Hydrogen-Rich Water Mitigates LPS-Induced Chronic Intestinal Inflammatory Response in Rats via Nrf-2 and NF-κB Signaling Pathways

Abstract

Long-term exposure to low-dose lipopolysaccharide can impair intestinal barriers, causing intestinal inflammation and leading to systemic inflammation. Hydrogen-rich water possesses antioxidant and anti-inflammatory functions and exerts inhibitory effects on various inflammatory diseases. In this study, we investigated whether oral hydrogen-rich water could prevent lipopolysaccharide-induced chronic intestinal inflammation. An experimental model was established by feeding hydrogen-rich water, followed by the injection of lipopolysaccharide (200 μg/kg) in the tail vein of rats after seven months. ELISA, Western blot, immunohistochemistry, and other methods were used to detect related cytokines, proteins related to the NF-κB and Nrf-2 signaling pathways, and tight-junction proteins to study the anti-inflammatory and antioxidant effects of hydrogen-rich water. The obtained results show that hydrogen-rich water significantly increased the levels of superoxide dismutase and structural proteins; activated the Nrf-2 signaling pathway; downregulated the expression of inflammatory factors cyclooxygenase-2, myeloperoxidase, and ROS; and decreased the activation of the NF-κB signaling pathway. These results suggest that hydrogen-rich water could protect against chronic intestinal inflammation in rats caused by lipopolysaccharide-induced activation of the NF-κB signaling pathway by regulating the Nrf-2 signaling pathway.

Keywords: LPS; NF-κB pathway; Nrf-2 pathway; gut inflammation; hydrogen-rich water; oxidative stress.

Figure 1

Effect of HRW on gut permeability. (A) Histopathological sections of rat ileum (150×). The black arrow indicates the villous state of the small intestine. (B) Immunohistochemical and quantitative analysis of ZO-1, occludin, and claudin-1 in ileal tissue. (C) OVA levels in the ileal tissue. # p < 0.05; **, ## p < 0.01.

Figure 2

Effects of HRW on inflammatory cytokines in rats with chronic gut inflammation. (A) Serum levels of IL-1β, IL-6, TNF-α, and MCP-1. (B) Levels of IL-1β, IL-6, TNF-α, and MCP-1 in ileal tissue. (C) mRNA expression and activity of IL-1β, IL-6, TNF-α, and MCP-1. (D) Level of COX-2 in ileal tissue. *, # p < 0.05; **, ## p < 0.01.

Figure 3

Effects of HRW on macrophage infiltration in rats with chronic gut inflammation. (A) Immunohistochemical analysis of macrophages in ileal tissue (200×). (B) Quantitative analysis of CD68 in ileum tissue. **, ## p < 0.01.

Figure 4

Effects of HRW on the NF-κB signaling pathway in rats with chronic gut inflammation. (A) Blot bands of protein expression related to the NF-κB signaling pathway. Protein expression of the NFκB signaling pathway was detected by Western blotting: (B) TLR4; (C) MyD88; (D) IκBα and p-IκBα; (E) p65 and p-p65 and their ratios; (F) p65 and p-p65 and their ratios. # p < 0.05; **, ## p < 0.01.

Figure 5

Effects of HRW on oxidative stress-related indicators in rats with chronic inflammation. (A) Immunofluorescence analysis and quantification of ROS in ileal tissue (200×). (B) Levels of MPO in ileal tissue. (C) Levels of SOD in ileal tissue. (D) Levels of MDA in ileal tissue. * p < 0.05; **, ## p < 0.01.

Figure 6

Effects of HRW on the Nrf-2 signaling pathway in rats with chronic gut inflammation. (A) Blot bands of protein expression related to the Nrf-2 signaling pathway. Protein levels of the Nrf-2 signaling pathway were detected by Western blotting: (B) Nrf-2; (C) HO-1; (D) NQO-1. * p < 0.05, ** p < 0.01 compared to the CON group; # p < 0.05, ## p < 0.01 compared to the LPS group. *, # p < 0.05; **, ## p < 0.01. Please view the original image in the File S1.