The role of JAK2/STAT3 pathway in non-cytotoxic concentrations of DON-induced aggravation of inflammatory response in IL-10 deficient RAW264.7 cells.

Deoxynivalenol (DON) as a mycotoxin was commonly found in food and cereals which can affect immune function and inflammatory response. The majority of foods contain DON at levels below the official limit. This study aimed to evaluate the effects of non-cytotoxic concentration of DON on inflammation and its mechanisms using the IL-10 gene-silenced RAW264.7 cell model. The results showed that a non-cytotoxic concentration of DON at 25 ng/ml aggravated IL-10 knockdown-induced inflammation, which was manifested by increasing IL-1ß and TNF-α mRNA expression, migration and phagocytosis, decreasing IL-10 mRNA expression, and enhancing JAK2/STAT3 phosphorylation. Adding JAK2 inhibitor AG490 attenuated the aggravating effect of DON on IL-10 knockdown-induced inflammation. In conclusion, a non-cytotoxic concentration of DON enhances the inflammatory response through the JAK2/STAT3 signaling pathway when inflammation occurs in the body. These results indicated that non-cytotoxic concentrations of DON could aggravate inflammation when inflammation was induced by IL-10 knockdown, which increases vigilance against DON contamination at low concentration especially when an animal's body has inflammation.

Arsenic Exposure-Induced Acute Kidney Injury by Regulating SIRT1/PINK1/Mitophagy Axis in Mice and in HK-2 Cells.

Groundwater resources are often contaminated by arsenic, which poses a serious threat to human and animal's health. Some studies have demonstrated that acute arsenic exposure could induce kidney injury because the kidney is a key target organ for toxicity, but the exact mechanism remains unclear. Hence, we investigated the effect of SIRT1-/PINK1-mediated mitophagy on NaAsO2-induced kidney injury in vivo and in vitro. In our study, NaAsO2 exposure obviously induced renal tubule injury and mitochondrial dysfunction. Meanwhile, NaAsO2 exposure could inhibit the mRNA/protein level of SIRT1 and activate the mitophagy-related mRNA/protein levels in the kidney of mice. In HK-2 cells, we also confirmed that NaAsO2-induced nephrotoxicity depended on the activation of mitophagy. Moreover, the activation of SIRT1 by resveratrol alleviated NaAsO2-induced acute kidney injury via the activation of mitophagy in vivo and in vitro. Interestingly, the inhibition of mitophagy by cyclosporin A (CsA) further exacerbated NaAsO2-induced nephrotoxicity and inflammation in HK-2 cells. Taken together, our study found that SIRT1-regulated PINK1-/Parkin-dependent mitophagy was implicated in NaAsO2-induced acute kidney injury. In addition, we confirmed that PINK1-/Parkin-dependent mitophagy played a protective role against NaAsO2-induced acute kidney injury. Therefore, activation of SIRT1 and mitophagy may represent a novel therapeutic target for the prevention and treatment of NaAsO2-induced acute renal injury.

Farnesoid X Receptor Plays a Key Role in Ochratoxin A-Induced Nephrotoxicity by Targeting Ferroptosis In Vivo and In Vitro.

The mycotoxin ochratoxin A (OTA) causes nephrotoxicity, hepatotoxicity, and immunotoxicity in animals and humans. The farnesoid X receptor (FXR) is a member of the NR family and is highly expressed in the kidney, which has an antilipid production function. Ferroptosis is an iron-dependent form of regulated cell death involved in several pathophysiological cell death and kidney injury. The present study aims to evaluate the role of FXR and ferroptosis in OTA-induced nephrotoxicity in mice and HK-2 cells. Results showed that OTA induced nephrotoxicity as demonstrated by inducing the histopathological lesions and neutrophil infiltration of the kidney, increasing serum BUN, CRE, and UA levels, increasing Ntn-1, Kim-1, and pro-inflammatory cytokine expression, and decreasing IL-10 expression and the cell viability of HK-2 cells. OTA treatment also induced FXR deficiency, ROS release, MDA level increase, GSH content decrease, and 4-HNE production in the kidney and HK-2 cells. OTA treatment induced ferroptosis as demonstrated by increasing labile iron pool and lipid peroxidation levels as well as Acsl4, TFR1, and HO-1 mRNA and protein levels, decreasing GPX4 and FTH mRNA and protein expressions, and inducing mitochondrial injury. The FXR activator (GW4064) rescued the accumulation of lipid peroxides, intracellular ROS, and Fe2+, inhibited ferroptosis, and alleviated OTA-induced nephrotoxicity. The ferroptosis inhibitor (Fer-1) prevented ferroptosis and attenuated nephrotoxicity. Collectively, this study elucidates that FXR played a critical role in OTA-induced nephrotoxicity via regulation of ferroptosis, which provides a novel strategy against OTA-induced nephrotoxicity.

