Pyxinol derivative suppresses LPS-induced inflammation by targeting the p65-p50 heterodimer

Pyxinol, the core pharmacophore of ocotillol-type ginsenosides, has derivatives with notable anti-inflammatory properties mediated via the NF-κB pathway; However, their precise molecular targets remain unidentified. In this study, a series of novel pyxinol derivatives were synthesized by introducing simple aromatic moieties into the C-3 hydroxyl group of pyxinol through short linkers. These derivatives were initially screened for anti-inflammatory activity based on their ability to inhibit lipopolysaccharide-induced nitric oxide production in RAW264.7 cells. Among them, derivative 2m demonstrated significant anti-inflammatory activity with minimal cytotoxicity. It effectively suppressed the release of key inflammatory cytokines, including interleukin-1β and tumor necrosis factor-α, and reduced the expression of inflammatory mediators such as inducible nitric oxide synthase and cyclooxgenase-2. In vivo evaluations revealed that 2m conferred robust protection against LPS-induced acute liver injury in septic mice. Mechanistic investigations indicated that 2m did not affect LPS-induced p65 phosphorylation or nuclear translocation. Instead, pull-down assays showed that 2m inhibited the binding of phosphorylated p65 to target DNA, a pivotal event in the transcriptional activation of proinflammatory genes. This interaction was further supported by cellular thermal shift assays, which confirmed that 2m directly binds to p65 in situ. Together, these findings highlight that 2m exerts its anti-inflammatory effects by targeting the classical NF-κB pathway through directly interaction with p65, thereby blocking its DNA-binding activity.