2,4′-Dihydroxybenzophenone: A Promising Anti-Inflammatory Agent Targeting Toll-like Receptor 4/Myeloid Differentiation Factor 2-Mediated Mitochondrial Reactive Oxygen Species Production during Lipopolysaccharide-Induced Systemic Inflammation

The biochemical properties of 2,4′-dihydroxybenzophenone (DHP) have not been extensively studied. Therefore, this study aimed to investigate whether DHP could alleviate inflammatory responses induced by lipopolysaccharide (LPS) and endotoxemia. The results indicated that DHP effectively reduced mortality and morphological abnormalities, restored heart rate, and mitigated macrophage and neutrophil recruitment to inflammatory sites in LPS-microinjected zebrafish larvae. Additionally, the expression of pro-inflammatory mediators, including inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), and interleukin-12 (IL-12), was significantly reduced in the presence of DHP. In RAW 264.7 macrophages, DHP inhibited the LPS-induced inflammatory response by downregulating pro-inflammatory mediators and decreasing the expression of myeloid differentiation primary response 88 (MyD88), phosphorylation of IL-1 receptor-associated protein kinase-4 (p-IRAK4), and nuclear factor-κB (NF-κB). Molecular docking analysis demonstrated that DHP occupies the hydrophobic pocket of myeloid differentiation factor 2 (MD2) and blocks the dimerization of Toll-like receptor 4 (TLR4). A molecular dynamics simulation confirmed that DHP stably bound to the hydrophobic pocket of MD2. Furthermore, the DHP treatment inhibited mitochondrial reactive oxygen species (mtROS) production during the LPS-induced inflammatory response in both RAW 264.7 macrophages and zebrafish larvae, which was accompanied by stabilizing mitochondrial membrane potential. In conclusion, our study highlights the therapeutic potential of DHP in alleviating LPS-induced inflammation and endotoxemia. The findings suggest that DHP exerts its anti-inflammatory effects by inhibiting the TLR4/MD2 signaling pathway and reducing the level of mtROS production. These results contribute to a better understanding of the biochemical properties of DHP and support its further exploration as a potential therapeutic agent for inflammatory conditions and endotoxemia.