Deoxynivalenol at No-Observed Adverse-Effect Levels Aggravates DSS-Induced Colitis through the JAK2/STAT3 Signaling Pathway in Mice.

The etiology of inflammatory bowel diseases (IBDs) involves complex genetic and environmental factors such as mycotoxin contamination. Deoxynivalenol (DON), a well-known mycotoxin, contaminates food and feed and can induce intestinal injury and inflammatory response. The dose of DON in many foods is also below the limit, although the dose of DON exceeds the limit. The present study aims to evaluate the effects of the nontoxic dose of DON on colitis induced by dextran sodium sulfate (DSS) and the mechanism in mice. The results showed a nontoxic dose of DON at 50 µg/kg bw per day exacerbated DSS-induced colitis in mice as demonstrated by increased disease activity index, decreased colon length, increased morphological damage, decreased occludin and mucoprotein 2 expression, increased IL-1ß and TNF-α expression, and decreased IL-10 expression. DON at 50 µg/kg bw per day enhanced JAK2/STAT3 phosphorylation induced by DSS. Adding JAK2 inhibitor AG490 attenuated the aggravating effects of DON on DSS-induced colitis by reversing the morphological damage, occludin and mucoprotein 2 expression increased, IL-1ß and TNF-α expression increased, and IL-10 expression decreased. Taken together, a nontoxic dose of DON could aggravate DSS-induced colitis via the JAK2/STAT3 signaling pathway. This suggests that DON, below the standard limit dose, is also a risk for IBD and may be harmful to the health of humans and animals, which could provide the basis for establishing limits for DON.

Lipopolysaccharide aggravates canine influenza a (H3N2) virus infection and lung damage via mTOR/autophagy in vivo and in vitro.

Influenza A (H3N2) accounts for the majority of influenza worldwide and continues to challenge human health. Disturbance in the gut microbiota caused by many diseases leads to increased production of lipopolysaccharide (LPS), and LPS induces sepsis and conditions associated with local or systemic inflammation. However, to date, little attention has been paid to the potential impact of LPS on influenza A (H3N2) infection and the potential mechanism. Hence, in this study we used canine influenza A (H3N2) virus (CIV) as a model of influenza A virus to investigate the effect of low-dose of LPS on CIV replication and lung damage and explore the underlying mechanism in mice and A549 and HPAEpiC cells. The results showed that LPS (25 µg/kg) increased CIV infection and lung damage in mice, as indicated by pulmonary virus titer, viral NP levels, lung index, and pulmonary histopathology. LPS (1 µg/ml) also increased CIV replication in A549 cells as indicated by the above same parameters. Furthermore, low doses of LPS reduced CIV-induced p-mTOR protein expression and enhanced CIV-induced autophagy-related mRNA/protein expressions in vivo and in vitro. In addition, the use of the mTOR activator, MHY1485, reversed CIV-induced autophagy and CIV replication in A549 and HPAEpiC cells, respectively. siATG5 alleviated CIV replication exacerbated by LPS in the two lines. In conclusion, LPS aggravates CIV infection and lung damage via mTOR/autophagy.

Isolation, identification and safety evaluation of OTA-detoxification strain Pediococcus acidilactici NJB421 and its effects on OTA-induced toxicity in mice.

