CAJ | 학술논문

目的评价磷酸二酯酶(PDE)4B蛋白在神经病理性痛大鼠脊髓炎症反应中的作用及其与ERK的关系.方法健康雄性SD大鼠60只,体重180 ～ 220 g,先行L5,6鞘内置管,第6天确定导管位置后,采用随机数字表法分为5组(n=12):假手术组(Sham组)、生理盐水组(NS组)、单纯载体组(Ⅴ组)、错配siRNA组(siR-M组)及PDE4B-siRNA组(siR-B组).结扎L5脊神经制备大鼠神经病理性痛模型.于结扎后即刻、第1、3、5、7天各组分别鞘内注射10 μl生理盐水、10μl生理盐水、Lipofecta-minTM RNAiMAX、错配siRNA和LipofectaminTM RNAiMAX包裹的PDE4B-siRNA(2 μg/10μl).术前1d及术后第2、4、6和8天分别测定大鼠机械痛阈；术后第8天行为学测试结束后处死大鼠取腰段脊髓,采用Western bolt法检测PDE4B、细胞外调节蛋白激酶(ERK)、磷酸化ERK(p-ERK)的蛋白表达,ELISA法测定TNF-α、IL-1β和IL-6水平.结果与Sham组比较,NS组、Ⅴ组、siR-M组和siR-B组术后2、4、6和8d时机械痛阈降低(P＜0.05),Ⅴ组和siR-M组术后2、4、6和8d时机械痛阈差异无统计学意义(P＞0.05),脊髓p-ERK、PDE4B蛋白、TNF-α、IL-1β和IL-6水平升高(P＜0.05)；与NS组相比,siR-B组术后2、4、6和8d时机械痛阈升高,脊髓p-ERK、PDE4B蛋白、TNF-α、IL-1β和IL-6水平降低(P＜0.05),Ⅴ组和siR-M组上述指标比较差异无统计学意义(P＞0.05).结论 PDE4B蛋白参与了大鼠神经病理性痛的形成,可能与促进脊髓ERK磷酸化,增强炎性反应有关.
목적 평개린산이지매(PDE)4B단백재신경병이성통대서척수염증반응중적작용급기여ERK적관계.방법 건강웅성SD대서60지,체중180 ～ 220 g,선행L5,6초내치관,제6천학정도관위치후,채용수궤수자표법분위5조(n=12):가수술조(Sham조)、생리염수조(NS조)、단순재체조(Ⅴ조)、착배siRNA조(siR-M조)급PDE4B-siRNA조(siR-B조).결찰L5척신경제비대서신경병이성통모형.우결찰후즉각、제1、3、5、7천각조분별초내주사10 μl생리염수、10μl생리염수、Lipofecta-minTM RNAiMAX、착배siRNA화LipofectaminTM RNAiMAX포과적PDE4B-siRNA(2 μg/10μl).술전1d급술후제2、4、6화8천분별측정대서궤계통역；술후제8천행위학측시결속후처사대서취요단척수,채용Western bolt법검측PDE4B、세포외조절단백격매(ERK)、린산화ERK(p-ERK)적단백표체,ELISA법측정TNF-α、IL-1β화IL-6수평.결과 여Sham조비교,NS조、Ⅴ조、siR-M조화siR-B조술후2、4、6화8d시궤계통역강저(P＜0.05),Ⅴ조화siR-M조술후2、4、6화8d시궤계통역차이무통계학의의(P＞0.05),척수p-ERK、PDE4B단백、TNF-α、IL-1β화IL-6수평승고(P＜0.05)；여NS조상비,siR-B조술후2、4、6화8d시궤계통역승고,척수p-ERK、PDE4B단백、TNF-α、IL-1β화IL-6수평강저(P＜0.05),Ⅴ조화siR-M조상술지표비교차이무통계학의의(P＞0.05).결론 PDE4B단백삼여료대서신경병이성통적형성,가능여촉진척수ERK린산화,증강염성반응유관.
Objective To evaluate the role of expression of phosphodiesterase 4B (PDE4B) in the spinal cord in inflammatory responses in a rat model of neuropathic pain and the relationship with extracellular signal-regulated kinase (ERK).Methods Sixty healthy male Sprague-Dawley rats,weighing 180-220 g,in which intrathecal catheters were implanted at L5,6 interspace,were used.The location of catheters was confirmed 6 days later.The rats were randomly divided into 5 groups (n =12 each):sham operation group (group Sham),normal saline (NS) group,vehicle group (group Ⅴ),mismatch siRNA group (group siR-M),and PDE4B-siRNA group (group siR-B).Neuropathic pain was induced by ligation of L5 spinal nerve (SNL).In Sham group,the L5 spinal nerve was only exposed,but not ligated.Immediately after ligation and on 1,3,5,and 7 days after ligation,10 μl NS,10 μl NS,LipofectaminTM RNAiMAX,PDE4B-siRNA (2 μg/10 μl) encapsulated in mismatch siRNA and PDE4B-siRNA (2 μg/10 μl) encapsulated in LipofectaminTM RNAiMAX were injected intrathecally in Sham,NS,V,siR-M,and siR-B groups,respectively.The mechanical pain threshold was measured at 1 day before and 2,4,6 and 8 days after operation.After behavioral testing on 8th day after operation,the rats were sacrificed and the lumbar segment of the spinal cord was removed for determination of PDE4B protein,ERK and phosphor-ERK (p-ERK)expression,and TNF-α,IL-1β and IL-6 levels.Results Compared with Sham group,the mechanical pain threshold was significantly decreased at 2,4,6 and 8 days after operation in NS,V,siR-M and siR-B groups (P ＜0.05),and no significant change was found in the mechanical pain threshold at 2,4,6 and 8 days after operation (P ＞ 0.05) and the expression of p-ERK and PDE4B protein,and levels of TNF-α,IL-1β and IL-6 were increased at 2,4,6 and 8 days after operation in V and siR-M groups (P ＜ 0.05).Compared with NS group,the mechanical pain threshold was significantly increased,and the expression of p-ERK and PDE4B protein and levels of TNF-α,IL-1β and IL-6 were decreased at 2,4,6 and 8 days after operation (P ＜ 0.05),and no significant change was found in the parameters mentioned above in V and siR-M groups (P ＞ 0.05).Conclusion Up-regulation of the expression of PDE4B protein in the spinal cord is involved in the development of neuropathic pain in rats,which may be related to promoted phosphorylation of ERK in the spinal cord and enhanced inflammatory responses.