瑞芬太尼诱发切口痛大鼠痛觉过敏时脊髓背角含GluR1亚基的AMPA受体与MnSOD硝基化的关系

Relationship between GluR1-eontaining AMPA receptors and MnSOD nitration in spinal cord dorsal horn during remifentanil-induced hyperalgesia in rats with incisional pain

目的 探讨瑞芬太尼诱发切口痛大鼠痛觉过敏时脊髓背角含GluR1亚基的AMPA受体与锰超氧化物歧化酶（MnSOD）硝基化的关系.方法 取鞘内置管和尾静脉置管成功的大鼠24只,体重240～ 260 g,2～3月龄,采用随机数字表法分为3组（n=8）,生理盐水对照组（C组）：鞘内注射生理盐水15μl,10 min后尾静脉输注与瑞芬太尼等容量的生理盐水60 min;瑞芬太尼＋切口痛组（RI组）：鞘内注射生理盐水15μl,10 min后建立切口痛模型,同时尾静脉输注瑞芬太尼1μg＆#183;kg-1＆#183;min-160 min;含GluR1亚基的AMPA受体抑制剂PhTX组（P组）：鞘内注射PhTX 1 mg/kg（10 μl）后用5μl生理盐水冲管,10 min后均建立切口痛模型,同时输注瑞芬太尼1μg＆#183;kg-1＆#183;min-1,60 min.于生理盐水或瑞芬太尼输注前24 h、输注停止后2、6、24和48 h（T0-T4）时,测定大鼠机械缩足反应阈（MWT）和热缩足潜伏期（TWL）.最后一次测定痛阈后处死大鼠并取L4-6脊髓背角组织,采用Western blot法测定脊髓背角总蛋白及膜蛋白含GluR1亚基的AMPA受体、MnSOD和硝基化MnSOD的表达水平.计算膜蛋白及总蛋白含GluR1亚基的AMPA受体比值（m/t比值）和硝化Mn-SOD/MnSOD比值.结果 与C组比较,RI组和P组T1-4时MWT降低,TWL缩短,脊髓背角总蛋白和膜蛋白含GluR1亚基的AMPA受体表达上调,m/t比值升高,硝化MnSOD表达上调,硝化MnSOD/MnSOD比值升高（P＜0.05）;与RI组比较,P组T1-4时MWT升高,TWL延长,脊髓背角膜蛋白含GluR1亚基的AMPA受体表达下调,m/t比值降低,硝化MnSOD表达下调,硝化MnSOD/MnSOD比值降低（P＜0.05）.结论 脊髓背角含GluR1亚基的AMPA受体向细胞膜转运后可促进MnSOD硝基化,该作用可能参与了瑞芬太尼诱发切口痛大鼠痛觉过敏形成的机制.

Ohjective To investigate the relationship between GluR1-containing AMPA receptors and manganese superoxide dismutase （MnSOD） nitration in the spinal cord dorsal horn during remifentanilinduced hyperalgesia in rats with incisional pain.Methods Twenty-four male Sprague-Dawley rats, aged 2-3 months, weighing 240-260 g, in which caudal and intrathecal catheters were successfully placed, were randomly divided into 3 groups （n =8 each） using a random number table ： normal saline control group （group C） , remifentanil ＋ incisional pain group （group RI） , and GluRl-containing AMPA receptor inhibitor PhTX group （group P）.In group C, normal saline 15 μ1 was injected intrathecally, and 10 min later normal saline equal to the volume of remifentanil was infused for 60 min via the caudal vein.In group RI, normal saline 15 μl was injected intrathecally, 10 min later incisional pain were produced in right hindpaws of anesthetized rats, and remifentanil was infused simultaneously at a rate of 1 μg ＆#183; kg 1 ＆#183; min-1 for 60 min.In group P, after PhTX 1 mg/kg （10 μl） was injected intrathecally, the catheter was flushed with normal saline 5 μ1, 10 min later incisional pain were produced, and remifentanil was infused simultaneously at a rate of 1 μg ＆#183; kg 1 ＆#183; min 1for 60 min.Mechanical paw withdrawal threshold （MWT） and thermal paw withdrawal latency （TWL） were measured at 24 h before normal saline or remifentanil administration and 2, 6,24 and 48 h after the end of administration （T0-T4）.The animals were sacrificed after the last measurement of pain threshold.The L4-6 segments of the spinal cord were isolated for determination of the expression of GluR1-containing AMPA receptor, MnSOD and nitrated MnSOD in the total （t） and membrane （m） proteins in the spinal cord dorsal horn by Western blot.The ratios of mGluR1/tGluR1 （m/t ratio） and nitrated MnSOD/MnSOD were calculated.Results Compared with group C, the MWT was significantly decreased, and TWL was shortened at T1-4, the expression of mGluR1, tGluR1 and nitrated MnSOD was up-regulated, and the ratios of m/t and nitrated MnSOD/MnSOD were increased in RI and P groups.Compared with group RI, the MWT was significantly increased, and TWL was prolonged at T1-4, the expression of mGluR1 and nitrated MnSOD was down-regulated, and the ratios of m/t and nitrated MnSOD/MnSOD were decreased in group P.Conclusion GluR1-containing AMPA receptors can enhance MnSOD nitration in the spinal cord dorsal horn after trafficking to the cell membrane, which may be involved in the mechanism underlying remifentanil-induced hyperalgesia in rats with incisional pain.