strong evidence for the protection of gut-derived liver inflammation.

Selenomethionine alleviated Ochratoxin A induced pyroptosis and renal fibrotic factors expressions in MDCK cells.

Ochratoxin A (OTA) is universally known to induce nephrotoxicity via inducing oxidative stress and apoptosis, inhibiting protein synthesis and activating autophagy. Our previous studies have proved that OTA induces nephrotoxicity in vitro and in vivo by adjusting the NOD-like receptor protein 3 (NLRP3) inflammasome activation and caspase-1-dependent pyroptosis. Based on these findings, we further investigated the protective role of selenomethionine (SeMet) on OTA-caused nephrotoxicity using the Madin-Darby canine kidney (MDCK) epithelial cells as an in vitro model, proposing to offer a new way for remedying OTA-induced nephrotoxicity by nutritional manipulation. We measured the cell vitality, lactate dehydrogenase (LDH) activity and the expression of renal fibrotic genes, NLRP3 inflammasome and pyroptosis related genes. MTT and LDH results indicated that SeMet supplementation significantly mitigated 2.0 µg/ml OTA-induced cytotoxicity in MDCK cells (p < 0.05). Meanwhile, SeMet alleviated OTA induced increase of reactive oxygen species in MDCK cells. Then, the expressions of α-SMA, Vimentin, and TGF-ß were detected both in mRNA and protein levels. The results indicated 8 µM SeMet supplementation could significantly downregulate the expression of OTA-induced renal fibrosis-related genes (p < 0.05). In addition, the upregulation of OTA-induced NLRP3 inflammasome and pyroptosis downstream genes was also significantly inhibited by 8 µM of SeMet (p < 0.05). In summary, SeMet could alleviate OTA-induced renal fibrotic genes expression and reduce NLRP3-caspase-1-dependent pyroptosis. Therefore, SeMet supplementation may become an effective approach for preserving animals from renal injury exposed to OTA.

Deoxynivalenol aggravates the immunosuppression in piglets and PAMs under the condition of PEDV infection through inhibiting TLR4/NLRP3 signaling pathway.

Mycotoxins are toxic metabolites produced by fungi, which are ubiquitous in cereals and feed worldwide and threaten human and animal health. Deoxynivalenol (DON) is one of the most prevalent mycotoxins and causes a series of toxicities, especially enterotoxicity and immunotoxicity. Porcine epidemic diarrhea virus (PEDV) is a destructive enteropathogenic animal coronavirus, is often accompanied with DON contamination in the swine herd. Previous studies have shown that PEDV infection leads severe immunosuppression in pigs. However, whether DON exposure aggravates the PEDV-induced immunosuppression remains unclear. In this study, weaned piglet and porcine alveolar macrophage cell (PAM) models were established to explore the effects of DON on the PEDV-induced immunosuppression and to clarify its underlying mechanism. The in vivo results showed that 2.25 mg/kg feed DON significantly exacerbated the immunosuppressive effects on the PEDV-infected piglets, as demonstrated by the decreases in growth performance, the numbers of goblet cells and CD3+T cells, as well as the protein expressions of ZO-1, Claudin1 and Muc2, in addition to the increases in anti-inflammatory factors levels and the intestinal injury. Similarly, the in vitro results demonstrated that 3-4 µM DON markedly aggravated apoptosis, enhanced the expressions of anti-inflammatory factors, but reduced the migration and phagocytosis abilities of the PEDV-infected PAMs. Furthermore, DON significantly suppressed the expressions of TLR4/NLRP3 in vivo and in vitro. To contrast, lipopolysaccharide (LPS), the corresponding activator, obviously alleviated the DON-exacerbated immunosuppression. Our findings suggest that DON could aggravate host immunosuppression under the condition of PEDV infection through inhibiting TLR4/NLRP3 signaling pathway, and provide novel theoretical insights into the further studies on the immunotoxicity of DON contamination and PEDV-induced immunosuppression.

PCV2 infection aggravates OTA-induced immunotoxicity in vivo and in vitro.

