慢病毒介导的DREAM沉默治疗在坐骨神经慢性压迫模型大鼠疼痛中的作用及其对GALR1的表达调控

Roles of GALR1 in curing neuropathic pain of rats suffered from CCI through DREAM silencing mediated by lentivirus

目的甘丙肽受体1(galanin receptor 1,GALR1)信号通路在痛觉调节中发挥重要作用,该研究利用坐骨神经慢性压迫(chronic constriction injury of the sciatic nerve,CCI)大鼠模型,探索慢病毒介导的DREAM沉默治疗在CCI模型大鼠疼痛中的作用及其对GALR1的表达调控。方法选择健康雄性SD大鼠24只,随机分为4组(n=6):即RNA干扰组、空白载体组、单纯CCI组和正常对照组。鞘内置管前后分别测定基础痛阈,RNA干扰组和空白载体组于CCI后经微导管给予pKCSHR-Puro/GFP-DREAM慢病毒和空白载体,并测定腰段脊髓内下游调控元件拮抗分子(downstream regulatory element antagonist,DREAM)和GALR1的蛋白表达。结果大鼠痛阈的变化:手术同侧热痛阈和机械痛阈测定结果表明,CCI处理后,RNA干扰组、空白载体组及单纯CCI组在各个时间点热痛阈和机械痛阈较正常对照组均显著降低(P<0.01);RNA干扰组鞘内注射后较注射前痛阈显著升高,治疗后相同时间点RNA干扰组较空白载体组及单纯CCI组痛阈亦显著升高。Western blotting结果表明,与其他实验组对比,RNA干扰组的DREAM蛋白表达水平显著下调,而GALR1蛋白表达水平较空白载体组、单纯CCI组亦有显著下调。结论 DREAM可以调控GALR1的表达,提示甘丙肽受体1信号通路参与DREAM基因调节大鼠神经病理性疼痛。

[Objective] To elaborate the mechanism of DREAM modulation pain further, the study ap proaches the roles of the galanin signal pathway in dorsal horn in the therapy of neuropathic pain of rats suf fered from CCI through DREAM silencing mediated by lentivirus. [Methods] 24 male and healthy rats were selected, then divide them into 4 groups （n =6 each）, that is, Group RNAi, Group blank vector, Group CCIand Group normal compared. Measure the basic pain threshold before inserting intrathecal cather and building the experiment model including paw withdraw thermal latency （PWTL） and paw withdraw mechanical threshold （PWMT）. And after CCI 7 day we had continuously given pKCSHR-Puro/ GFP- DREAM -LV or blank vector by intrathecal injection to Group RNAi, Group blank vector. After 14th day CCI, we took out the spinal cords （L4-5）, which were determined the protein expressions of DREAM and GALR1. [Results] Pain threshold： The results of PWTL and PWMT in the side of operation （the left side）： there were no differences among 4 groups with basic pain threshold in the days before inserting intrathecal cather and building the experiment model （P 〉0.05）. From the two days after CCI the PWTL and PWMT in Group RNAi, Group blank vector and Group CCI of every time point were much lower than Group normal compared （P 〈0.01）. After cured the pain threshold were higher than that of CCI after 6 days in Group RNAi （P〈0.O1）. However, after cured the pain threshold of Group RNAi in the same time point were also higher than that of Group blank vector and Group CCI （P 〈0.01）, There were no differences in the opposite side of operation （the right side） （P 〉0.05）. The ex periment results of Western blotting： the protein expressions of DREAM in dorsal horn in the day after 14th CCI of Group RNAi is lower than that of other Groups （P〈O.01）. And there were no differences in the other groups （P〉0.05）. The protein expressions of GALR1 of Group RNAi were lower than that of Group blank vector and Group CCI （P〈0.01）, but which were higher than that of Group normal compared （P〈O.01）. And there were no differences in Group blank vector and Group CCI （P〉0.05）. [Conclusion] DREAM may take part in the regulation of expressions of GALR1 gene and protein. The galanin signal pathway in dorsal horn may play an important role in the mechanism of DREAM participating in neuropathy pain.