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. 2024 Sep 23:14:1456356.
doi: 10.3389/fcimb.2024.1456356. eCollection 2024.

Relationship between pathogenic E.coli O78-induced intestinal epithelial barrier damage and Zonulin expression levels in yaks

Xiaoli Ren #  1 , Bin Shi #  1   2 , Zhenyu Chang  1 , Jingbo Zhang  1 , Shuo Wang  1 , Ruidong Liu  1 , Mudan Sang  1 , Hailong Dong  1 , Qingxia Wu  1
Affiliations

Relationship between pathogenic E.coli O78-induced intestinal epithelial barrier damage and Zonulin expression levels in yaks

Xiaoli Ren et al. Front Cell Infect Microbiol. .

Abstract

To explore whether the intestinal damage of yak colibacillosis resulted from the regulation of Zonulin expression by its pathogenic bacteria, the overexpression and interference plasmids of Zonulin were designed and cultured in Tranwell after cell transfection. Then qRT-PCR and Western blot were used to detect the results of cell transfection, 200 mL 1×105 CFU/mL E.coli O78 was added for 4 hours, transmembrane resistance was measured by transmembrane resistance meter, FD4 fluorescence concentration in the lower chamber was detected by enzyme labeling instrument, bacterial translocation was measured by CFU counting method, and epithelial mucin (MUC1, MUC2) and tight junction protein (FABP2, Occludin, ZO-1) were detected by qRT-PCR.

Results: The Zonulin gene overexpression and knockout cell lines were successfully constructed, the TEER value of the barrier of Zonulin overexpression cell lines began to decrease at 1 h after the addition of E.coli O78 and reached the lowest value at 4 h, and the TEER value of Zonulin interference cell lines decreased within 1-4 h after the addition of E.coli O78. At 4 h, the FD4 passing capacity of Zonulin overexpression cell lines was significantly higher than that of interfering cell lines, reaching twice as much as siRNA-1. The amount of bacterial translocation in overexpressed cell lines increased rapidly within 1-4 h, and the concentration of E.coli in the lower chamber was significantly higher than that in the siRNA-1 group at 4 h, but there was no significant change in the siRNA-1 group in the 1-4 h. There was no significant change in the mRNA level of MUC1 in Zonulin overexpression and interference cell lines after the addition of E.coli O78. In the overexpression group, the mRNA levels of MUC2, Occludin, and ZO-1 were significantly decreased, and the mRNA level of FABP2 was increased considerably. These results suggest stimulate epithelial cells to secrete Zonulin protein. Many Zonulin proteins regulate the opening of tight junction structures, reduce the transmembrane resistance of the cell barrier, and improve the permeability of the cell barrier and the amount of bacterial translocation.

Keywords: TEER; Zonulin; cell transfection; intestinal epithelial cell barrier; pathogenicity E.coli O78; yak.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Results of Zonulin overexpression and siRNA interference efficiency detection. (A) RNA integrity verification. (B) results of WB gray value of Zonulin protein. (C) overexpression and interference of Zonulin mRNA relative NC expression. (D) relative NC expression of overexpression and interference of Zonulin protein. *p<0.05, **p<0.01.
Figure 2
Figure 2
The changes of intestinal barrier function after Zonulin overexpression and interference. (A) E.coli O78 affected the change of transmembrane resistance of intestinal epithelial barrier during 1-4 h. (B) the transmembrane resistance of intestinal epithelial barrier at 4 h. (C) the content of FD4 in the lower chamber at 4 h. (D) the amount of E.coli translocation changed during 1-4 h. **p<0.01.
Figure 3
Figure 3
The effect of E.coli O78 on the expression of mucin and tight junction associated protein mRNA in intestinal epithelial barrier after Zonulin overexpression and interference. (A) RNA integrity verification. (B) the relative expression of MUC1 was compared. (C) the relative expression of MUC2 was compared. (D) the relative expression of FABP2 was compared. (E) the relative expression of Occludin was compared. (F) the relative expression of ZO-1 was compared. *p<0.05, **p>0.01, ns p < 0.05.
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Grants and funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This study is supported by the National Natural Science Foundation of China (3216190133), the Veterinary Key Disciplines Construction Project (XK2024-02), and the Xizang Institute of Agriculture and Animal Husbandry Postgraduate Education Innovation Project (YJS2024-15).

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