Ochratoxin A (OTA), frequently existing in the food and feeds, could induce immunotoxicity. Porcine circovirus type 2 (PCV2), as a primary causative agent of porcine circovirus-associated disease, also could induce immunosuppression. However, it is still unknown whether PCV2 infection impacts OTA-induced immunotoxicity. The pigs and porcine alveolar macrophages (PAMs) were used as the model in the present experiment. The results in vivo indicated that PCV2 infection exacerbated OTA-induced immunotoxicity, NF-κB p65 phosphorylation, and TLR4 and MyD88 mRNA and protein expression in spleen. The results in vitro showed that OTA at 7.0 and 9.0 µM decreased cell viability and increased LDH release of PAMs without PCV2 infection. However, with PCV2 infection, OTA at 5.0, 7.0 and 9.0 µM significantly decreased cell viability and increased LDH release compared with absence of PCV2 infection. In addition, OTA at 5.0 and 7.0 µM significantly increased Annexin V/PI-positive rate, apoptosis of nuclear, γ-H2AX foci, IL-1α and TNF-α expression in PAMs with PCV2 infection compared with absence of PCV2 infection. In addition, PCV2 infection enhanced OTA-induced TLR4 and MyD88 mRNA and protein expression and NF-κB p65 phosphorylation. Knockdown of TLR4 alleviated the exacerbating effects of PCV2 infection on OTA-induced cytotoxicity, apoptosis and DNA damage in PAMs. These results indicated that PCV2 infection aggravated OTA-induced immunotoxicity and reduced the dose of OTA-induced immunotoxicity via TLR4/NF-κB p65 signaling pathway, which could provide basis for establishing limits for OTA.

The therapeutic and preventive effects of a canine-origin VB12 -producing Lactobacillus on DSS-induced colitis in mice.

Vitamin B12 (VB12 ) plays vital roles as a cofactor in reactions related to biosynthesis and metabolic regulation. Animals with diarrhoea from intestinal inflammation are susceptible to VB12 deficiency due to dysfunctional absorption. No current medications for canine intestinal inflammation can simultaneously act as VB12 supplements. Here we have tested a strain of VB12 -producing Lactobacillus, to investigate its safety in healthy dogs and test for hypothesized therapeutic and preventive effects on murine colitis. Results from enzyme-linked immunosorbent assay, histopathological analysis, and quantitative polymerase chain reaction showed normal physical conditions of healthy dogs given Lactobacillus, and blood biochemical indices showed no significant differences in markers, indicating safety of Lactobacillus to healthy dogs. The microbiota in animals receiving VB12 -producing Lactobacillus probiotic exhibited decreased abundance of Escherichia coli and concomitant increase in Lactobacillus. The probiotic supplement also resulted in downregulation of proinflammatory cytokines in murine colon tissues, reduced myeloperoxidase activity and malondialdehyde level, and significantly increased serum VB12 level and decreased homocysteine in therapeutic and preventive experiments. Moreover, Lactobacillus supplement decreased colonic inflammation and injury, improved gut microbiota, and ameliorated VB12 deficiency as an adjunctive therapy. We conclude this product is potentially beneficial for efficient therapy and prevention of VB12 deficiency form intestinal inflammation in canine clinical practice.

Ochratoxin A induces nephrotoxicity in vitro and in vivo via pyroptosis.

Ochratoxin A (OTA), a prevalent nephrotoxic mycotoxin contaminant in food and feedstuff, has been reported to induce renal injury. To disclose the nephrotoxicity of continuous administration of OTA and to investigate potential mechanisms related to pyroptosis, male C57BL/6 mice were intraperitoneally injected with 1.0 and 2.0 mg/kg B.W. OTA every other day for 14 days. At 2.0 mg/kg B.W. OTA administration significantly increased histological injury and renal fibrosis molecules (α-SMA, Vimentin, TGF-ß) and activated the NOD-like receptor protein 3 (NLRP3) inflammasome and induced pyroptosis compared with control. In the in vitro tests, Madin-Darby canine kidney (MDCK) epithelial cells were exposed to 0-4.0 µg/ml OTA for 24 h in serum-free medium. Data showed that OTA dose-dependently affected cell viability and significantly up-regulated renal fibrosis genes (α-SMA, Vimentin, TGF-ß). 2.0 µg/ml OTA significantly induced NLRP3 inflammasome activation and caspase-1-dependent pyroptosis, increasing the expression and secretion of pro-inflammatory cytokines (IL-6, TNF-α) and pyroptosis-related genes (GSDMD, IL-1ß, IL-18) in MDCK cells. These outcomes were significantly abrogated after inhibiting NLRP3 activation with inhibitor MCC950 and silencing NLRP3 with small interfering RNA (siRNA). Furthermore, knockdown of caspase-1 also ameliorated OTA-induced renal fibrosis via the inhibition of pyroptosis. Collectively, the chosen doses of OTA-triggered nephrotoxicity through NLRP3 inflammasome activation and caspase-1-dependent pyroptosis both in vitro and in vivo.

