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. 2021 Dec 28;23(1):289.
doi: 10.3390/ijms23010289.

The Protective Effects of Lactoferrin on Aflatoxin M1-Induced Compromised Intestinal Integrity

Ya-Nan Gao  1   2   3   4 , Song-Li Li  1   2   3   4 , Xue Yang  1   2   3   4 , Jia-Qi Wang  1   2   3   4 , Nan Zheng  1   2   3   4
Affiliations

The Protective Effects of Lactoferrin on Aflatoxin M1-Induced Compromised Intestinal Integrity

Ya-Nan Gao et al. Int J Mol Sci. .

Abstract

Aflatoxin M1 (AFM1), the only toxin with maximum residue levels in milk, has adverse effects on the intestinal barrier, resulting in intestinal inflammatory disease. Lactoferrin (LF), one of the important bioactive proteins in milk, performs multiple biological functions, but knowledge of the protective effects of LF on the compromised intestinal barrier induced by AFM1 has not been investigated. In the present study, results using Balb/C mice and differentiated Caco-2 cells showed that LF intervention decreased AFM1-induced increased intestinal permeability, improved the protein expression of claudin-3, occludin and ZO-1, and repaired the injured intestinal barrier. The transcriptome and proteome were used to clarify the underlying mechanisms. It was found that LF reduced the intestinal barrier dysfunction caused by AFM1 and was associated with intestinal cell survival related pathways, such as cell cycle, apoptosis and MAPK signaling pathway and intestinal integrity related pathways including endocytosis, tight junction, adherens junction and gap junction. The cross-omics analysis suggested that insulin receptor (INSR), cytoplasmic FMR1 interacting protein 2 (CYFIP2), dedicator of cytokinesis 1 (DOCK1) and ribonucleotide reductase regulatory subunit M2 (RRM2) were the potential key regulators as LF repaired the compromised intestinal barrier. These findings indicated that LF may be an alternative treatment for the compromised intestinal barrier induced by AFM1.