Ochratoxin A (OTA) is a potent mycotoxin found in foods and feeds, posing a health risk to animals and humans. Biological detoxification of OTA is considered a promising method, and some bacteria and fungi which can degrade OTA are isolated. However, research on safety and alleviating toxic effects are scarce. This study aims to isolate OTA-detoxification probiotics from natural samples and evaluate their safety and protective effects in mice. Here, a new OTA-detoxification strain named Pediococcus acidilactici NJB421 (P. acidilactici NJB421) was isolated from cow manure, which exhibited a removal rate of OTA at 48.53% for 48 h. P. acidilactici NJB421 exhibited high temperature resistance, acid tolerance, 0.3% bile salt and 1.4% trypsin resistance. The safety evaluation showed that P. acidilactici NJB421 at 2 × 108 CFU/per mouse had no abnormalities in body weight, organ indices, ALT, AST and ALP activities, BUN, CRE and TP contents. And P. acidilactici NJB421 alleviated the decreases in body weight, organ indices and small intestinal length, and alleviated intestinal injury, liver injury and kidney injury. These results suggest P. acidilactici NJB421 is safe and has protection against OTA poisoning, which provides a new OTA-detoxification strain for livestock and food industries.

Gut microbiota-bile acid-intestinal Farnesoid X receptor signaling axis orchestrates cadmium-induced liver injury.

Cadmium (Cd) is a widely prevalent environmental pollutant that accumulates in the liver and induces liver injury. The mechanism of Cd-induced liver injury remains elusive. Our study aimed to clarify the mechanism by which changes in the gut microbiota contribute to Cd-induced liver injury. Here, a murine model of liver injury induced by chronic Cd exposure was used. Liver injury was assessed by biochemistry and histopathology. Expression profiles of genes involved in bile acid (BA) homeostasis, inflammation and injury were assessed via Realtime-PCR and Western-blot. 16S rRNA gene sequencing and mass spectrometry-based metabolomics were used to investigate changes in the gut microbiota and its metabolites in the regulation of Cd-induced liver injury. Here, we showed that Cd exposure induced hepatic ductular proliferation, hepatocellular damage and inflammatory infiltration in mice. Cd exposure induced gut microbiota dysbiosis and reduced the fecal bile salt hydrolase activity leading to an increase of tauro-ß-muricholic acid levels in the intestine. Cd exposure decreased intestine FXR/FGF-15 signaling and promoted hepatic BA synthesis. Furthermore, the mice receiving fecal microbiota transplantation from Cd-treated mice showed reduced intestinal FXR/FGF-15 signaling, increased hepatic BA synthesis, and liver injury. However, the depletion of the commensal microbiota by antibiotics failed to change these indices in Cd-treated mice. Finally, the administration of the intestine-restricted FXR agonist fexaramine attenuated the liver injury, improved the intestinal barrier, and decreased hepatic BA synthesis in the Cd-treated mice. Our study identified a new mechanism of Cd-induced liver injury. Cd-induced gut microbiota dysbiosis, decreased feces BSH activity, and increased intestinal T-ßMCA levels led to an inhibition of intestinal FXR/FGF-15 signaling and an increase in hepatic BA synthesis, ultimately facilitating the development of hepatic ductular proliferation, inflammation, and injury in mice. This study expands our understanding of the health hazards caused by environmental Cd pollution.

Biomechanical Evaluation of Intervertebral Fusion Process After Anterior Cervical Discectomy and Fusion: A Finite Element Study.

Introduction: Anterior cervical discectomy and fusion (ACDF) is a widely accepted surgical procedure in the treatment of cervical radiculopathy and myelopathy. A solid interbody fusion is of critical significance in achieving satisfactory outcomes after ACDF. However, the current radiographic techniques to determine the degree of fusion are inaccurate and radiative. Several animal experiments suggested that the mechanical load on the spinal instrumentation could reflect the fusion process and evaluated the stability of implant. This study aims to investigate the biomechanical changes during the fusion process and explore the feasibility of reflecting the fusion status after ACDF through the load changes borne by the interbody fusion cage. Methods: The computed tomography (CT) scans preoperatively, immediately after surgery, at 3 months, and 6 months follow-up of patients who underwent ACDF at C5/6 were used to construct the C2-C7 finite element (FE) models representing different courses of fusion stages. A 75-N follower load with 1.0-Nm moments was applied to the top of C2 vertebra in the models to simulate flexion, extension, lateral bending, and axial rotation with the C7 vertebra fixed. The Von Mises stress at the surfaces of instrumentation and the adjacent intervertebral disc and force at the facet joints were analyzed. Results: The facet contact force at C5/6 suggested a significantly stepwise reduction as the fusion proceeded while the intradiscal pressure and facet contact force of adjacent levels changed slightly. The stress on the surfaces of titanium plate and screws significantly decreased at 3 and 6 months follow-up. A markedly changed stress distribution in extension among three models was noted in different fusion stages. After solid fusion is achieved, the stress was more uniformly distributed interbody fusion in all loading conditions. Conclusions: Through a follow-up study of 6 months, the stress on the surfaces of cervical instrumentation remarkably decreased in all loading conditions. After solid intervertebral fusion formed, the stress distributions on the surfaces of interbody cage and screws were more uniform. The stress distribution in extension altered significantly in different fusion status. Future studies are needed to develop the interbody fusion device with wireless sensors to achieve longitudinal real-time monitoring of the stress distribution during the course of fusion.