Compound Lactobacillus sp. administration ameliorates stress and body growth through gut microbiota optimization on weaning piglets.

The composition of bacteria in the gastrointestinal tract of piglets is easily affected by environmental changes, particularly during the weaning period. Compound strains of Lactobacillus reuteri and Lactobacillus salivarius were supplemented to piglets during pre- and post-weaning to determine their effects in improving the growth performance and ameliorating the diarrhea rate and stress caused by antioxidation in piglets. A larger number of L. reuteri and L. salivarius colonized the distal segment of the ileum and the total numbers of Lactobacillus spp. and Bifidobacteria were higher in the ileal mucous membrane and cecal lumen with probiotics supplementation. The numbers of antioxidants and immune molecules increased, levels of cortisol and endotoxin reduced, and growth hormone and insulin-like growth factor 1 improved in the plasma following compound bacteria (CL) supplementation. Spearman's and KEGG analysis of the bacterial operational taxonomic unit and antioxidative and immune indices and metabolic genes indicated that the body growth modulation by CL supplementation could be attributed to optimization of the intestinal bacterial composition; functional strains of L. delbrueckii, L. salivarius, L. formicilis, L. reuteri, and L. mucosae were positively correlated with body antioxidation and immunity derived by CL supplementation. Strains of L. agilis and L. pontis were diverse and negatively correlated with body antioxidation and immunity. Collectively, these results suggest that supplementation with CL could reduce stress and improve the growth performance of piglets during weaning by optimizing the intestinal bacterial composition. KEY POINTS: • The colonization of L. reuteri and L. salivarius in ileal mucous membrane optimize bacterial composition of GIT, mainly some functional strains of Lactobacillus, L. delbrueckii, L. salivarius, L. formicilis, L. reuteri, and L. mucosae. • The optimized bacterial composition of piglets in both ileal mucous membrane and cecal content improves body growth hormone level, immunity, and antioxidation, which is helpful to defend the stress. These benefits induce to increased growth performance of animal model piglets during weaning.

PCV2 replication promoted by oxidative stress is dependent on the regulation of autophagy on apoptosis.

Porcine circovirus type 2 (PCV2) is an economically important swine pathogen but some extra trigger factors are required for the development of PCV2-associated diseases. By evaluating cap protein expression, viral DNA copies and the number of infected cells, the present study further confirmed that oxidative stress can promote PCV2 replication. The results showed that oxidative stress induced autophagy in PCV2-infected PK15 cells. Blocking autophagy with inhibitor 3-methyladenine or ATG5-specific siRNA significantly inhibited oxidative stress-promoted PCV2 replication. Importantly, autophagy inhibition significantly increased apoptosis in oxidative stress-treated PK15 cells. Suppression of apoptosis by benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone in conditions of autophagy inhibition restored PCV2 replication. Taken together, autophagy protected host cells against potential apoptosis and then contributed to PCV2 replication promotion caused by oxidative stress. Our findings can partly explain the pathogenic mechanism of PCV2 related to the oxidative stress-induced autophagy.

Nephrotoxicity instead of immunotoxicity of OTA is induced through DNMT1-dependent activation of JAK2/STAT3 signaling pathway by targeting SOCS3.

