Protective effect of zinc gluconate on intestinal mucosal barrier injury in antibiotics and LPS-induced mice

Abstract

Objective: The aim of the study is to investigate the function and mechanism of Zinc Gluconate (ZG) on intestinal mucosal barrier damage in antibiotics and Lipopolysaccharide (LPS)-induced mice.

Methods: We established a composite mouse model by inducing intestinal mucosal barrier damage using antibiotics and LPS. The animals were divided into five groups: Control (normal and model) and experimental (low, medium, and high-dose ZG treatments). We evaluated the intestinal mucosal barrier using various methods, including monitoring body weight and fecal changes, assessing pathological damage and ultrastructure of the mouse ileum, analyzing expression levels of tight junction (TJ)-related proteins and genes, confirming the TLR4/NF-κB signaling pathway, and examining the structure of the intestinal flora.

Results: In mice, the dual induction of antibiotics and LPS led to weight loss, fecal abnormalities, disruption of ileocecal mucosal structure, increased intestinal barrier permeability, and disorganization of the microbiota structure. ZG restored body weight, alleviated diarrheal symptoms and pathological damage, and maintained the structural integrity of intestinal epithelial cells (IECs). Additionally, ZG reduced intestinal mucosal permeability by upregulating TJ-associated proteins (ZO-1, Occludin, Claudin-1, and JAM-A) and downregulating MLCK, thereby repairing intestinal mucosal barrier damage induced by dual induction of antibiotics and LPS. Moreover, ZG suppressed the TLR4/NF-κB signaling pathway, demonstrating anti-inflammatory properties and preserving barrier integrity. Furthermore, ZG restored gut microbiota diversity and richness, evidenced by increased Shannon and Observed features indices, and decreased Simpson's index. ZG also modulated the relative abundance of beneficial human gut bacteria (Bacteroidetes, Firmicutes, Verrucomicrobia, Parabacteroides, Lactobacillus, and Akkermansia) and harmful bacteria (Proteobacteria and Enterobacter), repairing the damage induced by dual administration of antibiotics and LPS.

Conclusion: ZG attenuates the dual induction of antibiotics and LPS-induced intestinal barrier damage and also protects the intestinal barrier function in mice.

Keywords: 16S rRNA; LPS; TLR4/NF-κB; antibiotics; gut microbiome; intestinal mucosal barrier; tight junction; zinc gluconate.

Figure 1

Experimental design protocol and changes in feces and body weight of mice during the experiment. (A) Animal experiment protocol and design. (B) Changes in feces of mice: (a) normal stool (b) Wet stools (c) Pasty stools (d) Semiliquid stools (e) Watery diarrhea. (C) Changes in body weight of mice. Compared with group NC: ^*p < 0.05, ^****p < 0.0001; Compared with group NS + ABX/LPS: ^##p < 0.01, ^###p < 0.001.

Figure 2

Representative images of histopathological and morphological changes in the ileum of each group of mice (HE staining, A,B: ×200, C,D: ×400). The detachment of intestinal villous epithelium is indicated by yellow arrows, while separation from the lamina propria and widening of the interstitial space are denoted by red arrows. Cytoplasmic laxity of intestinal villous epithelial cells is highlighted by blue arrows, granulocytic infiltration by green arrows, and disruption of the muscularis propria structure by black arrows. (E) Villus length (F) crypt depth (G) VCR. Compared with group NC: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001; Compared with group NS + ABX/LPS: ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001.

Figure 3

Representative images of ultrastructural changes of ileum in mice of each group (×8,000). Mv, Microvilli; TJ, Tight junction; ZA, Zonula Adherens; De, Desmosome; N, Nuclear; M, Mitochondria; Ly, Lysosome; ASS, Autolysosome; AP, Autophagosome; RER, Rough endoplasmic reticulum.

Figure 4

ZO-1 mRNA and protein relative expression levels in the ileum of mice in each group. Compared with group NC: ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001; Compared with group NS + ABX/LPS: ^#p < 0.05, ^###p < 0.001,^####p < 0.0001. (A-C) The protein expression of tight junction protein (ZO-1) of ileum tissue in each group. (B) The mRNA expression of tight junction protein (ZO-1) of ileum tissues in each group.