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.

Phenethyl isothiocyanate as an anti-nutritional factor attenuates deoxynivalenol-induced IPEC-J2 cell injury through inhibiting ROS-mediated autophagy.

Deoxynivalenol (DON) is considered to be the most harmful mycotoxin that affects the intestinal health of animals and humans. Phenethyl isothiocyanate (PEITC) in feedstuff is an anti-nutritional factor and impairs nutrient digestion and absorption in the animal intestinal. In the current study, we aimed to explore the effects of PEITC on DON-induced apoptosis, intestinal tight junction disorder, and its potential molecular mechanism in the porcine jejunum epithelial cell line (IPEC-J2). Our results indicated that PEITC treatment markedly alleviated DON-induced cytotoxicity, decreasing the apoptotic cell percentage and pro-apoptotic mRNA/protein levels, and increasing zonula occludens-1 (ZO-1), occludin and claudin-1 mRNA/protein expression. Meanwhile, PEITC treatment ameliorated DON-induced an increase of the inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) mRNA levels and intracellular reactive oxygen species (ROS) level, and a decrease of glutathione peroxidase 1 (GPx1), superoxide dismutase 2 (SOD2), catalase (CAT) and heme oxygenase 1 (HO-1) mRNA levels. Additionally, PEITC treatment significantly down-regulated autophagy-related protein 5 (ATG5), beclin-1 and microtubule-associated protein 1 light chain 3B (LC3-Ⅱ) mRNA/protein levels, decreased the number of green fluorescent protein-microtubule-associated protein 1 light-chain 3 (GFP-LC3) puncta and phosphatidylinositol 3 kinase (PI3K) protein expression, and up-regulated phospho-protein kinase B (p-Akt) and phospho-mammalian target of rapamycin (p-mTOR) protein expression against DON. However, the activation of autophagy by rapamycin, an autophagy agonist, abolished the protective effects of PEITC against DON-induced cytotoxicity, apoptosis and intestinal tight junction disorder. Collectively, PEITC could confer protection against DON-induced porcine intestinal epithelial cell injury by suppressing ROS-mediated autophagy.

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.

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.

Effects of Selenium-enriched probiotics on ochratoxin A-induced kidney injury and DNMTs expressions in piglets.

Effects of Selenium-enriched probiotics (SP) on ochratoxin A-induced kidney injury, growth performance, antioxidant injury, selenoprotein and DNA methylation transferases (DNMTs) expression of piglets were investigated in the article. A total of 48 piglets were randomly divided into 4 groups and fed with basal diet (Con, 0.15 mg Se/kg and OTA at 0.00 mg/kg), basal diets added with OTA (OTA, 0.40 mg OTA/kg), SP and OTA (SP1, 0.15 mg Se/kg and 0.40 mg OTA/kg), SP and OTA (SP2, 0.30 mg Se/kg and 0.40 mg OTA/kg) respectively for 42 days. From each group, six piglets were randomly selected for blood collection on Days 0 and 42 and three piglets were selected for tissue collection on Day 42.The results showed that OTA at 0.40 mg /kg significantly decreased growth performance of pigs, induced the histopathological lesions of kidney and increased urea and creatine levels of serum, decreased GPx and SOD activities, and increased MDA levels. OTA decreased GPx1, GPx4 and SelS expressions, and increased TR1, DNMT 1, DNMT3a and SOCS3 expressions. Both SP1 and SP2 improved OTA-induced poor growth performance, kidney injury, poor antioxidant statues, GPx1, SelS, TR1, SOCS3, DNMT1 and DNMT3a expressions in kidney of pigs. The effects of SP2 on the above parameters changes were better than that of SP1. SP increased GPx and SOD activities and decreased MDA levels changes induced by OTA treatment. These results suggest that SP may serve as a better feed additive for piglets under mycotoxin contamination environments.

