Effect of Bacillus subtilis isolated from yaks on D-galactose-induced oxidative stress and hepatic damage in mice

Abstract

Acute hepatic injury is a severe condition that is always accompanied by oxidative stress and inflammation, seriously threatening the health of the host. Probiotics have been shown to be involved in the regulation of antioxidant system and gut microbiota activity, but studies on the effects of yak derived Bacillus subtilis (B. subtilis) on acute liver injury and oxidative stress remain scarce. Here, we aim to explore the ameliorative effects of B. subtilis isolated from yaks on oxidative stress and hepatic injury caused by D-galactose, as well as the underlying processes. Results indicated that B. subtilis administration, particularly the BS3, significantly mitigated hepatic damage induced by D-galactose in mice as evidenced by ameliorating liver tissue damage as well as decreasing ALT (p < 0.05) and AST (p < 0.05) levels. Additionally, the B. subtilis intervention was demonstrated to enhance the antioxidant system in D-galactose-exposed mice, as manifested by increased T-AOC and SOD, alongside a decrease in MDA levels (p < 0.05). Meanwhile, B. subtilis intervention could effectively mitigate oxidative damage via modulating the Keap1/Nrf2 signaling pathway. Importantly, B. subtilis exhibited a pronounced protective effect against D-galactose-induced intestinal barrier dysfunction through improving tight junction proteins. The gut microbiota results suggest that BS3 alters the abundance of some gut flora such as Firmicutes phylum and Oscillibacter and Lachnospiraceae_NK4A136 genera, which affects the composition of the gut microbiota and reverses the decrease in the microbial richness index in mice. In summary, these findings demonstrated that B. subtilis isolated from yaks serve as a promising candidate to ameliorate oxidative damage and hepatic injury. Meanwhile, the positive regulation effect of B. subtilis on gut microbiota and intestinal mucosal barrier may be one of its underlying mechanisms to alleviate oxidative stress and hepatic injury.

Keywords: Bacillus subtilis; Keap1/Nrf2 signaling pathway; gut microbiota; hepatic injury; oxidative stress; yak.

Figure 1

The intervention of B. subtilis significantly alleviated the negative effects induced by D-galactose. (A) Changes in body weight across seven groups. The organ indices for the liver, kidney, and spleen are depicted in B–D, respectively. (E,F) Represent the serum concentrations of ALT and AST, respectively. The serum antioxidant capacity, as illustrated in G–K, encompasses the levels of T-AOC, CAT, SOD, GSH-Px, and MDA. C: the control group, DG: the D-galactose-induced model group, VC: vitamin C treatment group, BSA: the BS3 supplement group, BSB: BS7 supplement group, TBSA: the BS3 intervention group (i.e., BS3 treatment + D-galactose), TBSB: the BS7 intervention group (i.e., BS7 treatment + D-galactose). The symbol # indicates comparison with the C group, while * represents comparison with the D-galactose induced oxidative injury group (DG group).

Figure 2

The histological morphology of the liver in groups C, VC, BSA, and BSB demonstrated a regular and healthy structure, while the DG group exhibited evident pathological changes, such as the red arrow indicating vacuolar degeneration. In contrast, the TBSA and TBSB group showed a significant alleviation of vacuolar degeneration. The scale bars are 100 μm and 50 μm, respectively.

Figure 3

The HE staining of colon tissue in different groups. The colon structure of groups C, BSA, and BSB appeared normal and healthy, without any obvious pathological changes. In contrast, the DG group exhibited significant pathological changes, such as the red arrow indicating inflammatory cell infiltration. However, the intervention of VC (VC), BS3 (TBSA), and BS7 (TBSB) resulted in a notable decrease in inflammatory cell infiltration. Scale bars: 100 μm or 50 μm.

Figure 4

(A–C) Western blot analysis of occludin and claudin-1 proteins in various experimental groups. (D–H) Western blot analysis of key proteins in the Keap1/Nrf2 signaling pathway, including KEAP1, NRF2, HO-1, and NQO1. The symbol # indicates comparison with the C group, while * represents comparison with the D-galactose induced oxidative injury group (DG group).

Figure 5

The impact of BS3 on alterations in microbial richness and composition in response to D-galactose exposure. (A) The OTUs count in each sample. (B) The Venn diagram displayed the distinct and overlapping operational taxonomic units (OTUs) among the five groups. (C) The rank abundance curve. (D) The rarefaction curve. (E–H) The alpha diversity analysis. (I,J) The PCoA analysis was conducted using the weight and unweighted UniFrac distance. The symbol # indicates comparison with the C group, while * represents comparison with the D-galactose induced oxidative injury group (DG group).

Figure 6

(A,B) The relative abundance of gut microbiota at the phylum and genus levels in five groups. (C) The heatmap displays the distribution of bacterial genera across five groups.

Figure 7

Statistical evaluation of differentially abundant bacterial taxa was conducted across five distinct groups, with specific focus on both the phylum (marked with ★) and genus levels. *p < 0.05 and **p < 0.01. The symbol # indicates comparison with the C group, while * represents comparison with the D-galactose induced oxidative injury group (DG group).

Figure 8

LEfSe analysis combined with LDA scores revealed the differential biomarkers among five groups. (A) The cladogram illustrates the phylogenetic distribution of diverse bacteria. (B) The linear discriminant analysis (LDA) scores, with thresholds exceeding 4, were indicated statistically significant differential bacteria.