Christoph Thomas, Gillen Clemens, Mika Joanna, Grünweller Arnold, Schäfer Martin K-H, Schiene Klaus, Frank Robert, Jostock Ruth, Bahrenberg Gregor, Weihe Eberhard, Erdmann Volker A, Kurreck Jens
Research & Development, Grünenthal GmbH, Zieglerstr. 6, 52078 Aachen, Germany.
Neurochem Int. 2007 Jan;50(1):281-90. doi: 10.1016/j.neuint.2006.08.017. Epub 2006 Oct 12.
To examine the role of the vanilloid receptor TRPV1 in neuropathic pain, we assessed the effects of the receptor antagonist thioxo-BCTC and antisense oligonucleotides against the TRPV1 mRNA in a rat model of spinal nerve ligation. In order to identify accessible sites on the mRNA of TRPV1, the RNase H assay was used, leading to the successful identification of binding sites for antisense oligonucleotides. Cotransfection studies using Cos-7 cells were employed to identify the most effective antisense oligonucleotide efficiently inhibiting the expression of a fusion protein consisting of TRPV1 and the green fluorescent protein in a specific and concentration-dependent manner. In an in vivo rat model of spinal nerve ligation, intravenous application of the TRPV1 antagonist thioxo-BCTC reduced mechanical hypersensitivity yielding an ED(50) value of 10.6mg/kg. Intrathecal administration of the antisense oligonucleotide against TRPV1, but not the mismatch oligonucleotide or a vehicle control, reduced mechanical hypersensitivity in rats with spinal nerve ligation in a similar manner. Immunohistochemical analysis revealed neuropathy- and antisense-associated regulation of TRPV1 protein expression in spinal cord and dorsal root ganglia. Our data demonstrate comparative analgesic effects of a TRPV1 anatagonist and a rationally designed TRPV1 antisense oligonucleotide in a spinal nerve ligation model of neuropathic pain and thus, lend support to the validation of TRPV1 as a promising target for the treatment of neuropathic pain.
为研究香草酸受体TRPV1在神经性疼痛中的作用，我们在大鼠脊神经结扎模型中评估了受体拮抗剂硫代-BCTC和针对TRPV1 mRNA的反义寡核苷酸的作用。为了确定TRPV1 mRNA上的可结合位点，我们采用了RNase H分析方法，成功鉴定出了反义寡核苷酸的结合位点。利用Cos-7细胞进行共转染研究，以特异性和浓度依赖性方式高效鉴定出最有效的反义寡核苷酸，该反义寡核苷酸能有效抑制由TRPV1和绿色荧光蛋白组成的融合蛋白的表达。在大鼠脊神经结扎的体内模型中，静脉注射TRPV1拮抗剂硫代-BCTC可降低机械性超敏反应，半数有效剂量（ED50）值为10.6mg/kg。鞘内注射针对TRPV1的反义寡核苷酸，而非错配寡核苷酸或溶剂对照，以类似方式降低了脊神经结扎大鼠的机械性超敏反应。免疫组织化学分析显示，脊髓和背根神经节中TRPV1蛋白表达存在与神经病变和反义相关的调节。我们的数据表明，在神经性疼痛的脊神经结扎模型中，TRPV1拮抗剂和合理设计 的TRPV1反义寡核苷酸具有相当的镇痛作用，因此，支持将TRPV1验证为治疗神经性疼痛的一个有前景的靶点。