Keywords: aflatoxin M1; intestinal barrier; lactoferrin; proteome; tight junction; transcriptome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The effects of LF and AFM1 on mice body weight (n = 10 animals) and serum indicators. (A) Body weight, (B) Cit, (C) I-FABP and (D) D-lactate. Control represents the mice fed with physiological saline. DMSO represents the mice fed with the solution of 1% DMSO/99% corn oil. AFM1 represents the mice exposed to individual AFM1. LF represents the mice exposed to individual LF. LF + AFM1 represents the mice exposed to combined LF and AFM1. Values represent the mean ± SEM. * represents p < 0.05, *** represents p < 0.001.
Figure 2
Figure 2
The effects of LF and AFM1 on mice ileum histology. Histology of the ileum was assessed by hematoxylin-eosin staining (HE, 200×). (A) Control mice (physiological saline), (B) DMSO-treated mice (1%DMSO/99% corn oil), (C) LF-treated mice, (D) AFM1 treated mice, (E) LF + AFM1 treated mice, (F) Villus height and crypt depth in mice ileum, (G) the ratio of villus height/crypt depth. Values represent the means ± SEM (n = three animals). * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001.
Figure 3
Figure 3
The effects of LF and AFM1 on claudin-3, occludin and ZO-1 expression and distribution in ileum measured by immunofluorescence staining (200×). Control represents the mice fed with physiological saline. DMSO represents the mice fed with the solution of 1% DMSO/99% corn oil. AFM1 represents the mice exposed to individual AFM1. LF represents the mice exposed to individual LF. LF + AFM1 represents the mice exposed to combined LF and AFM1. n = 3 animals.
Figure 4
Figure 4
The effects of LF and AFM1 on intestinal permeability in differentiated Caco-2 cells. (A) TEER assay, (B) LY paracellular permeability. Data represent the mean ± SEM of three independent experiments (n = 4). Control represents the differentiated Caco-2 cells exposed to culture medium. LF represents the differentiated Caco-2 cells exposed to individual LF. AFM1 represents the differentiated Caco-2 cells exposed to individual AFM1. LF + AFM1 represents the differentiated Caco-2 cells exposed to combined LF and AFM1. * represents p < 0.05, ** represents p < 0.01.
Figure 5
Figure 5
Transcriptome analysis of differentiated Caco-2 cells exposed to LF and AFM1. (A) The number of differentially expressed genes (DEGs). (B) Unsupervised PCA analysis. (C) Venn diagram depicting the DEGs regulated by LF and AFM1 treatment. (D) The intestinal integrity related KEGG pathways enriched by DEGs. (E) The gene expression level measured by qRT-PCR and RNA-seq. These selected genes were not DEGs between Control and individual LF treatment. LF represents individual 100 μg/mL LF, AFM1 represents individual 8 μg/mL AFM1, LM represents the combination of 100 μg/mL LF and 8 μg/mL AFM1. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001.
Figure 5
Figure 5
Transcriptome analysis of differentiated Caco-2 cells exposed to LF and AFM1. (A) The number of differentially expressed genes (DEGs). (B) Unsupervised PCA analysis. (C) Venn diagram depicting the DEGs regulated by LF and AFM1 treatment. (D) The intestinal integrity related KEGG pathways enriched by DEGs. (E) The gene expression level measured by qRT-PCR and RNA-seq. These selected genes were not DEGs between Control and individual LF treatment. LF represents individual 100 μg/mL LF, AFM1 represents individual 8 μg/mL AFM1, LM represents the combination of 100 μg/mL LF and 8 μg/mL AFM1. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001.
Figure 5
Figure 5
Transcriptome analysis of differentiated Caco-2 cells exposed to LF and AFM1. (A) The number of differentially expressed genes (DEGs). (B) Unsupervised PCA analysis. (C) Venn diagram depicting the DEGs regulated by LF and AFM1 treatment. (D) The intestinal integrity related KEGG pathways enriched by DEGs. (E) The gene expression level measured by qRT-PCR and RNA-seq. These selected genes were not DEGs between Control and individual LF treatment. LF represents individual 100 μg/mL LF, AFM1 represents individual 8 μg/mL AFM1, LM represents the combination of 100 μg/mL LF and 8 μg/mL AFM1. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001.
Figure 6
Figure 6
Proteome analysis of differentiated Caco-2 cells exposed to LF and AFM1. (A) The number of differentially expressed proteins (DEPs). (B) Venn diagram depicting the DEGs regulated by LF and AFM1 treatment. (C) The intestinal integrity-related KEGG pathways enriched by DEPs. (D) The protein expression level measured by Western blotting and expressed as the mean ± SEM of three independent experiments. LF represents individual treatment with 100 μg/mL LF, AFM1 represents individual treatment with 8 μg/mL AFM1, and LM represents combined treatment with of 100 μg/mL LF and 8 μg/mL AFM1. ** represents p < 0.01, *** represents p < 0.001.
Figure 6
Figure 6
Proteome analysis of differentiated Caco-2 cells exposed to LF and AFM1. (A) The number of differentially expressed proteins (DEPs). (B) Venn diagram depicting the DEGs regulated by LF and AFM1 treatment. (C) The intestinal integrity-related KEGG pathways enriched by DEPs. (D) The protein expression level measured by Western blotting and expressed as the mean ± SEM of three independent experiments. LF represents individual treatment with 100 μg/mL LF, AFM1 represents individual treatment with 8 μg/mL AFM1, and LM represents combined treatment with of 100 μg/mL LF and 8 μg/mL AFM1. ** represents p < 0.01, *** represents p < 0.001.
Figure 7
Figure 7
Correlation between identified transcripts and proteins in differentiated Caco-2 cells. (A) Cells exposed to LF, (B) cells exposed to AFM1 and (C) cells exposed to the combination of LF and AFM1. Black points (area e) represent no significant changes at both mRNA and protein levels. Blue points (area d, f) represent the significant changes at the protein level. Green points (area b, h) represent significant changes only at the transcript level. Red points (area a, c, g and i) represent significant changes in mRNA and protein levels. Gray points (area a, b, c, d, f, g, h and i) represent the transcripts or proteins meeting the criterion of p > 0.05 and fold change > 1.5.
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