核转录因子-κB拮抗剂对大鼠海马CA1区局灶性脑缺血再灌注损伤的影响

The effect of the nuclear factor-κB inhibitor on the focal cerebral ischemia reperfusion injury in the hippocampal CA1 region of rats

目的观察局灶性脑缺血再灌注后大鼠右侧海马CA1区的远隔损害变化及核转录因子（NF）-KB拮抗剂的调节作用，探讨远隔损害区内的炎性变化及NF-κB拮抗剂抗炎调节作用的可能机制。方法将Sprague-Dawley（SD）雄性大鼠按照随机数字表法随机分为假手术组（n=24）、模型组（缺血再灌注组；n=38）、药物组[NF-κB必需调节蛋白结合区（NBD）多肽组；n=38]及对照组（NBD转化多肽组；n=38），按再灌注后的时间点24h和7d再将上述4组各分为2个亚组。利用改良线栓法制备右侧大脑中动脉闭塞再灌注模型。利用右侧海马注射NF-κB拮抗剂NBD多肽及NBD转化多肽干预后检测白细胞介素1β和白细胞介素1-Ra的含量变化，采用苏木素-伊红（HE）及Fluoro-JadeB（FJB）染色法观察神经元的损伤及表达变化，利用免疫荧光双标及蛋白质印迹法检测神经细胞内NF-KBp65及NF-KB抑制蛋白α的表达量及表达部位。结果模型组大鼠海马CA1区细胞胞核内NF-κBp65蛋白的表达（24h，2．340±0．101；7d，2．440±0．081）较药物组（24h，0．206±0．013；7d，0．090±0．012）及假手术组（24h，0．120±0．007；7d，0．100±0．014）明显升高（q=64．431、66．704、67．747、56．624，均P〈0．05），同时模型组的IL-1β含量[24h（1．850±0．192）ng／ml，7d（1．000±0．178）ng／m1]较药物组[24h（1．250±0．211）ng／ml，7d（0．560±0．183）ng／m1]显著升高（q=10．730、9．710，均P〈0．05）。模型组（24h，27．50％±3．59％；7d，28．10％±4．46％）及对照组（24h，27．30％±4．53％；7d，26．30％±5．03％）中HE染色显示神经细胞存活百分比相较药物组（24h，58．90％±3．46％；7d，68．40％±4．20％）明显降低（q=19．949、19．731、2．139、22．249，均P〈0．05），FJB染色显示模型组（24h，28．10±2．13；7d，29．50±2．45）神经细胞损伤数目明显高于药物组（24h，12．50±2．41；7d，9．30±2．52，q=3．211、4．521，均P〈0．05），模型组与对照组上述结果差异无统计学意义（P〉0．05）；与其他3组比较（假手术组24h，0．130±0．008；7d，0．150±0．010；模型组24h，1．340±0．213；7d，1．750±0．119，对照组24h，1．250±0．114；7d，1．620±0．097），药物组大鼠海马细胞胞核内IκBα蛋白表达（24h，1．680±0．148；7d，2．010±0．085，q=6．348、9．139、9．414、1．711、5．277、5．555，均P〈0．05）明显升高。与模型组[24h（0．570±0．028）ng／ml，7d（0．430±0．039）ng／m1]及对照组[24h（0．490±0．042）ng／ml，7d（0．380±0．018）ng／m1]比较，药物组内IL-1Ra含量[24h（1．390±0．055）ng／ml，7d（1．250±0．043）ng／ml，q=4．577、6．205、9．683、6．389，均P〈0．05]明显升高。结论脑缺血再灌注能引起大鼠海马区的远隔损害并诱发严重的炎性反应，NBD多肽能通过改变NF-κBp65及IκBα蛋白的表达变化有效抑制炎性反应，从而缓解脑缺血再灌注诱发的远隔损害。

