Functional dissection of Nrf2-dependent phase II genes in vascular inflammation and endotoxic injury using Keap1 siRNA

Keap1 is a cytoplasmic repressor of the transcription factor Nrf2, and its degradation induces Nrf2 activation, leading to upregulation of antioxidant phase II genes. We investigated the roles of phase II genes in vascular inflammation and septic injury using Keap1 siRNA and elucidated its underlying mechanism. Selective knockdown of Keap1 with siRNA promoted Nrf2-dependent expression of phase II genes in endothelial cells, such as heme oxygenase-1 (HO-1), glutamate–cysteine ligase (GCL), and peroxiredoxin-1 (Prx1), resulting in the elevation of cellular glutathione levels and suppression of tumor necrosis factor (TNF)-α-induced intracellular H2O2 accumulation. Keap1 knockdown inhibited TNF-α-induced expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by suppressing NF-κB activation via inhibition of its upstream modulators, Akt, NIK, and IKK, resulting in the elevation of monocyte adhesion to endothelial cells. Importantly, these events were reversed by HO-1 and GCL inhibitors and Prx1-specific siRNA. Keap1 knockdown also inhibited endotoxin-induced expression of inducible nitric oxide synthase (iNOS) and TNF-α by upregulating HO-1, GCL, and Prx1 expression in macrophages. Moreover, in vivo Keap1 knockdown increased the expression of phase II genes and suppressed the expression of ICAM-1, VCAM-1, iNOS, and TNF-α in an endotoxemic mouse model, resulting in significant protection against liver and lung injuries and lethality. Our results indicate that Keap1 knockdown prevents NF-κB-mediated vascular inflammation and endotoxic shock by suppressing NF-κB-mediated inflammatory gene expression via upregulation of Nrf2-mediated antioxidant genes. Thus, siRNA targeting Keap1 may provide a new therapeutic approach for inflammation-associated vascular diseases and sepsis.

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► Keap1 siRNA induces Nrf2-mediated expression of phase II genes in vitro and in vivo.
► Keap1 siRNA blocks ROS generation, NF-κB activation, and inflammatory gene expression.
► These effects are reversed by functional inhibition of HO-1, GCL, and Prx1.
► Keap1 siRNA blocks inflammation, organ injury, and lethality in an endotoxemic model.
► The target effect of Keap1 siRNA may protect against inflammatory disorders.