延髓头端腹内侧部NADPH氧化酶2激活导致活性氧簇释放在皮肤/肌肉切开和牵拉引起的慢性术后疼痛中的作用

NADPH oxidase 2-derived release of reactive oxygen species in rostral ventromedial medulla contributes to chronic postoperative pain after skin/muscle incision and retraction

目的:探讨延髓头端腹内侧部(RVM)内NADPH氧化酶2(NOX2)激活导致活性氧簇(ROS)释放在皮肤/肌肉切开和牵拉(SMIR)引起的慢性术后疼痛中的作用并探讨其机制。方法:SD大鼠随机分为对照组、SMIR(1 d和7 d)组、SMIR+术前RVM内注射NOX2阻断肽gp91ds-tat组、SMIR+术前RVM内注射无序肽scrambled ds-tat(NOX2阻断肽的对照肽)组、SMIR+术后RVM内注射gp91ds-tat组、SMIR+术后RVM内注射scrambled ds-tat阻断组、SMIR+RVM注射ROS清除剂N-叔丁基-α-苯基硝酮(PBN)组和SMIR+RVM内注射生理盐水组,以up-down方法测量其50%机械刺激撤足阈值,免疫组化染色检测RVM内NOX2的激活情况及核酸氧化产物8-羟基脱氧鸟苷(8-OHdG)的水平,ELISA法检测RVM及脊髓背角内5-羟色胺(5-HT)含量。结果:SMIR引起机械痛敏,并可激活RVM内神经元和星形胶质细胞中的NOX2。NOX2阻断肽gp91ds-tat微量注射入RVM(于SMIR术前30 min开始,每天1次,共3次)可阻断SMIR引起的机械痛敏,并可降低RVM及脊髓背角5-HT含量。术后给予gp91ds-tat对机械痛敏及5-HT含量无显著影响。SMIR也可引起RVM内核酸氧化产物8-OHdG的生成增多,SMIR术后7 d RVM内微量注射ROS清除剂PBN可短暂翻转已经产生的机械痛敏。结论:RVM内NOX2-ROS途径的激活参与了SMIR后慢性术后疼痛的发生发展,ROS增多导致的脊髓背角5-HT含量增高可能介导了此作用。

AIM:To investigate the role of reactive oxygen species(ROS)derived from NADPH oxidase 2(NOX2)in chronic postsurgical pain(CPSP)induced by skin/muscle incision and retraction(SMIR).METHODS:The rats were randomly divided into the following groups:sham group,SMIR group,SMIR+preemptive injection of gp91 ds-tat(NOX2 blocking peptide)into RVM group,SMIR+preemptive injection of scrambled ds-tat into RVM group,SMIR+postoperative injection of gp91 ds-tat into RVM group,SMIR+postoperative injection of scrambled ds-tat into RVM group,SMIR+postoperative injection of phenyl-N-tert-butylnitrone(PBN;ROS scavenger)into RVM group,SMIR+postoperative injection of saline into RVM group.The 50%paw withdrawal threshold of the rats was measured by the up-down method.Immunofluroscience was used to detect the activation of NOX2 and the level of 8-hydroxyguanine(8-OHdG),an oxidized stress marker that reflect the production of ROS in RVM.5-Hydroxytryptamine(5-HT)level in RVM and spinal cord was measured by ELISA.RESULTS:The SMIR induced mechanical allodynia,and activated NOX2 in RVM neurons and astrocytes.Microinjection of gp91 ds-tat(started 30 min before SMIR,then once daily for to-tally 3 d)into RVM attenuated the development of mechanical allodynia,and decreased the 5-HT level in RVM and spi-nal dorsal horn.In contrast,postoperative application of the same dose of gp91 ds-tat had no effect on either mechanical allodynia or 5-HT level following SMIR.The SMIR also induced increase in 8-OHdG.Microinjection of PBN into RVM on day 7 after SMIR also transiently alleviated the established mechanical allodynia.CONCLUSION:The SMIR acti-vates NOX2-ROS pathway in RVM,and increases 5-HT level both in RVM and spinal cord.The NOX2-derived ROS might contribute to the development of CPSP.