The box a domain of high mobility group box-1 protein as an efficient siRNA carrier with anti-inflammatory effects

Abstract

High mobility group box-1 (HMGB-1) is a DNA binding nuclear protein and pro-inflammatory cytokine. The box A domain of HMGB-1 (rHMGB-1A) exerts an anti-inflammatory effect, inhibiting wild-type HMGB-1 (wtHMGB-1). In this study, HMGB-1A was evaluated as an siRNA carrier with anti-inflammatory effects. HMGB-1A was expressed and purified by consecutive nickel chelate chromatography, cationic exchange chromatography, and polymixin B chromatography. Purified rHMGB-1A demonstrated an anti-inflammatory effect, reducing tumor necrosis factor-a (TNF-a) in wtHMGB-1 or lipopolysaccharide (LPS) activated macrophages. In gel retardation assay, rHMGB-1A formed a stable complex with siRNA at or above a 1:2 weight ratio (siRNA:rHMGB-1A). A heparin competition assay showed that an siRNA/rHMGB-1A complex released siRNA more easily than an siRNA/polyethylenimine (PEI, 25 kDa) complex. Luciferase siRNA/rHMGB-1A reduced firefly luciferase expression at a similar level as luciferase siRNA/PEI complex. Furthermore, TNF-a siRNA/rHMGB-1A synergistically reduced TNF-a expression in LPS activated macrophages. Therefore, rHMGB-1A may be useful as an siRNA carrier with anti-inflammatory effects in siRNA therapy for various inflammatory diseases.