高迁移率族蛋白 B1 盒 A 域作为一种具有抗炎作用的高效 siRNA 载体。
The box A domain of high mobility group box-1 protein as an efficient siRNA carrier with anti-inflammatory effects.
机构信息
Department of Bioengineering, Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, Seoul, 133-791, Korea.
出版信息
J Cell Biochem. 2012 Jan;113(1):122-31. doi: 10.1002/jcb.23334.
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.
高迁移率族蛋白 B1（HMGB-1）是一种 DNA 结合核蛋白和促炎细胞因子。HMGB-1 的盒 A 结构域（rHMGB-1A）发挥抗炎作用，抑制野生型 HMGB-1（wtHMGB-1）。在本研究中，HMGB-1A 被评估为具有抗炎作用的 siRNA 载体。HMGB-1A 通过连续的镍螯合层析、阳离子交换层析和多粘菌素 B 层析进行表达和纯化。纯化的 rHMGB-1A 表现出抗炎作用，可降低 wtHMGB-1 或脂多糖（LPS）激活的巨噬细胞中的肿瘤坏死因子-α（TNF-α）。在凝胶阻滞实验中，rHMGB-1A 以 1:2 或更高的重量比（siRNA：rHMGB-1A）与 siRNA 形成稳定的复合物。肝素竞争实验表明，siRNA/rHMGB-1A 复合物比 siRNA/聚乙烯亚胺（PEI，25kDa）复合物更容易释放 siRNA。荧光素酶 siRNA/rHMGB-1A 降低荧光素酶表达的水平与荧光素酶 siRNA/PEI 复合物相似。此外，TNF-α siRNA/rHMGB-1A 协同降低 LPS 激活的巨噬细胞中 TNF-α 的表达。因此，rHMGB-1A 可能是一种有用的 siRNA 载体，可在针对各种炎症性疾病的 siRNA 治疗中发挥抗炎作用。
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