Thymosin as a possible therapeutic drug for COVID-19: A case report.

BACKGROUND: There are no effective antiviral therapies for coronavirus disease 2019 (COVID-19) at present. Although most patients with COVID-19 have a mild or moderate course of disease, up to 5%-10% of patients may have a serious and potentially life-threatening condition, indicating an urgent need for effective therapeutic drugs. The therapeutic effect of thymosin on COVID-19 has not been previously studied. In this paper, for the first time we report a case of thymosin treatment of COVID-19. CASE SUMMARY: A 51-year-old man with imported COVID-19 was admitted with definite symptoms of chest tightness, chest pain, and fatigue. The polymerase chain reaction results for severe acute respiratory syndrome coronavirus 2 were negative. The antibody test was positive, confirming the diagnosis of COVID-19. As many orally administered drugs were not well tolerated due to gastrointestinal symptoms, an emergency use of thymosin, a polypeptide consisting of 28 amino acids, was administered by injection. Finally, after the implementation of the treatment program, symptoms and lung imaging improved significantly. CONCLUSION: In this case report, it is confirmed that thymosin may help alleviate the severity of COVID-19 symptoms.

Non-cytotoxic dosage of fumonisin B1 aggravates ochratoxin A-induced nephrocytotoxicity and apoptosis via ROS-dependent JNK/MAPK signaling pathway.

Ochratoxin A (OTA) and fumonisin B1 (FB1), two of the most toxicologically important mycotoxins, often coexist in a variety of foodstuff and feed in humans and animals. Because of the low content of FB1 in foodstuff and feed, alone harmfulness of FB1 is often ignored. However, it is unknown whether the lower dosage of FB1 aggravates the toxicity of other mycotoxins. In this article, we aimed to investigate the effects of the lower dosage of FB1 on OTA-induced nephrotoxicity and apoptosis, and its underlying mechanism in porcine kidney cells (PK-15). Our current study showed that the non-cytotoxic concentration of FB1 (8 µM) could enhance OTA(5 µM)-induced nephrocytotoxicity and the expression of pro-apoptosis-associated genes in PK-15 cells. We also observed that the production of reactive oxygen species (ROS) was increased. However, the expression of pro-apoptosis-associated genes were down-regulated when the N-acetylcysteine (NAC), a ROS scavenger, was used in our experiment. Besides, we found that the combined toxins could increase the protein expression of p-JNK instead of p-p38 and p-ERK. Pretreatment with SP600125, a JNK inhibitor, could significantly block the promotion effects of FB1 on OTA-induced nephrocytotoxicity and apoptosis. The protein expression of p-JNK was also inhibited and the promotion effects of FB1 were significantly alleviated when NAC was used. In conclusion, the non-cytotoxic dosage of FB1 could aggravate the nephrocytotoxicity and apoptosis caused by OTA via ROS-dependent JNK/MAPK signaling pathway.

Nontoxic dose of Phenethyl isothiocyanate ameliorates deoxynivalenol-induced cytotoxicity and inflammation in IPEC-J2 cells.

