Citric Acid Promotes Immune Function by Modulating the Intestinal Barrier

Abstract

Amidst increasing concern about antibiotic resistance resulting from the overuse of antibiotics, there is a growing interest in exploring alternative agents. One such agent is citric acid, an organic compound commonly used for various applications. Our research findings indicate that the inclusion of citric acid can have several beneficial effects on the tight junctions found in the mouse intestine. Firstly, the study suggests that citric acid may contribute to weight gain by stimulating the growth of intestinal epithelial cells (IE-6). Citric acid enhances the small intestinal villus-crypt ratio in mice, thereby promoting intestinal structural morphology. Additionally, citric acid has been found to increase the population of beneficial intestinal microorganisms, including Bifidobacterium and Lactobacillus. It also promotes the expression of important protein genes such as occludin, ZO-1, and claudin-1, which play crucial roles in maintaining the integrity of the tight junction barrier in the intestines. Furthermore, in infected IEC-6 cells with H9N2 avian influenza virus, citric acid augmented the expression of genes closely associated with the influenza virus infection. Moreover, it reduces the inflammatory response caused by the viral infection and thwarted influenza virus replication. These findings suggest that citric acid fortifies the intestinal tight junction barrier, inhibits the replication of influenza viruses targeting the intestinal tract, and boosts intestinal immune function.

Keywords: H9N2 influenza virus; citric acid; gut microorganisms; intestinal barrier; metabolomics.

Figure 1

CA promotes body weight and affects IEC-6 cell growth and cycle in mice. (A): Mouse body weight growth curve at 14 days; (B): IEC-6 cell growth curve at 48 h. (OD value on the vertical axis); (C): Significant changes in cells after 24 h at each cell concentration; (D): Analysis of G1 and G2 phases in IEC-6 cells using Beckman Coulter Ultra-High-Speed Flow Sorting System; (E): Relative expression of cell numbers in G1 phase; (F): Relative expression of cell numbers in G2 phase. * Significant difference (p < 0.05), ** highly significant difference (p < 0.01).

Figure 2

HE-stained image of small intestine (200×). (A): 0 g/L; (B): 0.05 g/L; (C): 0.1 g/L; (D): 0.15 g/L; (E): 0.2 g/L. Scale bar = 50 μm.

Figure 3

Citric acid promotes expression of intestinal tight junction genes in mice. (A) RT-PCR for detection of relative mRNA expression of mouse intestinal tight junction genes occludin, ZO-1, and claudin-1; (B–D) RT-qPCR for detection of relative mRNA expression of mouse intestinal tight junction genes occludin, ZO-1, and claudin-1; (E) RT-PCR to detect the relative expression levels of mRNA of the tight junction genes occludin, ZO-1, and claudin-1 in IEC-6 cells. (F–H) RT-qPCR to detect the relative expression levels of mRNA of the tight junction genes occludin, ZO-1, and claudin-1 in IEC-6 cells. * Significant difference (p < 0.05), ** highly significant difference (p < 0.01).

Figure 4

Multivariate statistical analysis. (A) Peca score, (B) PLS-DA score, (C) 200 displacement tests, and (D) S-plot test.

Figure 5

Analysis of significantly different metabolites in serum. (A) Heat maps of significantly different metabolite clusters, (B) significantly up-regulated metabolite enrichment maps, (A: linoleic acid metabolism B: taurine and subtaurine metabolism; C: arachidonic acid metabolism; the abscissa represents the pathway impact value calculated by path topology analysis, the ordinate −log10 (P) represents the significance level of metabolic pathway enrichment analysis, and the circle size represents the ratio of pathway impact to −log10 (P). The larger the ratio, the larger the circle diameter.). (C) KEGG pathways for taurine and hypotaurine, (D) KEGG pathways for arachidonic acid, and (E) KEGG pathways for cysteine and methionine metabolism.

Figure 6

H9N2 AIV infection induces mRNA expression levels in the IEC-6 inflammatory response. (A) Relative mRNA expression levels of TNF-α, IL-6, IFN-β, ISG15, and viral NP detected by RT-PCR; (B–F) Relative mRNA expression levels of TNF-α, IL-6, IFN-β, ISG15, and viral NP detected by RT-qPCR. * Significant difference (p < 0.05), ** highly significant difference (p < 0.01).

Figure 7

CA promotes expression of tight junction genes in IAV infection to attenuate inflammatory response and inhibit viral replication. (A) Relative mRNA expression levels of occludin detected by RT-qPCR and RT-PCR; (B) Relative mRNA expression levels of ZO-1 detected by RT-qPCR and RT-PCR; (C) Relative mRNA expression levels of claudin-1 detected by RT-qPCR and RT-PCR; (D) RT-qPCR and RT-PCR to detect the relative mRNA expression level of IL-6; (E) RT-qPCR and RT-PCR to detect the relative mRNA expression level of TNF-α; (F) RT-qPCR and RT-PCR to detect the relative mRNA expression level of IFN-β; (G) RT-qPCR and RT-PCR to detect the relative mRNA expression level of ISG15; and (H) RT-qPCR and RT-PCR to detect the relative mRNA expression level of NP. * Significant difference (p < 0.05), ** highly significant difference (p < 0.01).