鞘内注射PDE4B-siRNA对L5脊神经结扎大鼠痛觉高敏的影响

Intrathecal injection of siRNA targeting phosphodiesterase 4B attenuated hyperalgesia in rats with L_5 spinal nerve ligation

目的观察鞘内注射针对磷酸二酯酶4B(PDE4B)基因的小干扰RNA(siRNA)对L5脊神经结扎(SNL)大鼠的热痛觉及机械痛觉高敏的影响。方法雄性SD大鼠70只,随机均分为假手术组(Sham组)、生理盐水组(NS组)、单纯载体组(V组)、错配siRNA组(siRNA-M组)和PDE4B-siRNA组(siRNA-B组),分别于神经结扎术后即刻以及术后1、3、5、7d鞘内注射生理盐水、生理盐水、LipofectamineRNAiMAX、错配siRNA 2μg和PDE4B-siRNA 2μg,容量均为10μl。PDE4B-siRNA、错配siRNA与载体均在注射前30min混匀。于术前1d及术后2、4、6、8d分别测定五组大鼠的机械缩足阈值(MWT)和热缩足潜伏期(TWL);术后4、8d行为学测试结束后每组各处死6只大鼠,取腰段脊髓,采用实时定量PCR(real-time PCR,RT-PCR)和western bolt分别测定PDE4B的mRNA和蛋白表达。结果与术前1d比较,术后2、4、6、8dNS组、V组、siRNA-M组和siRNA-B组大鼠MWT明显缩短、TWL明显减小(P<0.05)。与NS组比较,术后2、4、6、8dsiRNA-B组大鼠MWT明显延长、TWL明显增加(P<0.05)。与Sham组比较,术后4、8dNS组、V组、siRNA-M组、siRNA-B组大鼠脊髓组织的PDE4B mRNA、PDE4B蛋白表达升高(P<0.05)。与NS组比较,术后4、8dB组大鼠脊髓PDE4B mRNA、PDE4B蛋白表达明显降低(P<0.05)。结论鞘内注射PDE4B-siRNA可显著抑制L5SNL大鼠腰段脊髓PDE4B的mRNA和蛋白表达,有效缓解机械和热痛觉高敏。

Objective To investigate the effect of intrathecal injection of siRNA targeting phosphodiesterase 4B（PDE4B）on mechanical and thermal hyperalgesia in rats with Ls spinal nerve [igation（SNL）. Methods Seventy male Sprague-Dawley rats were randomly divided into five groups （n=14）： the sham group（group Sham）, saline group （group NS）, vehicle group （group V）, mismatch siRNA group （group siRNA-M） and PDE4B-siRNA group （group siRNA-B）. The PDE4Pr siRNA（2μg/10 μ1） was administered intrathecally just after ligation and at 1,3,5 and 7 days after surgery. Other four groups were injected intrathecally with saline, lipofectamin RNAiMAX and mismatch siRNA in 10 ~1 respectively. Both thermal and mechanical hyperalgesia were measured at 1 day before and at 2,4,6 and 8 days after operation. After behavioral testing at 4 and 8 days after operation, the lumbar spinal cord was harvested and the expressions of PDE4Bts mRNA and protein were assessed by real-time PCR （RT PCR） and western bolt respectively. Results Compared with baseline, MWT and TWL decreased at 2,4,6, and 8 d in group NS, group V, group siRNA-M and group siRNA-B（P〈0. 05）. Compared with group NS, MWT and TWL increases at 2,4,6, and 8 d in group siRNA-B （P 〈 0.05）. Compared with group Sham, spinal PDE4B＇ s mRNA and protein increased at 4 and 8 d in group NS, group V, group siRNA-M and group siRNA-B（P〈0.05）. Compared with group NS, spinal PDE4B＇ s mRNA and protein decreased at 4 and 8 d in group siRNA-B（P〈0.05）. Conclusion Intrathecal injection of PDE4B-siRNA can effectively inhibit the expression of PDE4B＇ s mRNA and protein in the lumbar spinal cord and remarkably alleviate mechanical and thermal hyperalgesia in rats with SNL.