Uncovering the mechanism of Clostridium butyricum CBX 2021 to improve pig health based on in vivo and in vitro studies

Abstract

Introduction: As a symbiotic probiotic for the host, Clostridium butyricum (CB) has the potential to strengthen the body's immune system and improve intestinal health. However, the probiotic mechanism of CB is not completely understood. The Clostridium butyricum CBX 2021 strain isolated by our team from a health pig independently exhibits strong butyric acid production ability and stress resistance. Therefore, this study comprehensively investigated the efficacy of CBX 2021 in pigs and its mechanism of improving pig health.

Methods: In this study, we systematically revealed the probiotic effect and potential mechanism of the strain by using various methods such as microbiome, metabolites and transcriptome through animal experiments in vivo and cell experiments in vitro.

Results: Our in vivo study showed that CBX 2021 improved growth indicators such as daily weight gain in weaned piglets and also reduced diarrhea rates. Meanwhile, CBX 2021 significantly increased immunoglobulin levels in piglets, reduced contents of inflammatory factors and improved the intestinal barrier. Subsequently, 16S rRNA sequencing showed that CBX 2021 treatment implanted more butyric acid-producing bacteria (such as Faecalibacterium) in piglets and reduced the number of potentially pathogenic bacteria (like Rikenellaceae RC9_gut_group). With significant changes in the microbial community, CBX 2021 improved tryptophan metabolism and several alkaloids synthesis in piglets. Further in vitro experiments showed that CBX 2021 adhesion directly promoted the proliferation of a porcine intestinal epithelial cell line (IPEC-J2). Moreover, transcriptome analysis revealed that bacterial adhesion increased the expression of intracellular G protein-coupled receptors, inhibited the Notch signaling pathway, and led to a decrease in intracellular pro-inflammatory molecules.

Discussion: These results suggest that CBX 2021 may accelerate piglet growth by optimizing the intestinal microbiota, improving metabolic function and enhancing intestinal health.

Keywords: Clostridium butyricum CBX 2021; IPEC-J2 cells; intestinal health; intestinal microbiota; weaned piglets.

Figure 1

Effects of Clostridium butyricum CBX 2021 on fecal microbiota in weaned piglets (n = 6). (A) Principal coordinate analysis (PCoA) scatter diagram based on genus level. Column chart of species composition at the phylum (B) and genus (C) level. (D) Histogram of bacterial differences at the phylum and genus level. (E) Linear discriminant analysis (LDA) histogram of differential microbial community in the CON and CB group. Data are displayed as the mean ± SEM. *** Indicates p < 0.001, ** indicates p < 0.01, * indicates p < 0.05, and 0.05 ≥ p > 0.10 indicates a significant trend in comparison with the CON piglets.

Figure 2

Effects of Clostridium butyricum CBX 2021 on serum metabolome in weaned piglets (n = 6). Orthogonal partial least squares discriminant analysis (OPLS-DA) score chart under positive (A) and negative (B) ion modes. Permutation test chart under positive (C) and negative (D) ion modes. Difference statistics volcano plot under positive (E) and negative (F) ion modes. (G) KEGG enrichment pathway diagram of different metabolites in two groups of piglets. (H) Histogram of the differential metabolites (L-tryptophan and tryptamine) that cause KEGG enrichment. Data are displayed as the mean ± SEM. ** Indicates p < 0.01 in comparison with the CON piglets.

Figure 3

Correlation of microbiota, metabolites, and blood biochemical indicators (n = 6). (A) Heatmap of correlations between the top 20 bacterial genera and serum differential metabolites; The abscissa is the fecal bacteria with relative abundance of top 20; The ordinate is 12 serum differential metabolites. (B) Heatmap of correlations between serum differential metabolites and blood indicators; The abscissa is 12 serum differential metabolites; The ordinate is the blood biochemical indicators (immunoglobulin A/G/M, ghrelin, interleukin-10, growth hormone, gastrin, tumor necrosis factor-α, Glucagon-like peptide-1, diamine oxidase, interleukin-1β, peptide YY, D-lactic acid).

Figure 4

Effects of Clostridium butyricum CBX 2021 adhesion on the proliferation and inflammatory factors in IPEC-J2 cells (n = 25 or 3). (A) Gram staining microscopy of IPEC-J2 cells (scale bar = 20 μm). (B) Adhesion quantity of the CBX 2021 strain cocultured with IPEC-J2 cells at different time. (C) Line chart of cell proliferation activity. (D) Histogram showing the difference in inflammatory cytokine levels in the cell supernatant between two groups. Data are displayed as the mean ± SEM. *** Indicates p < 0.001, ** indicates p < 0.01, and * indicates p < 0.05 in comparison with the CON group.

Figure 5

Effects of Clostridium butyricum CBX 2021 adhesion on the transcriptomics in IPEC-J2 cells (n = 5). (A) Principal component analysis (PCA) plot between the CON and CB RNA-seq samples. (B) The volcano plot displays differentially expressed genes (DEGs) from CON vs. CB. Bubble diagram of GO (C) and KEGG (D) enrichment analysis of differentially expressed genes.

Figure 6

Gene set enrichment analysis (GSEA) pathway analysis and qRT-PCR validation (A–F) GSEA signal pathway analysis. (G) Histograms of target gene expression levels showed by qRT-PCR. Data are displayed as the mean ± SEM. *** Indicates p < 0.001 and ** indicates p < 0.01 in comparison with the CON group.