Ochratoxin A (OTA) is reported to induce nephrotoxicity and immunotoxicity in animals and humans. However, the underlying mechanism and the effects of OTA on DNA damage have not been reported until now. The present study aims to investigate OTA-induced cytotoxicity and DNA damage and the underlying mechanism in PK15 cells and PAMs. The results showed that OTA at 2.0-8.0 µg/mL for 24 h induced cytotoxicity and DNA damage in PK15 cells and PAMs as demonstrated by decreasing cell viabilities and mRNA levels of DNA repair genes (OGG1, NEIL1 and NEIL3), increasing LDH release, Annexin V staining cells, apoptotic nuclei and the accumulation of γ-H2AX foci. OTA at 2.0-8.0 µg/mL increased DNMT1 and SOCS3 mRNA expressions about 2-4 fold in PK15 cells or 1.3-2 fold in PAMs. OTA at 2.0-8.0 µg/mL increased DNMT1, SOCS3, JAK2 and STAT3 protein expressions in PK15 cells or PAMs. DNMT inhibitor (5-Aza-2-dc), promoted SOCS3 expression, inhibited JAK2 and STAT3 expression, alleviated cytotoxicity, apoptosis and DNA damage induced by OTA at 4.0 µg/mL in PK15 cells. While, in PAMs, 5-Aza-2-dc had no effects on SOCS3 expression induced by OTA at 4.0 µg/mL, but inhibited JAK2 and STAT3 expression, and alleviated cytotoxicity, apoptosis and DNA damage induced by OTA. JAK inhibitor (AG490) or STAT3-siRNA alleviated OTA-induced cytotoxicity and DNA damage in PK15 cells or PAMs. Taken together, nephrotoxicity instead of immunotoxicity of OTA is induced by targeting SOCS3 through DNMT1-mediated JAK2/STAT3 signaling pathway. These results provide a scientific and new explanation of the underlying mechanism of OTA-induced nephrotoxicity and immunotoxicity.

Low-Level Aflatoxin B1 Promotes Influenza Infection and Modulates a Switch in Macrophage Polarization from M1 to M2.

BACKGROUND/AIMS: Swine influenza virus (SIV) is a major pathogen of both animals and humans. Afatoxin B1 (AFB1) is one of the most common mycotoxins in feed and food. However, the central contribution of AFB1 to SIV infection remains unclear. METHODS: Here, TCID50 assays, fluorescence-based quantitative real-time PCR, western blotting, immunofluorescence staining, histopathological examination, flow cytometry and scanning electron microscopy were performed to investigate the involvement and underlying mechanism of AFB1 in SIV infection in vivo and in vitro using mouse models and porcine alveolar macrophage (PAM) models, respectively. RESULTS: The in vivo study showed that low levels of AFB1 promoted SIV infection and increased its severity, as demonstrated by the increased mRNA expression of viral matrix protein (M); by the increased protein expression of nucleoprotein (NP), matrix protein 1 and ion channel protein; and by animal weight loss, lung index and lung histologic damage. In addition, the increased occurrence of SIV infection accompanied by increases in the level of IL-10 in sera and lungs, in the spleen index and in the number of CD206-positive mouse alveolar macrophages but decreases in the level of TNF-α in sera and lungs, in the thymus index and in the number of CD80-positive mouse alveolar macrophages was observed in SIV-infected mice after low-level AFB1 exposure. The in vitro study showed that low concentrations of AFB1 promoted SIV infection, as demonstrated by the increases in viral titers and viral M mRNA and NP expression levels in SIV-infected PAMs as well as by the number of cells positive for NP protein expression. Furthermore, AFB1 promoted the polarization of SIV-infected PAMs to the M1 phenotype at 8 hpi and to the M2 phenotype at 24 hpi, as measured by the increases in IL-10 expression and in the number of CD206-positive PAMs as well as by the morphological changes observed by scanning electron microscopy. The administration of the immune stimulant lipopolysaccharide (LPS) reversed the switch in PAM polarization from M2 to M1 and thereby counteracted the promotion of influenza virus infection induced by AFB1. CONCLUSION: Our results are the first to confirm that low-level exposure to AFB1 promotes SIV infection and modulates a switch in macrophage polarization from M1 to M2. The work reported here provides important data that point to a role for AFB1 in SIV infection, and it opens a new field of study.

