Department of Orthopaedics, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan.
J Gene Med. 2010 Feb;12(2):219-27. doi: 10.1002/jgm.1378.
Drug resistance mediated by P-glycoprotein (P-gp) is one of the major reasons for the failure of rheumatoid arthritis (RA) therapy with disease modifying anti-rheumatic drugs and glucocorticoids. In the present study, we aimed to investigate the in vitro effectiveness of small interfering RNA (siRNA) to render rat fibroblast-like synoviocytes (FLS) susceptible to drugs. We also attempted the electroporation-mediated transfer of siRNA against multidrug resistance (MDR) genes into rat knee joints.
FLS were transfected with siRNAs corresponding to MDR1a and MDR1b genes. FLS were treated with dexamethasone (DEX) and lipopolysaccharide. The mRNA and protein levels of tumor necrosis factor-alpha, interleukin (IL)-6 and IL-1beta were measured. Both siRNAs were co-transduced into rat knee joints by an electroporation method and evaluated the target gene expressions in the synovium.
Each siRNA could sequence-specifically reduce the target gene expression by over 70% and effectively suppressed P-gp expression and function in the FLS. Both gene expression and protein production of the inflammatory cytokines in the cells transfected with siRNA were reduced by a greater amount compared to in control cells. The in vivo electroporation-mediated transduction of siRNA could significantly inhibit the target gene expressions.
MDR1a/1b gene silencing by siRNA could effectively inhibit P-gp in rat FLS, resulting in a significant enhancement of the anti-inflammatory effects of DEX. The in vivo siRNA transduction could successfully silence MDR gene expression in the rat synovium. These findings indicate that the siRNA targeting MDR gene could be a useful tool for treating refractory arthritis in RA.
P-糖蛋白（P-gp）介导的耐药性是疾病修饰抗风湿药物和糖皮质激素治疗类风湿关节炎（RA）失败的主要原因之一。在本研究中，我们旨在研究小干扰 RNA（siRNA）使大鼠成纤维样滑膜细胞（FLS）对药物敏感的体外有效性。我们还尝试用电穿孔介导的方法将针对多药耐药（MDR）基因的 siRNA 转染到大鼠膝关节中。
将 siRNA 转染到 MDR1a 和 MDR1b 基因对应的 FLS 中。用地塞米松（DEX）和脂多糖处理 FLS。测量肿瘤坏死因子-α、白细胞介素（IL）-6 和 IL-1β的 mRNA 和蛋白水平。通过电穿孔方法将两种 siRNA 共转染到大鼠膝关节中，并评估滑膜中的靶基因表达。
每种 siRNA 都能特异性地使靶基因表达降低 70%以上，并有效地抑制 FLS 中的 P-gp 表达和功能。与对照细胞相比，转染 siRNA 的细胞中的炎症细胞因子的基因表达和蛋白产生均减少了更多。体内电穿孔介导的 siRNA 转导可显著抑制靶基因表达。
siRNA 对 MDR1a/1b 基因的沉默可有效抑制大鼠 FLS 中的 P-gp，从而显著增强 DEX 的抗炎作用。体内 siRNA 转导可成功抑制大鼠滑膜中 MDR 基因的表达。这些发现表明，针对 MDR 基因的 siRNA 可能是治疗 RA 中难治性关节炎的有用工具。
