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. 2020 Nov:145:111712.
doi: 10.1016/j.fct.2020.111712. Epub 2020 Aug 30.

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

Lei Ge  1 , Ziman Lin  1 , Guannan Le  1 , Lili Hou  1 , Xinru Mao  1 , Shuiping Liu  1 , Dandan Liu  1 , Fang Gan  1 , Kehe Huang  2
Affiliations

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

Lei Ge et al. Food Chem Toxicol. 2020 Nov.

Abstract

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.

Keywords: Deoxynivalenol; Inflammation; LC3B; LPS; NF-κB signaling pathway; Tight junction.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Image 1
Graphical abstract
Fig. 1
Fig. 1
Cytotoxic effect of DON combined with LPS for 12 h and 24 h of IPEC-J2 cell monolayers. Monolayers of IPEC-J2 cells were exposed with different concentrations of DON (0, 1, 2, 4, 8, 16 μM) and LPS (1 μg/mL) for 12 h and 24 h. The cell viability was assessed by MTT assay (A for 12 h and C for 24 h) and LDH activity assay (B for 12 and D for 24 h) respectively. Date were presented as mean ± SEM (n = 6). *P < 0.05 and **P < 0.01 vs. control group.
Fig. 2
Fig. 2
Effects of LPS combined with nontoxic-dose DON on inflammatory cytokines of IPEC-J2 cell monolayers. IPEC-J2 cells were exposed to DON (2 μM) and/or LPS (1 μg/mL) for 12 h and 24 h. The expression of TNF-α, IL-6 and TGF-β mRNA was measured by RT-qPCR after corresponding treatments for 12 h (A) and 24 h (B) respectively. Date were presented as mean ± SEM (n = 3). *P < 0.05 and **P < 0.01 vs. control group. #P < 0.05 and ##P < 0.01 vs. LPS group.
Fig. 3
Fig. 3
Effects of LPS combined with nontoxic-dose DON on protein expression of NLRP3 and procaspase-1 of IPEC-J2 cell monolayers. After corresponding treatment using DON and LPS for 24 h, the protein expression of NLRP3 and procaspase-1 of IPEC-J2 cells was measured by western blotting. Date were presented as mean ± SEM (n = 3). *P < 0.05 and **P < 0.01 vs. control group. #P < 0.05 and ##P < 0.01 vs. LPS group.
Fig. 4
Fig. 4
Effects of LPS combined with nontoxic-dose DON on expression and distribution of tight junction proteins of IPEC-J2 cell monolayers. After corresponding treatment using DON and LPS, the mRNA expression of ZO-1, Occludin and Claudin-1 was assessed using qRT-PCR (A for 12 h and B for 24 h). The expression of tight junction protein (ZO-1 and Occludin) of IPEC-J2 cells was measured by western blotting (C for 12 h and D for 24 h). After treatment for 24 h, the distribution of ZO-1 (green) was measured by immunofluorescence (DAPI, nucleus, blue) (E). Date were presented as mean ± SEM (n = 3). *P < 0.05 and **P < 0.01 vs. control group. #P < 0.05 and ##P < 0.01 vs. LPS group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 5
Fig. 5
Role of NF-κB in inflammation and intestinal epithelial barrier dysfunction of IPEC-J2 monolayers induced by LPS combined with nontoxic-dose DON. After corresponding treatment using DON and LPS, the expression of IκB-α, pp65 and p65 protein was determined by western blotting (A). The distribution of p65 inside and outside the nucleus was measured by immunofluorescence (B). Cells were immunostained with p65 (green) antibody and DAPI (nucleus, blue). After addition of inhibitor BAY 11–7082, the expression of TNF-α, IL-6 and TGF-β was measured by RT-qPCR (C) and the expression of NLRP3 (D) and tight junction protein (ZO-1 and Occludin) (E)was measured by western blotting. *P < 0.05 and **P < 0.01 vs. control group. #P < 0.05 and ##P < 0.01 vs. LPS group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 6
Fig. 6
Role of LC3B in inflammation and intestinal epithelial barrier dysfunction of IPEC-J2 cell monolayers induced by nontoxic-dose DON and LPS. After corresponding treatment using DON and LPS, the expression of LC3B was determined by western blotting (A). LC3B-/- IPEC-J2 cell was constructed by CRISPR/Cas9 system (B). The expression of TNF-α, IL-6 and TGF-β mRNA was measured by RT-qPCR (C), and the protein expression of NLRP3 (D) and tight junction protein (ZO-1 and Occludin) (E) was measured by western blotting after corresponding treatment in IPEC-J2 cells and LC3B-/- IPEC-J2 cells. *P < 0.05 and **P < 0.01 vs. IPEC-J2 control group. #P < 0.05 and ##P < 0.01 vs. IPEC-J2 LC3B-/- control group. !P < 0.05 and !!P < 0.01 vs. IPEC-J2 DON + LPS group.
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