NMDA受体2B亚基模拟表位对慢性神经病理性痛大鼠的镇痛效应

Analgesic effect of the epitope of NMDA receptor 2B subunit on chronic neuropathic pain in rats

目的评价N-甲基-D-天冬氨酸受体2B亚基（NR2B）模拟表位对慢性神经病理性痛大鼠的镇痛效应。方法成年雄性SD大鼠25只,体重200～250g,随机分为5组（n=5）,C组不进行任何处理;神经损伤（SNI）组行左后肢保留性SNI术;NR2B单纯免疫组（pNR2B组）于皮下两点注射携带NR2B模拟表位的特异性噬菌体（pNR2B）4×10^12pfu/ml,每点100μl,间隔1周后重复注射,共重复2次;无关噬菌体＋SNI组（pSNI组）和pNR2B＋SNI组（pNR2B/SNI组）于相应时点分别注射无关噬菌体或携带NR2B模拟表位的pNR2B,于首次注射后1周时行左后肢SNI术。持续观察大鼠痛行为学,分别于SNI术前1d、术后1d、1、2和3周测定左后爪机械刺激缩足反应阈值（MWT）。首次注射后4周,采用间接ELISA法测定血清NR2B抗体和噬菌体抗体水平。结果与C组和pNR2B组比较,SNI组和pSNI组术后1d出现痛行为学症状,MWT降低,并持续至术后3周;pNR2B/SNI组术后1d、1、2周痛行为学症状无明显缓解,术后3周痛行为学症状明显缓解,MWT差异无统计学意义（P〉0.05）。与SNI组和pSNI组比较,pNR2B/SNI组术后1周痛行为学症状明显缓解,MWT升高,持续至术后3周。pNR2B组、pSNI组和pNR2B/SNI组血清噬菌体抗体水平高于C组和SNI组（P〈0.05）,pNR2B组和pNR2B/SNI组血清NR2B抗体水平明显高于其他3组（P〈0.05）。结论NR2B模拟表位通过诱导SNI致慢性神经病理性痛大鼠产生NR2B抗体,产生明显的镇痛效应。

Objective To investigate the analgesic effect of the mimic epitope of NMDA receptor 2B subunit （NR2B） screened from a 12-mer Ph.D.random peptide library on chronic neuropathic pain.Methods Twenty-five male SD rats weighing 200-250 g were randomly divided into 5 groups （n=5 each）：groupⅠblank control;groupⅡsciatic nerve injury （SNI）;groupⅢspecific phase expressing a mimic epitope of NR2B （pNR2B）;groupⅣpNR2B＋SNI and groupⅤnon-specific phage＋SNI （pSNI）.In SNI group （Ⅱ） spared nerve injury,was performed.The common fibular nerve and tibial nerve of the left leg were ligated and cut at 2-4 mm distal to the ligature according to Decosterd and Woolf.In pNR2B group （Ⅲ） pNR2B was amplified to 4×10^12pfu/ml,100μl of which was injected at 2 sites subcutaneously 3 times at one week intervals.In groupⅣand V pNR2B and non-specific phage were injected respectively 3 times at one week intervals as in groupⅢ,and SNI was performed at one week after first inoculation.The behavior of the rats was evaluated continuously and the withdrawal threshold to mechanical yon Frey stimulation （MWT） was measured on the day after surgery and once a week for 3 weeks.The animals were killed at 4 weeks after first inoculation and blood was collected for determination of serum levels of the specific antibody against NR2B and antibody against non-specific phage （EHSA）.Results Allodynia and decrease in MWT were observed from one day to the 3rd week after SNI in groupⅡ（SNI） andⅣ（pSNI） as compared with control group （Ⅰ）.Allodynia was significantly alleviated and MWT increased starting from the 1st week and reached baseline level at the 3rd week after SNI in the pNR2B＋ SNI group （groupⅣ）.The specific antibody against NR2B was induced in pNR2B （Ⅲ） and pNR2B＋SNI group （Ⅳ）.The antibody against non-specific phase was induced in groupⅢ,ⅣandⅤ.Conclusion The mimic epitope of NR2B screened from the 12-mer Ph.D.random peptide library can elicit the specific antibody against NR2B in rats with chronic neuropathic pain induced by SNI and alleviate hyperalgesia.