Gallic Acid Alleviates Gut Dysfunction and Boosts Immune and Antioxidant Activities in Puppies Under Environmental Stress Based on Microbiome-Metabolomics Analysis

Abstract

Early-life exposure to environmental stress disrupts the gut barrier and leads to inflammatory responses and changes in gut microbiota composition. Gallic acid (GA), a natural plant polyphenol, has received significant interest for its antioxidant, anti-inflammatory, and antimicrobial properties that support the maintenance of intestinal health. To assess whether dietary supplementation of GA alleviates environmental stress, a total of 19 puppies were randomly allocated to the following three dietary treatments for 2 weeks: 1) basal diet (control (CON)); 2) basal diet + transportation (TS); and 3) basal diet with the addition of 500 mg/kg of GA + transportation (TS+GA). After a 1-week supplementation period, puppies in the TS and TS+GA groups were transported from a stressful environment to another livable location, and puppies in the CON group were then left in the stressful environment. Results indicated that GA markedly reduced the diarrhea rate in puppies throughout the trial period and caused a moderate decline of serum cortisol and HSP-70 levels after transportation. Also, GA alleviated the oxidative stress and inflammatory response caused by multiple environmental stressors. Meanwhile, puppies fed GA had a higher abundance of fecal Firmicutes and Lactobacillus and lower Proteobacteria, Escherichia-Shigella, and Clostridium_sensu_stricto_1 after transportation. As a result, the TS+GA group had the highest total short-chain fatty acids and acetic acid. Also, the fecal and serum metabolomics analyses revealed that GA markedly reversed the abnormalities of amino acid metabolism, lipid metabolism, carbohydrate metabolism, and nucleotide metabolism caused by stresses. Finally, Spearman's correlation analysis was carried out to explore the comprehensive microbiota and metabolite relationships. Overall, dietary supplementation of GA alleviates oxidative stress and inflammatory response in stressed puppies by causing beneficial shifts on gut microbiota and metabolites that may support gut and host health.

Keywords: antioxidant; environmental stress; gallic acid; inflammatory response; metabolomics; microbiome; puppy.

Figure 1

Schematic representation of the study design. BT7, the 7th day before transportation; BT1, the 1st day before transportation; AT1, the 1st day after transportation; AT7, the 7th day after transportation. The CON group was fed basal diet with no transportation (n = 6), the TS group was fed basal diet with transportation (n = 6), and the TS+GA group was fed basal diet+500 mg/kg of gallic acid (GA) with transportation (n = 7).

Figure 2

Effect of gallic acid (GA) on fecal score (FS) (A, B), serum hormone (C–E), HSP-70 (F), antioxidant capacity (G–J), and inflammatory factors (K–N) in puppies (n = 6 or 7). The symbol (*) indicates statistically significant differences between two groups (*p < 0.05, **p < 0.01, and ***p < 0.001), and the symbol (#) represents difference tendency (#p < 0.10). BT1, the 1st day before transportation; AT1, the 1st day after transportation; AT7, the 7th day after transportation. TDR (total diarrhea rate, %) = [cases of diarrhea during 14 days/(14 days × total puppies for each group)] × 100. COR, cortisol; ACTH, adrenocorticotropic hormone; GC, glucocorticoid; HSP-70, heat stress protein 70; GSH-Px, glutathione peroxidase; MDA, malondialdehyde; T-AOC, total antioxidant capacity; SOD, superoxide dismutase; IgG, immunoglobulin G; TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ; IL-4, interleukin 4.

Figure 3

Effect of gallic acid (GA) on gut microbial composition and structure in puppies (n = 6 or 7). Alpha diversity (A), principal coordinate analysis (PCoA) based on weighted UniFrac distances (B), predominant fecal microbial communities, and different bacteria at the phylum, family, and genus levels (C). The symbol (*) indicates statistically significant differences between two groups (*p < 0.05 and **p < 0.01), and the symbol (#) represents difference tendency (#p < 0.10). BT1, the 1st day before transportation; AT1, the 1st day after transportation; AT7, the 7th day after transportation.

Figure 4

The linear discriminant analysis effect size (LEfSe) analysis identified gut bacterial biomarkers in puppies on BT1 and AT1 (A). Spearman’s correlation network of fecal microbiota at genus level on AT1 (purple solid line, positive correlation; gray dotted line, negative correlation; thick line, significant correlation, p < 0.05) (B). BT1, the 1st day before transportation; AT1, the 1st day after transportation.

Figure 5

Effect of gallic acid (GA) on fecal short-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs) in puppies on BT1 (A), AT1 (B), and AT7 (C) (n = 6 or 7). The symbol (*) indicates statistically significant differences between two groups (*p < 0.05), and the symbol (#) represents difference tendency (#p < 0.10). BT1, the 1st day before transportation; AT1, the 1st day after transportation; AT7, the 7th day after transportation.

Figure 6

Multivariate statistical analysis on BT1, AT1, and AT7 (n = 6 or 7). Score plots from the principal component analysis (PCA) model among three groups on BT1, AT1, and AT7 (A). Score plots from the orthogonal partial least-squares discriminant analysis (OPLS-DA) model among three groups on BT1, AT1, and AT7 (B). BT1, the 1st day before transportation; AT1, the 1st day after transportation; AT7, the 7th day after transportation.

Figure 7

Bar charts of the metabolic pathway analysis of differential fecal metabolites on BT1 (A), AT1 (B), and AT7 (C) (n = 6 or 7). The pathway enrichment analysis shows all matched pathways, and the green boxes indicate significant metabolic pathways (p < 0.05). BT1, the 1st day before transportation; AT1, the 1st day after transportation; AT7, the 7th day after transportation.

Figure 8

Multivariate statistical analysis on BT1, AT1, and AT7 (n = 6 or 7). Score plots from the principal component analysis (PCA) model among three groups on BT1, AT1, and AT7 (A). Score plots from the orthogonal partial least-squares discriminant analysis (OPLS-DA) model among three groups on BT1, AT1, and AT7 (B). BT1, the 1st day before transportation; AT1, the 1st day after transportation; AT7, the 7th day after transportation.

Figure 9

Bar charts of the metabolic pathway analysis of differential serum metabolites on BT1 (A), AT1 (B), and AT7 (C) (n = 6 or 7). The pathway enrichment analysis shows all matched pathways, and the green boxes indicate significant metabolic pathways (p < 0.05). BT1, the 1st day before transportation; AT1, the 1st day after transportation; AT7, the 7th day after transportation.

Figure 10

Spearman’s correlation analysis between the differential feces metabolites and fecal microbiota (A), and the differential serum metabolites and fecal microbiota (B) on AT1 and AT7. The symbol (*) indicates a significant association between metabolite and microbiota (*P < 0.05, **P < 0.01, and ***P < 0.001). Red color indicates a positive correlation, and blue color indicates a negative correlation. AT1, the 1st day after transportation; AT7, the 7th day after transportation.