ERK1/2-mediated autophagy is essential for cell survival under Ochratoxin A exposure in IPEC-J2 cells.

The intestinal epithelium represents the first physical barrier against food contaminations. Ochratoxin A (OTA), one of the most deleterious mycotoxins, is commonly detected in food and feed stuff. The purpose of this study is to explore the adaptive cyto-protection of intestinal epithelium against OTA exposure and relevant regulation mechanisms. The intestinal porcine epithelial cell line (IPEC-J2) was applied as in vitro models for intestinal epithelium. Western blot and immunofluorescence analysis confirmed that OTA induced extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activation in IPEC-J2 cells. Hoechst 33258 staining and Annexin V/PI analysis exhibited that U0126, the ERK1/2 inhibitor, aggravated OTA-induced apoptosis. Then, we observed that OTA could induce autophagy by western blot. Furthermore, OTA-induced autophagy could be inhibited by U0126. Chloroquine (CQ), the autophagy inhibitor, enhanced OTA-induced apoptosis in IPEC-J2 cells. In addition, CQ aggravated the production of mitochondrial reactive oxygen species, the release of cytochrome c release, and the activation of caspase-3. Taken together, these results suggest that ERK1/2-mediated autophagy is required for porcine intestinal epithelial cell survival against OTA toxicity.

Thioredoxin reductase from Toxoplasma gondii: an essential virulence effector with antioxidant function.

Thioredoxin reductase (TR) can help pathogens resist oxidative-burst injury from host immune cells by maintaining a thioredoxin-reduction state during NADPH consumption. TR is a necessary virulence factor that enables the persistent infection of some parasites. We performed bioinformatics analyses and biochemical assays to characterize the activity, subcellular localization, and genetic ablation of Toxoplasma gondii TR (TgTR), to shed light on its biologic function. We expressed the TgTR protein with an Escherichia coli expression system and analyzed its enzyme activity, reporting a Km for the recombinant TgTR of 11.47-15.57 µM, using NADPH as a substrate, and 130.48-151.09 µM with dithio-bis-nitrobenzoic acid as a substrate. The TgTR sequence shared homology with that of TR, but lacked a selenocysteine residue in the C-terminal region and was thought to contain 2 flavin adenine dinucleotide (FAD) domains and 1 NADPH domain. In addition, immunoelectron microscopy results showed that TgTR was widely dispersed in the cytoplasm, and we observed that parasite antioxidant capacity, invasion efficiency, and proliferation were decreased in TR-knockout (TR-KO) strains in vitro, although this strain still stimulated the release of reactive oxygen species release in mouse macrophages while being more sensitive to H2O2 toxicity in vitro Furthermore, our in vivo results revealed that the survival time of mice infected with the TR-KO strain was significantly prolonged relative to that of mice infected with the wild-type strain. These results suggest that TgTR plays an important role in resistance to oxidative damage and can be considered a virulence factor associated with T. gondii infection.-Xue, J., Jiang, W., Chen, Y., Gong, F., Wang, M., Zeng, P., Xia, C., Wang, Q., Huang, K. Thioredoxin reductase from Toxoplasma gondii: an essential virulence effector with antioxidant function.

SeMet attenuates OTA-induced PCV2 replication promotion by inhibiting autophagy by activating the AKT/mTOR signaling pathway.

Porcine circovirus type 2 (PCV2) is recognized as the causative agent of porcine circovirus-associated diseases. PCV2 replication could be promoted by low doses of ochratoxin A (OTA) as in our previous study and selenium has been shown to attenuate PCV2 replication. However, the underlying mechanism remains unclear. The aim of the study was to investigate the effects of selenomethionine (SeMet), the major component of organic selenium, on OTA-induced PCV2 replication promotion and its potential mechanism. The present study demonstrates that OTA could promote PCV2 replication as measured by cap protein expression, viral titer, viral DNA copies and the number of infected cells. In addition, OTA could activate autophagy as indicated by up-regulated light chain 3 (LC3)-II and autophagy-related protein 5 expressions and autophagosome formation. Further, OTA could down-regulate p-AKT and p-mTOR expressions and OTA-induced autophagy was inhibited when insulin was applied. SeMet at 2, 4 and 6 µM had significant inhibiting effects against OTA-induced PCV2 replication promotion. Furthermore, SeMet could attenuate OTA-induced autophagy and up-regulate OTA-induced p-AKT and p-mTOR expression inhibition. Rapamycin, an inhibitor of AKT/mTOR, could reverse the effects of SeMet on OTA-induced autophagy and the PCV2 replication promotion. In conclusion, SeMet could block OTA-induced PCV2 replication promotion by inhibiting autophagy by activating the AKT/mTOR pathway. Therefore, SeMet supplementation could be an effective prophylactic strategy against PCV2 infections and autophagy may be a potential marker to develop novel anti-PCV2 drugs.

