NL-1在瑞芬太尼诱发切口痛小鼠痛觉过敏时脊髓背角含GluR1亚基的AMPA受体膜转运中的作用

Role of NL-1 in trafficking of GluR1-containing AMPA receptor to cell membrane in spinal cord dorsal horn during remifentanil-induced hyperalgesia in mice with incisional pain

目的评价神经连接蛋白-1(NL-1)在瑞芬太尼诱发切口痛小鼠痛觉过敏时脊髓背角含GluRl亚基的AMPA受体膜转运中的作用。方法SPF级健康雄性C57BL/6J小鼠40只,8~10周龄,体重18~22 g。采用随机数字表法分为5组(n=8):对照组(C组)、NL-1-shRNA质粒组(NL组)、切口痛+瑞芬太尼组(I+R组)、切口痛+瑞芬太尼+空白载体组(I+R+B组)和切口痛+瑞芬太尼+NL-1-shRNA质粒组(I+R+NL组)。I+R+B组鞘内注射阴性慢病毒,NL组和I+R+NL组鞘内注射NL-1-shRNA慢病毒,1×10^8 IFU/ml,10μl,1次/d,连续3 d;C组和I+R组于相同时点鞘内注射10μl生理盐水。待稳定转染后,C组和NL组尾静脉注射生理盐水0.1 ml,连续4次,间隔15 min;I+R组、I+R+B组和I+R+NL组尾静脉注射瑞芬太尼10μg/kg,0.1 ml,连续4次,间隔15 min,并于第1次给药后制备切口痛模型。分别于输注生理盐水或瑞芬太尼前24 h(T0)、输注停止后3、6、24和48 h(T1-4)时测定机械缩足反应阈(MWT)和热甩尾潜伏期(TFL)。于T4时测定痛阈后处死各组小鼠,取脊髓背角L4-6节段,测定NL-1及其mRNA和AMPA受体的表达水平,计算膜蛋白和总蛋白AMPA受体表达的比值(m/t比值)。结果与C组比较,I+R组和I+R+B组T1-4时MWT降低,TFL缩短,脊髓背角NL-1及其mRNA、脊髓总蛋白及膜蛋白含GluR1的AMPA受体表达上调,m/t比值增加(P<0.05);与I+R组和I+R+B组比较,I+R+NL组T1-4时MWT增加,TFL延长,脊髓背角NL-1及其mRNA、脊髓膜蛋白和总蛋白AMPA受体表达下调,m/t比值降低(P<0.05)。结论脊髓背角NL-1可促进含GluRl亚基的AMPA受体向细胞膜转运,参与了瑞芬太尼诱发切口痛小鼠痛觉过敏形成和维持的过程。

Objective To evaluate the role of neuroligin 1(NL-1)in trafficking of GluR1-containing AMPA receptor to cell membrane in spinal cord dorsal horns during remifentanil-induced hyperalgesia in mice with incisional pain.Methods Forty SPF healthy male C57BL/6J mice,aged 8-10 weeks,weighing 18-22 g,were divided into 5 groups(n=8 each)using a random number table method:control group(group C),NL1-shRNA plasmid group(group NL),incisional pain plus remifentanil group(group I+R),incisional pain plus remifentanil plus blank vector group(group I+R+B),and incisional pain plus remifentanil plus NL-1-shRNA plasmid group(group I+R+NL).Negative lentivirus was intrathecally injected in group I+R+B.In NL and I+R+NL groups,10μl NL-1-shRNA lentivirus at 1×10^8 IFU/ml was intrathecally injected once a day for 3 consecutive days.Normal saline 10μl was intrathecally injected at the same time point in C and I+R groups.After transfection was stable,normal saline 0.1 ml was injected via the caudal vein for 4 consecutive times at 15 min intervals in C and NL groups.In I+R,I+R+B and I+R+NL groups,0.1 ml remifentanil 10μg/kg was injected via the caudal vein for 4 consecutive times at 15 min intervals,and the model of incisional pain was established after the first administration.The mechanical paw withdrawal threshold(MWT)and tail-flick latency(TFL)were measured at 24 h before normal saline or remifentanil administration(T0)and at 3,6,24 and 48 h after the end of administration(T1-4).The animals were sacrificed after measurement of pain threshold at T4,and L4-6 segments of the spinal dorsal horn were then collected for determination of the expression of NL-1 protein and mRNA and AMPA receptors,and the ratio of AMPA receptor expression in the membrane protein to that in the total protein(m/t ratio)was calculated.Results Compared with group C,the MWT was significantly decreased,and TFL was shortened at T1-4,the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was up-regulated,and m/t ratio was increased in I+R and I+R+B groups(P<0.05).Compared with I+R and I+R+B groups,the MWT was significantly increased and TFL was prolonged at T1-4,the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was down-regulated,and m/t ratio was decreased in group I+R+NL(P<0.05).Conclusion NL-1 in spinal cord dorsal horns can promote the trafficking of GluR1-containing AMPA receptors to cell membrane,which is involved in the development and maintenance of remifentanil-induced hyperalgesia in mice with incisional pain.