Millet Bran Bound Phenolic Compounds Suppresses LPS-Induced Inflammatory Response in Macrophages and Liver Injury Mice via TLR4/NF-κB Signaling Pathway

Millet bran, rich in bioactive phenolic compounds, holds potential for both nutritional and therapeutic applications. In this study, bound phenolic compounds were isolated from millet bran, yielding a potent fraction named BPS-2. UPLC-MS/MS detected 16 major phenolic compounds in BPS-2. In vitro assays revealed that BPS-2 exerted a significant anti-inflammatory activity in lipopolysaccharide (LPS)-induced RAW 264.7 macrophage, as manifested by reduced production of inflammatory mediators (IL-1β, IL-6, and TNF-α) and downregulation of the expression levels of the pro-inflammatory enzymes Cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS). Network pharmacological analysis identified the suppression of the TLR4/NF-κB pathway as the primary mechanism mediating the anti-inflammatory activity of BPS-2, which was validated using the LPS-induced RAW 264.7 macrophage model and liver injury mice model. Western blot analysis revealed that BPS-2 significantly decreased the phosphorylation of IκBα and p65 to regulate the TLR4/NF-κB signaling pathway, thereby exerting anti-inflammatory activity. Molecular docking studies revealed strong interactions between the active compounds of BPS-2 and TLR4 through key amino acid residues, including Pro116, Thr114, and Arg105. These results underscore the potential application of millet bran bound phenolic compounds as naturally occurring anti-inflammatory substances.