Zearalenone can relieve dextran sulfate sodium-induced inflammatory reaction.

In this study, we investigated the influence of zearalenone (ZEA) on the dextran sulfate sodium (DSS)-induced colitis model both in vitro and in vivo. Our results show that the mRNA levels of IL-1ß, IL-18, NLRP3, ASC, and caspase-1 in the DSS+ZEA-treated group are lower than those in either the DSS or ZEA group, and the protein expression trends are similar. Furthermore, colitis, which is characterized by body weight loss, stool consistency, and the presence of bloody feces, was significantly alleviated in the DSS+ZEA group when compared with that in the DSS group. In addition, histological analysis showed that inflammatory cell infiltration and tissue damage of the colon in the DSS+ZEA group were recovered compared with that in the DSS-treated group. These results suggest that, instead of aggravating DSS-induced colitis, ZEA relieves the inflammatory reaction in colon tissue, which may be related to its estrogenic activity.

Zearalenone induces ROS-mediated mitochondrial damage in porcine IPEC-J2 cells.

In this study, the mitochondrial damage effect and mechanism of zearalenone (ZEA) in swine small intestine IPEC-J2 cells in vitro were comprehensively characterized. The analyses revealed that ZEA at high doses (8 and 7 µg/mL) can significantly increase P < 0.05 the malondialdehyde levels and decrease antioxidant enzymes activities after 48 h of exposure. Meanwhile, the reactive oxygen species (ROS) accumulation increased in high dose ZEA-treated groups after 2 h treatment, but decreased due to the ROS-induced mitochondrial damage and the caused cell apoptosis after 48 h of high does ZEA treatment. Moreover, the decreasing of mitochondrial membrane potential (MMP; ΔΨ) in high dose ZEA exposure was observed in line with the increasing ROS production in mitochondria. Results suggest that ZEA exposure can induce mitochondrial damage by reducing antioxidant enzyme activities, accumulation of ROS, and decreasing MMP. The mitochondrial damage had a dramatic concentration-effects relationship with ZEA.

Effects of ochratoxin A on ER stress, MAPK signaling pathway and autophagy of kidney and spleen in pigs.

Ochratoxin A (OTA), a worldwide mycotoxin found in food and feeds, is a potent nephrotoxin and immunotoxin in animals and humans. This research was conducted to evaluate whether endoplasmic reticulum (ER) stress, MAPK signaling pathway and autophagy were induced by OTA in kidney and spleen of pigs. Twenty-seven crossbred pigs randomly allocated to 3 groups were fed for 42 days ad libitum a basal diet without (Con group, 0.00 µg OTA/kg) and with supplementation of OTA at 400 (OTA-L group) and 800 µg/kg (OTA-H group). From each group, 6 pigs were randomly selected for blood collection on days 0, 21, and 42 and 3 pigs were randomly selected for tissue collection on day 42. The results showed that OTA at 400 and 800 µg/kg diets significantly increased OTA concentrations in serum and kidney and spleen induced the histopathological lesions of kidney and spleen, decreased TCR-stimulated T lymphocyte viabilities and IL-2 concentration, increased TNF-α concentration, and decreased T-AOC levels. OTA increased glucose regulated protein 78, p38, and ERK1/2 phosphorylation, and LC3 II and Atg5 protein expression in kidney and spleen of pigs. These results provide new insights into the relationship between OTA and ER stress, p38 and ERK1/2 MAPK signaling pathway and autophagy in pigs.