Combined Supplementation of Clostridium butyricum and Bifidobacterium infantis Diminishes Chronic Unpredictable Mild Stress-Induced Intestinal Alterations via Activation of Nrf-2 Signaling Pathway in Rats

Abstract

Introduction

Chronic stress is a significant factor contributing to various gastrointestinal disorders, including irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) . The gut-brain axis, a bidirectional communication system between the central nervous system and the gastrointestinal tract, plays a crucial role in stress-induced gut alterations .

Chronic unpredictable mild stress (CUMS) is a well-established animal model that mimics human depression and anxiety-related disorders, often accompanied by gastrointestinal symptoms . CUMS exposure leads to intestinal barrier dysfunction, increased permeability, and low-grade inflammation, which are hallmark features of stress-related gut disorders .

The nuclear factor erythroid 2-related factor 2 (Nrf-2) signaling pathway is a critical cellular defense mechanism against oxidative stress . Under physiological conditions, Nrf-2 is sequestered in the cytoplasm by its inhibitor, Keap1. Upon oxidative stress, Nrf-2 dissociates from Keap1, translocates to the nucleus, and activates the transcription of antioxidant response element (ARE)-dependent genes, including heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1) .

Probiotics, defined as live microorganisms that confer health benefits when administered in adequate amounts, have shown promise in ameliorating stress-induced gastrointestinal alterations . Clostridium butyricum, a butyrate-producing bacterium, and Bifidobacterium infantis, a common gut commensal, have demonstrated anti-inflammatory and antioxidant properties in previous studies .

This study aimed to investigate whether combined supplementation of C. butyricum and B. infantis could alleviate CUMS-induced intestinal alterations through activation of the Nrf-2 signaling pathway in rats.

Methods

Results

Effects on Intestinal Permeability and Barrier Function

CUMS exposure significantly increased intestinal permeability, as evidenced by elevated serum FITC-dextran levels (P < 0.01). Probiotic supplementation significantly reduced intestinal permeability compared to the CUMS group (P < 0.05) .

Western blot analysis revealed that CUMS exposure decreased the expression of tight junction proteins occludin and ZO-1 (P < 0.01). Probiotic treatment restored the expression of these proteins to near-normal levels (P < 0.05) .

Effects on Inflammatory Cytokines

CUMS exposure significantly increased the levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in colonic tissues (P < 0.01). Probiotic supplementation markedly reduced these cytokine levels (P < 0.05) .

Effects on Oxidative Stress Markers

CUMS exposure increased malondialdehyde (MDA) levels and decreased superoxide dismutase (SOD) activity and glutathione (GSH) content (P < 0.01). Probiotic treatment reversed these changes, reducing MDA levels and increasing SOD activity and GSH content (P < 0.05) .

Activation of Nrf-2 Signaling Pathway

CUMS exposure decreased nuclear Nrf-2 protein expression and its downstream targets HO-1 and NQO1 (P < 0.01). Probiotic supplementation significantly increased the expression of these proteins (P < 0.05), indicating activation of the Nrf-2 signaling pathway .

Discussion

This study demonstrates that combined supplementation of C. butyricum and B. infantis ameliorates CUMS-induced intestinal alterations in rats. The protective effects are associated with reduced intestinal permeability, decreased inflammation, attenuated oxidative stress, and activation of the Nrf-2 signaling pathway.

The gut barrier dysfunction observed in CUMS-exposed rats is consistent with previous studies showing that stress disrupts tight junction proteins, leading to increased intestinal permeability . Our findings that probiotic treatment restored the expression of occludin and ZO-1 suggest that C. butyricum and B. infantis can strengthen the intestinal barrier, potentially through their effects on tight junction protein expression and distribution.

The anti-inflammatory effects of the probiotic combination are evidenced by the reduced levels of TNF-α, IL-1β, and IL-6 in colonic tissues. These cytokines are key mediators of inflammation in the gut and are elevated in various gastrointestinal disorders . The ability of C. butyricum and B. infantis to suppress these pro-inflammatory cytokines suggests their potential therapeutic value in inflammatory gut conditions.

Oxidative stress plays a crucial role in the pathogenesis of stress-induced intestinal damage. Our results show that probiotic treatment reduced lipid peroxidation (as indicated by decreased MDA levels) and enhanced antioxidant defense (as shown by increased SOD activity and GSH content). These findings are consistent with previous reports on the antioxidant properties of probiotics .

The most significant finding of this study is the activation of the Nrf-2 signaling pathway by probiotic supplementation. Nrf-2 is a master regulator of cellular defense against oxidative stress, and its activation leads to the expression of various antioxidant and detoxifying enzymes . Our results demonstrate that C. butyricum and B. infantis increased nuclear translocation of Nrf-2 and upregulation of its downstream targets HO-1 and NQO1. This suggests that the Nrf-2 pathway is a key mechanism through which these probiotics exert their protective effects against CUMS-induced intestinal alterations.

The combination of C. butyricum and B. infantis may have synergistic effects. C. butyricum produces butyrate, a short-chain fatty acid with known anti-inflammatory and antioxidant properties, while B. infantis has been shown to modulate immune responses and improve gut barrier function . Further studies are needed to elucidate the specific contributions of each strain and their potential synergistic interactions.

Conclusion

In conclusion, combined supplementation of C. butyricum and B. infantis ameliorates CUMS-induced intestinal alterations by improving intestinal barrier function, reducing inflammation, attenuating oxidative stress, and activating the Nrf-2 signaling pathway in rats. These findings suggest that this probiotic combination may represent a promising therapeutic approach for stress-related gastrointestinal disorders.

References