Hydroxychavicol suppresses early TNF-α-driven cytokines in activated endothelial cells via canonical NF-κB-independent pathways

Abstract

Atherosclerosis is a chronic inflammatory vascular disease, partly driven by tumor necrosis factor-α (TNF-α)-mediated endothelial activation, leading to increased proinflammatory cytokine release and subsequently leukocyte recruitment. Hydroxychavicol, a phenolic compound derived from Piper betle, has been reported to exert anti-inflammatory effects, but its precise mechanisms in endothelial inflammatory response remain underexplored. This study investigated whether hydroxychavicol modulates early TNF-α-induced inflammatory responses in activated human endothelial cells, with a focus on canonical NF-κB signaling independence. Human umbilical vein endothelial cells (HUVECs) were pre-treated with hydroxychavicol prior to TNF-α exposure. After 4 hours, pro-inflammatory cytokine production, including interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1), was quantified using ELISA. NF-κB pathway involvement was assessed by evaluating IκB degradation via Western blot analysis. TNF-α significantly upregulated IL6, IL-8, and MCP-1 secretion, consistent with endothelial activation relevant to atherosclerotic inflammation. Hydroxychavicol treatment markedly suppressed TNF-α-induced cytokine release in a dose-dependent manner. Notably, Western blot analysis revealed preservation of IκB levels following hydroxychavicol exposure, indicating inhibition of early inflammatory signaling without canonical IκB degradation and NF-κB activation. These findings suggest that hydroxychavicol attenuates endothelial inflammatory responses through NF-κB-independent mechanisms. In conclusion, hydroxychavicol effectively suppresses early TNF-α-driven cytokine production in endothelial cells implicated in atherosclerosis, potentially via alternative signaling pathways independent of canonical NF-κB activation. This supports its therapeutic potential as a vascular-protective anti-inflammatory agent targeting early endothelial dysfunction in atherosclerotic disease.