Figure 5

Relative expression levels of Occludin mRNA and protein in the ileum of mice in each experimental group. Compared with group NC: ^**p < 0.01, ^****p < 0.0001; Compared with group NS + ABX/LPS: ^#p < 0.05, ^##p < 0.01,^####p < 0.0001. (A-C) The protein expression of tight junction protein (Occludin) of ileum tissue in each group. (B) The mRNA expression of tight junction protein (Occludin) of ileum tissues in each group.

Figure 6

Claudin-1 mRNA and protein relative expression levels in the ileum of mice in each group. Compared with group NC: ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001; Compared with group NS + ABX/LPS: ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001.

Figure 7

Representative images of JAM-A immunohistochemical staining of mouse ileum tissue (A: ×200, B: ×400). JAM-A mRNA (C) and protein (D) relative expression levels in the ileum of mice in each group. Compared with group NC:^**p < 0.01, ^***p < 0.001, ^****p < 0.0001; Compared with group NS + ABX/LPS: ^##p < 0.01, ^####p < 0.0001.

Figure 8

Representative images of MLCK immunohistochemical staining of mouse ileum tissue (A: ×200, B: ×400). MLCK mRNA (C) and protein (D) relative expression levels in the ileum of mice in each group. Compared with group NC: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001; Compared with group NS + ABX/LPS: ^#p < 0.05, ^##p < 0.01, ^####p < 0.0001.

Figure 9

Comparison of variations in the relative expression levels of TLR4 and NF-κB protein in ileum tissues of mice in various groups. Compared with group NC: ^*p < 0.05, ^****p < 0.0001; Compared with group NS + ABX/LPS: ^#p < 0.05,^##p < 0.01,^####p < 0.0001. (A-B) The protein expression of TLR4/NF-κB signaling pathway protein (TLR4) of ileum tissue in each group. (C-D) The protein expression of TLR4/NF-κB signaling pathway protein (NF-κB/p65) of ileum tissue in each group.

Figure 10

Changes of DAO, D-LA and ET levels in serum of mice in each group. Compared with group NC: ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001; Compared with group NS + ABX/LPS: ^#p < 0.05,^##p < 0.01,^####p < 0.0001. (A-C) Concentration of DAO, D-LA and ET in mouse serum. They are the main biomarkers of intestinal mucosal permeability and indirectly reflect intestinal mucosal barrier damage.

Figure 11

Diversity and composition of gut microbiota. (A,B) Species diversity rarefaction curve. (C–E) Alpha diversity was evaluated by the Shannon, Simpson, and Observed features index. Weighted UniFrac analysis was utilized to distinguish bacterial clustering. (F) PCoA, (G) NMDS. Compared with group NC: ^*p < 0.05, ^**p < 0.01, ^****p < 0.0001; Compared with group NS + ABX/LPS: ^##p < 0.01, ^####p < 0.0001.

Figure 12

The distribution of cecal content microflora in each group of mice categorized at the phylum level. (A) Histogram of relative abundance ratios. (B) Heat map of absolute abundance clustering. (C–F) Histogram of relative abundance of the dominant bacterial phylum. Compared with group NC: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001; Compared with group NS + ABX/LPS: ^##p < 0.01, ^###p < 0.001,^####p < 0.0001.

Figure 13

The distribution of cecal content microflora in each group of mice classified at the genus level. (A) Histogram of relative abundance ratios. (B) Heat map of absolute abundance clustering. (C–H) Histogram of relative abundance of dominant bacterial genera. Compared with group NC: ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001; Compared with group NS + ABX/LPS: ^#p < 0.05,^##p < 0.01, ^###p < 0.001,^####p < 0.0001.

Figure 14

LEfSe analysis of the microflora of the mouse cecum. (A) Cladogram. (B) Histogram. LDA coefficient cutoff values of 2.