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.

As the most toxic mycotoxin of all of the fungal toxins, aflatoxin B1 (AFB1) has carcinogenesis, heptotoxicity, and immunotoxicity. DNA methylation plays a critical role in gene expression regulation of the pathological process. However, the relationship between DNA methylation and AFB1-induced immunotoxicity was not yet reported. Therefore, the objectives of this study were to verify AFB1-induced immunotoxicity and investigate the potential role of the DNA methyltransferase (DNMT) family in AFB1-induced immunotoxicity and the pathway mechanism in 3D4/21 cells. The results showed that AFB1 could induce cytotoxicity, apoptosis, pro-inflammatory cytokine expression, DNA damage, and oxidative stress and decrease phagocytotic capacity. Meanwhile, the levels of DNMT1 and DNMT3a were significantly increased in 0.04 and 0.08 μg/mL AFB1 compared to the control. Inhibition of DNMT1 and DNMT3a by 5-Aza-2dc could reverse changes of the above parameters. Further, the JAK2/STAT3 pathway was significantly activated in 0.04 μg/mL AFB1. Inhibition of p-JAK2 and p-STAT3 by AG490 could alleviate AFB1-induced immunotoxicity. Moreover, inhibition of DNMT1 and DNMT3a by 5-Aza-2dc could suppress the phosphorylation of JAK2 and STAT3. Taken together, AFB1-induced immunotoxicity is related to the JAK2/STAT3 pathway mediated by DNMTs in 3D4/21 cells.

Cyclosporine A (CsA) extracted from the products of fungal fermentation is used to develop a chronic nephropathy model. However, it has numerous side effects. Ochratoxin A (OTA) is a mycotoxin that induces renal injury. We developed a chronic nephropathy model to lessen the side effects of CsA by administration of nontoxic dosage of OTA, and investigated the underlying mechanism. C57BL/10 wild-type mice, toll-like receptor 4 (TLR4) −/− mice, and HK-2 cells were used in this study. The nontoxic dosage (0.25 mg/kg, qod) of OTA could significantly decrease the dosage of CsA from 30 to 20 mg/kg per day, and combination of them induced chronic nephropathy model and alleviated the side effects of onefold CsA in vivo, including cardiotoxicity, hepatotoxicity, and immunosuppression. The nontoxic concentration (0.5 μg/ml) of OTA could significantly decrease the concentration of CsA from 10 to 6 μg/ml that induced cytotoxicity, oxidative stress, and nephrotoxicity in vitro. Nontoxic concentration of OTA and low dosage of CsA activated TLR4 and autophagy. These toxic effects induced by OTA and CsA could be reversed by knockdown of TLR4 and autophagy inhibitor 3-methyladenine in vitro. Furthermore, the renal injury and autophagy induced by OTA and CsA could be attenuated in TLR4 −/− mice. It suggested that a chronic nephropathy model had been successfully developed by administration of nontoxic concentration of OTA and low dosage of CsA via TLR4-mediated autophagy. The side effects of current model were significantly lesser than those of the previous model induced by onefold CsA. It provided a new tool for exploring the pathogenesis and treatment of chronic kidney disease.

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