The intestinal tract is a target for the deoxynivalenol (DON), which has adverse effects in animals and humans' health by affecting intestinal functions. Phenethyl isothiocyanate (PEITC) is an important degradation product of glucosinolates (GSLs), belonging to an anti-nutritional factor that affects the digestion and absorption of nutrients in the animals' intestinal. However, little attention has been paid to the interaction and its mechanism between DON and PEITC. Therefore, the purpose of this study was to assess the effects of PEITC on DON-induced cytotoxicity and inflammation, and explore the potential mechanisms in IPEC-J2 cells. Our results showed that DON exposure could decrease the cell viability and pro-inflammatory cytokine expression in IPEC-J2 cells in a dose-dependent manner. PEITC treatment at the concentrations of 1.25-5 µM had no significant effect on IPEC-J2 cells viability, but above 10 µM of PEITC treatment significantly reduced the cell viability. Interestingly, 1.25-5 µM of PEITC treatment could suppress 4 µM of DON-induced decrease in cell viability and increase in pro-inflammatory cytokine expression. Meanwhile, the protein ratios of p-p65/p-65 and p-IκBα/IκBα were markedly decreased in the groups treated with 1.25-5 µM PEITC compared to DON exposure alone. However, the protective effects of PEITC treatment were significantly blocked after pre-treatment with LPS, NF-κB activator, in IPEC-J2 cells. In conclusion, these findings indicated that the nontoxic dose of PEITC could alleviate DON-induced cytotoxicity and inflammation responses via suppressing the NF-κB signaling pathway in IPEC-J2 cells. Our results provide a new theoretical basis for the rational addition of rapeseed meal in animal feedstuff.

Fumonisin B1 induces nephrotoxicity via autophagy mediated by mTORC1 instead of mTORC2 in human renal tubule epithelial cells.

Fumonisin B1 (FB1), a worldwide contaminating mycotoxin, can cause global food issue. It has been reported that FB1 is related to chronic kidney disease of unknown etiology. However, the study of FB1-induced nephrotoxicity in vitro is very limited and the mechanism is unknown. Human renal tubule epithelial (HK-2) cells were used in this study. The results showed that FB1 exposure could decrease cell viability, induce cell apoptosis and up-regulate the expression of Kim-1, collagen I, α-SMA and TGF-ß1. In addition, autophagy was activated after FB1 exposure, including the conversion of LC3 and up-regulation of ATGs. Furthermore, autophagy inhibitor 3-MA could block FB1-induced abnormalities. And antioxidant enzymes (Gpx1 and Gpx4) were obviously down-regulated and intracellular ROS levels displayed an ascent trend as FB1 exposure concentrations increased. Employing of antioxidant NAC could suppress FB1-induced nephrotoxicity and autophagy. FB1 inhibited the phosphorylation of p70 S6k, a downstream protein of mTORC1. Also, oxidative stress, autophagy and phosphorylation of p70 S6k induced by FB1 was inhibited by MHY1485, an activator of mTOR. But the phosphorylation of AKT, a downstream protein of mTORC2 showed no change with or without MHY1485. Taken together, FB1 induced nephrotoxicity via autophagy mediated by mTORC1 instead of mTORC2 in HK-2 cells.

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.

Nontoxic-dose deoxynivalenol aggravates lipopolysaccharides-induced inflammation and tight junction disorder in IPEC-J2 cells through activation of NF-κB and LC3B.

Lipopolysaccharide (LPS) is the key factor in various intestinal inflammation which could disrupt the epithelial barrier function. Deoxynivalenol (DON), a well-known mycotoxin, can induce intestinal injury. However, the combined enterotoxicity of LPS and DON has rarely been studied. In this study, IPEC-J2 cell monolayers were exposed to LPS and nontoxic-dose DON for 12 and 24 h to investigate the effects of DON on LPS-induced inflammatory response and tight junction variation, and specific inhibitor and CRISPR-Cas9 were used to explore the underlying mechanisms. Our results showed that nontoxic-dose DON aggravated LPS-induced cellular inflammatory response, reflecting on more significant changes of inflammatory cytokines mRNA expression, higher protein expression of NOD-like receptor protein 3 (NLRP3) and procaspase-1. Moreover, nontoxic-dose DON aggravated LPS-induced mRNA and protein expression decreased, and distribution confused of tight junction proteins. We found that DON further enhanced LPS-induced phosphorylation and nucleus translocation of p65, and expression of LC3B-Ⅱ. NF-κB inhibitor and CRISPR-Cas9-mediated knockout of LC3B attenuated the effects of combination which indicated nontoxic-dose DON aggravated LPS-induced intestinal inflammation and tight junction disorder through activating NF-κB signaling pathway and autophagy-related protein LC3B. It further warns that ingesting low doses of mycotoxins may exacerbate the effects of intestinal pathogens on the body.