Objective To observe the effect of the nuclear factor （NF）-κB inhibitor on the inflammatory injury and the secondary remote damage in remote areas of the CA1 region in the right hippocampus of the focal cerebral ischemie/reperfusion rats, and the NF-κB essential modifier binding domain （NBD） peptide was used to inhibit the activation of NF-κB signaling pathway to explore the function and mechanism of the NBD peptide in restraining inflammatory injury and reducing secondary remote damage in the hippocampus CA1 region. Methods According to the random number table, the male Sprague- Dawley （SD） rats were randomly divided into a sham group （n = 24 ）, an ischemia/reperfusion （I/R） group （ n = 38 ） , a NBD group （ n = 38 ） and a modified type peptide （ MT-NBD ） group （ n = 38 ） , then at the time point of 24 h and 7 d after reperfusion, the above 4 groups were divided into 2 subgroups. The experimental models were made by middle cerebral artery occlusion （modified line plug method） for 2 hours. The NBD peptide and the MT-NBD peptide were respectively injected into the right hippocampus of the experimental groups. The injury of neurons was examined by the methods of H＆E and Fluoro-Jade B-（FJB） staining. The levels of interleukin-1β （IL-1β） and IL-1Ra were detected by using enzyme-linked immunosorbent assay. The protein expressions of NF-κB 1365 and IκBα were analyzed by Western blotting and the double-labelling immunofluorescence. Results Compared with the NBD group （ 24 h 0. 206 ± 0. 013, 7 d 0. 090 ± 0. 012） and the sham group （24 h 0. 120 ± 0. 007, 7 d 0. 100 ± 0. 014）, the NF-κB 1365 protein expression in the I/R group （ 24 h 2. 340 ± 0. 101, 7 d 2. 440± 0. 081 ） was increased significantly （ q = 64. 431, 66. 704, 67.747, 56. 624, all P 〈 0. 05 ）. The level of IL-1β was remarkably increased in the I/R group （24 h （ 1. 850 ±0. 192） ng/ml, 7 d （ 1. 000 ±0. 178） ng/ml） compared with the NBD group （24h （1.250±0.211） ng/ml, 7 d （0.560±0.183） ng/ml, q=10.730, 9. 710, P〈 0.05）. The percent of survival neurons was significantly lower in the I/R group （24 h 27. 50%± 3.59% , 7 d 28. 10%±4.46% ） and the MT-NBD group （24 h 27.30% ±4. 53% ,7 d 26. 30% ±5.03% ）than the NBD group （24 h 58.90% ±3.46%, 7 d 68.40% ±4. 20%, q = 19. 949, 19. 731, 2. 139, 22. 249, all P 〈0.05）. The FJB staining showed that the number of neuron degeneration in the I/R group （24 h 28.10 ±2. 13, 7 d 29. 50 ±2. 45） was higher than the NBD group （24 h 12. 50 s2.41, 7 d 9. 30 s2. 52, q = 3. 211,4. 521, P 〈 0. 05 ）. Compared with the other three groups （ sham group- 24 b 0. 130 ± 0. 008, 7 d 0. 150 ±0. 010; I/R group： 24 h 1. 340±0. 213, 7 d 1. 750 ±0. 119; MT-NBD group： 24 h 1. 250 ± 0. 114, 7 d 1. 620± 0. 097） , the IκBα protein expression in the NBD group （24 h 1. 680 ± 0. 148, 7 d 2. 010 ±0. 085） was significantly increased （q = 6. 348, 9. 139, 9. 414, 1. 711, 5. 277, 5. 555, all P 〈 0.05）. Compared with the I/R group （24 h （0. 570±0. 028） ng/ml, 7 d （0. 430 ±0. 039） ng/ml） and the MT-NBD group （24 h （0. 490 ±0. 042） ng/ml, 7 d （0. 380 ±0. 018） ng/ml）, the level of IL-1Ra in the NBD group （24 h （ 1. 390 ± 0. 055 ） ng/ml, 7 d （ 1. 250 ±0. 043 ） ng/ml） was remarkably increased （q =4. 577, 6. 205, 9. 683,6. 389, all P 〈 0. 05）. The results between the I/R group and the MT-NBD group were not significantly different. Conclusions The research shows that NBD peptide treatment contributes to altering the NF-κB p65/IκBα expression in nucleus effectively. And it directly regulates the NF-KB activation to alleviate the inflammatory injury in the hippoeampus CA1 region after the secondary remote damage.