Nucleotides and β-glucan alleviates lipopolysaccharide-induced inflammatory responses in intestinal fibroblasts of grass carp (Ctenopharyngodon idella) in vitro through inhibiting Toll/NF-κB signaling pathways.

Abstract

Intestinal inflammation severely restricts the healthy culture of grass carp. Although nucleotides (NT) and β-glucan (BG) exert immunoregulatory effects separately, their combined immunomodulatory mechanism in fish intestinal fibroblasts remains poorly understood. The present study aimed to investigate the effects of NT, BG and their combination on intestinal inflammatory responses and oxidative stress in lipopolysaccharide (LPS)-induced inflammatory models, and further evaluate their impacts on the Toll/NF-κB signaling pathway using grass carp (Ctenopharyngodon idella) intestinal fibroblasts. Fibroblasts were subjected to LPS-induced inflammation followed by individual or combined NT and BG supplementation. Results showed that NT and BG, either individually or in combination, significantly reduced the LPS-induced up-regulation of TNF-α and IL-6 gene expression and secretion, and reversed the LPS-induced reductions in SOD activity and T-AOC level, and inhibited the elevation of MDA content in intestinal fibroblasts (P < 0.05). Meanwhile, LPS stimulation significantly inhibited nrf2 expression but increased keap1 expression (P < 0.05). However, NT and/or BG significantly down-regulated LPS-induced keap1 expression (P < 0.05), yet failed to induce significant change in nrf2 levels (P > 0.05). Furthermore, NT and/or BG reversed LPS-regulated expression of multiple pro- and anti-inflammatory cytokines and suppressed excessive activation of the Toll/NF-κB signalling pathway. Notably, co-application of NT and BG produced combinatory protective benefits superior to single-agent treatments. Quantitative analysis showed that compared with NT-alone treatment, combined NT and BG treatment further improved the mitigation of inflammatory and oxidative-related indicators by 7.26% - 47.65%; compared with BG-alone treatment, the combined treatment yielded an additional improvement of 5.38% - 38.95% for these biomarkers. Collectively, these findings verify that NT and BG alleviate LPS-induced inflammatory injury in grass carp intestinal fibroblasts via restraining Toll/NF-κB signaling, and their combined treatment achieves more prominent protective effects, offering theoretical support for developing compound anti-inflammatory diet additives in aquaculture.