Necrostatin-1对大鼠脑缺血再灌注损伤后小胶质细胞和炎症因子的影响

Influence of necrostatin-1 in microglia activation and inflammatory factor levels after cerebral ischemia/reperfusion damage in rats

目的探讨干预程序性坏死途径对大鼠脑缺血再灌注损伤后小胶质细胞活化及相关炎症因子的影响及意义。方法采用改良线栓法构建大鼠右侧大脑中动脉缺血再灌注模型。（1）SD大鼠按随机数字表法分为脑缺血再灌注6h组、脑缺血再灌注12h组、脑缺血再灌注24h组、脑缺血再灌注72h组，每组6只。免疫组化染色检测不同时间点小胶质细胞激活标志离子型钙接头蛋白-1（iba-1）的表达，根据实验结果选取iba-1表达相对明显的时间点。（2）SD大鼠按随机数字表法分为假手术组、脑缺血再灌注组、Necrostatin-1（Nec-1）（80nm01）干预组、Nec-1（160nmol）干预组．每组6只，并于脑缺血后2h给予Nec-1干预。采用Longa法进行神经功能损伤评分、TTC法检测梗死体积，根据实验结果选取Nec-1干预的适宜给药剂量。（3）SD大鼠按随机数字表法分为假手术组、脑缺血再灌注组、溶剂组、Nec-1干预组，每组6只，并于脑缺血后2h给予Nec．1或DMS0溶剂干预。HE染色观察梗死周围组织神经元存活情况，免疫组化染色检测梗死周围组织iba-l的表达．免疫组化染色及Westernblotting分别检测炎症因子肿瘤坏死因子-α（TNF-α）、白介素-1β（IL-1β）、胶质源性神经营养因子（GDNF）免疫阳性细胞及蛋白的表达。结果（1）iba-1表达在脑缺血再灌注24h后升高明显，与其他各时间点比较差异有统计学意义（（P〈0．05）。（2）与脑缺血再灌注组相比，两Nec．1干预组神经功能损伤评分明显降低、梗死体积明显减小，差异均有统计学意义（P〈0．05）；与Nec-1（80nmoll干预组相比，Nec-1（160nmol）干预组神经功能损伤及梗死体积的改善效果更为明显，差异均有统计学意义（氏0．05）。（3）HE染色显示脑缺血再灌注组梗死周围组织炎症细胞浸润、神经元减少、胞体皱缩、细胞核固缩；与脑缺血再灌注组相比，Nec-1干预组神经元增多、核固缩减轻、胞质染色较深。与脑缺血再灌注组相比，Nec-1干预组iba-1、TNF-α、IL-1β、GDNF免疫阳性细胞及蛋白表达均明显减少，差异均有统计学意义（P〈0．05）。结论程序性坏死途径参与大鼠脑缺血再灌注损伤后小胶质细胞的活化；Nec-1干预可抑制小胶质细胞的活化，并通过对炎症因子的调控从而减轻神经元损伤。

Objective To investigate the influence of necroptosis pathways in microglia activation and inflammatory factor levels in cerebral ischemia/reperfusion （I/R） injury in rats and their significances. Methods A modified suture method was used to establish the models of middle cerebral artery FR in rats. （1） SD rats according to the random number table were divided into cerebral UR 6 h group, cerebral I/R 12 h group, cerebral FR 24 h group, and cerebral I/R 72 h group （n=6）; the expression of ionized calcium binding adaptor molecule-1 （iba-1） was detected by immunohistochemical staining, and time points enjoyed obvious iba-1 expression were selected according to the experimental results. （2） SD rats were randomly divided into sham operation group, cerebral I/R group, 80 nmolnecrostatin-1 （Nec-1） intervention group and 160 nmol Nec-1 intervention group （n=6）, and the Nec-1 intervention was given 2 h after ischemia; Longa method was used to evaluate the neurological function scores and TTC method was used to detect the infarct volume; and the appropriate dosages of Nec-1 were selected according to these results. （3） SD rats were randomly divided into sham operation group, cerebral I/R group, solvent group and Nec-1 intervention group （n=6）, and Nec-1 or DMSO intervention was given 2 h after ischemia. HE staining was used to observe the survival and proliferation of microglias around the infarction tissues; immunohistochemical staining was used to detect the iba-1 expression surrounding the infarction tissues; immunohistochemistry and Western blotting were employed to observe the cytokine tumor necrosis factor （TNF）-α and interleukin （IL）-1β and glia-derived neurotrophic factor （GDNF） expressions. Results （1） The iba-1 expression at cerebral I/R 24 h group was significantly increased as compared with that in other groups （P〈0.05）. （2） As compared with those in the cerebral I/R group, the neurological function scores and infarct volume were significantly decreased in the 80 nmol Nec-1 intervention group and 160 nmol Nec-1 intervention group （P〈0.05）; more obvious changes were noted in the 160 nmol Nee-1 intervention group as compared with those in the 80 nmol Nec-1 intervention group, with significant difference （P〈0.05）. （3） HE staining showed peri-infarct tissue inflammatory cell infiltration, reduced neuron number, cell body shrinkage and nuclear pyknosis in the cerebral I/R group, while these changes were less obvious in the Nec-1 intervention group; as compared with cerebral FR group, the Nec-1 intervention group had significantly decreased expressions of iba-1, TNF-α, IL-1β and GDNF （P〈0.05）. Conclusion Necroptosis pathway involves in the activation of microglias after I/R injury in rat brains; Nec-1 inhibits the activation ofmicroglia, and reduces the neuronal damage by regulating inflammatory cytokines.