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‐α (TNF‐α) 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‐α siRNA/rHMGB‐1A synergistically reduced TNF‐α 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. J. Cell. Biochem. 113: 122–131, 2012.