Alfalfa Flavonoids Mitigate Salmonella-Induced Colitis via the Keap1-Nrf2 and TLR4/NF-κB/COX-2 Pathways

Abstract

Alfalfa is rich in flavonoid compounds, which are known for their antioxidative and anti-inflammatory properties, suggesting therapeutic potential for alfalfa flavonoids (AF) in inflammation-related diseases. This study investigated the effects of AF on Salmonella-induced colitis, a severe inflammatory bowel disorder characterized by oxidative damage and inflammatory response. In vitro, antioxidant assays revealed AF's concentration-dependent radical scavenging, significantly reducing electron paramagnetic resonance (EPR) signals for HO^• and O₂^•− by 42% and 54%, respectively. In vivo, AF treatment significantly mitigated body weight (BW) loss by 6%, increased colon length by 11%, and reduced liver and spleen weights by 19% and 81%, respectively, compared to the colitis group. Mechanistically, AF suppressed inflammation by downregulating the Toll-like receptor 4 (TLR4)/IκB/nuclear factor-κB (NF-κB)/cyclooxygenase-2 (COX-2) pathway and inhibiting nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome activation, thereby lowering levels of pro-inflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin 6 [IL-6], interleukin 1 beta [IL-1β]). Concurrently, AF enhanced antioxidant defense via the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, reducing reactive oxygen species (ROS) and malondialdehyde (MDA) levels while increasing catalase (CAT) (69%), glutathione peroxidase (GPx) (83%), and superoxide dismutase (SOD) (21%) activities. Moreover, AF preserved epithelial and mucosal barriers by reducing apoptosis and upregulating tight junction proteins (Claudin1, ZO-1, E-cadherin) and goblet cell marker Ulex europaeus (Gorse) Agglutinin I (UEA-1). Microbiota analysis revealed that AF significantly enriched beneficial bacteria, including Akkermansia, Oscillibacter, and butyrate-producing taxa, thereby counteracting Salmonella-induced dysbiosis. Furthermore, AF restored the disrupted profile of short-chain fatty acids (SCFAs), strengthening the relationship between symbiotic microbiota and mucosal defense. Overall, AF exerted multifaced protection against Salmonella-induced colitis by alleviating oxidative stress, stabilizing intestinal homeostasis, and thus attenuating inflammation. These findings make AF a promising phytopharmaceutical for the prevention and treatment of inflammatory diseases.