纳布啡改善瑞芬太尼诱导的大鼠痛觉过敏的机制

Mechanism of nalbuphine in the improvement of remifentanil induced hyperalgesia in rats

目的探讨纳布啡对瑞芬太尼诱导的大鼠痛觉过敏的改善作用及对脊髓背角常染色质组蛋白赖氨酸N-甲基转移酶2(G9a)和钠激活钾通道(Slack)表达的影响。方法55只大鼠随机抽取30只作为对照组、瑞芬太尼组和切口痛组,每组10只,其余25只建造瑞芬太尼诱发大鼠术后痛觉过敏反应模型,20只建模成功大鼠随机分为模型组、纳布啡组,每组10只。瑞芬太尼组、模型组和纳布啡组静脉输注瑞芬太尼,对照组、切口痛组输注等体积生理盐水,输注5 min后,切口痛组、模型组、纳布啡组接受左后足底切口术,对照组、瑞芬太尼组仅仰卧固定到手术台相同时间。纳布啡组泵注瑞芬太尼前3 min静注纳布啡,其余各组静注等体积的氯化钠注射液。大鼠造模后2~48 h进行痛觉行为学测定;用酶联免疫吸附法测定脊髓背角细胞炎症因子;用免疫荧光法检验脊髓背角的星形胶质细胞激活状态;用蛋白免疫印迹法测定造模后48 h脊髓背角G9a、Slack蛋白的表达水平。结果随造模时间延长,瑞芬太尼组、切口痛组、模型组大鼠的机械缩足反应阈(paw withdrawal mechanical threshold,PWMT)、热缩足反应潜伏期(paw withdrawal thermal latency,PWTL)值持续减小,而纳布啡组持续增大(P<0.05),对照组无明显变化;与对照组比较,瑞芬太尼组、切口痛组、模型组、纳布啡组大鼠的PWMT和PWTL值均减小(P<0.05);与瑞芬太尼组比较,模型组大鼠的PWMT和PWTL值减小、纳布啡组PWMT和PWTL值增大(P<0.05),切口痛组与之比较,差异无统计学意义;与模型组比较,纳布啡组PWMT和PWTL值增大(P<0.05)。与对照组比较,造模组肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)水平升高(P<0.05);与瑞芬太尼组比较,模型组TNF-α、IL-1β水平较高,纳布啡组TNF-α、IL-1β水平降低(P<0.05),切口痛组与之比较差异无统计学意义;与模型组比较,纳布啡组TNF-α、IL-1β水平降低(P<0.05)。与对照组比较,瑞芬太尼组、切口痛组的星形胶质细胞明显激活,模型组激活星形胶质细胞密度最大,纳布啡组与之相比差异无统计学意义。与对照组比较,模型组G9a蛋白相对表达量升高,Slack总蛋白与膜蛋白的相对表达量降低(P<0.05);与瑞芬太尼组比较,模型组G9a蛋白的相对表达量升高,Slack总蛋白与膜蛋白的相对表达量降低(P<0.05),纳布啡组G9a蛋白的相对表达量降低,Slack总蛋白与膜蛋白的相对表达量升高(P<0.05),切口痛组与之比较差异无统计学意义;与模型组比较,纳布啡组G9a蛋白的相对表达量降低,Slack总蛋白与膜蛋白的相对表达量升高(P<0.05)。结论对瑞芬太尼诱导的术后痛觉过敏反应大鼠模型进行纳布啡预处理,可有效改善大鼠的痛觉过敏,可能是通过下调G9a、上调Slack的表达,达到镇痛的效果。

Objective To investigate the improvement effect of nalbuphine on remifentanil induced hyperalgesia in rats and the expression of histone lysine N-methyltransferase 2(G9a)and sodium activated potassium channel(slack)in spinal dorsal horn.Methods 30 rats were randomly selected from 55 rats as control group,remifentanil group and incision pain group,with 10 rats in each group.The remaining 25 rats were established with remifentanil induced postoperative hyperalgesia model.20 rats were randomly divided into model group and nalbuphine group,with 10 rats in each group.Remifentanil group,model group and nalbuphine group were given remifentanil intravenously,while the control group and incision pain group were infused with equal volume of normal saline.After 5 minute infusion,the incision pain group,model group and nalbuphine group received left posterior plantar incision,while the control group and remifentanil group were only supine fixed to the operating table for the same time.The rats in the nalbuphine group were intravenously injected 3 minutes before remifentanil injection,and the other groups were given equal volume of sodium chloride solution.Pain behavior was measured 2-48 hours after modeling.Enzyme linked immunosorbent assay(ELISA)was used to detect inflammatory factors in spinal dorsal horn cells.The activation of astrocytes in spinal dorsal horn was detected by immunofluorescence.The expression levels of G9a and slack protein in spinal dorsal horn were measured by Western blot.Results With the prolongation of modeling time,the paw withdrawal mechanical threshold(PWMT)and paw withdrawal thermal latency(PWTL)values of the rats in the remifentanil group,the incision pain group and the model group persistly,decreasd,but continued to increase in the nalbuphine group(P<0.05).Meanwhile,there was no significant change in control group(P>0.05).Compared with the control group,the levels of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in the modeling group were higher(P<0.05).Compared with the fentanyl group,the levels of TNF-αand IL-1βin the model group were higher,and the levels of TNF-αand IL-1βin the nalbuphine group were lower(P<0.05),and there was no statistical difference;Compared with the model group,the levels of TNF-αand IL-1βin the nalbuphine group were lower(P<0.05).Compared with the control group,the astrocytes in the remifentanil group and the incision pain group were significantly activated,and the density of activated astrocytes in the model group was the highest,and there was no significant difference in the nalbuphine group.Compared with the control group,the relative expression of G9a protein in the modeling group was higher,and the relative expression of total Slack protein and membrane protein was lower(P<0.05).Compared with the remifentanil group,the relative expression of G9a protein in the model group was higher,the relative expression of Slack total protein and membrane protein was lower(P<0.05),the relative expression of G9a protein in nalbuphine group was lower,and the relative expression of Slack total protein and membrane protein was higher(P<0.05).Compared with the model group,the relative expression of G9a protein was lower in the nalbuphine group,and the relative expression of total Slack protein and membrane protein was higher in the nalbuphine group(P<0.05).Conclusion Pretreatment with nalbuphine on remifentanil induced hyperalgesia in rats can effectively improve hyperalgesia,which may be achieved by down regulating G9a and up regulating the expression of Slack.