摘要
目的观察神经调节素-1β(Neuregulin-1β，NRG-1β)对大鼠大脑中动脉缺血(MCAO)性损伤的预防和治疗作用，探讨MCAO的病理生理学机制及NRG-1β治疗的最佳时机和神经保护作用。
方法成年健康Wistar大鼠475只，随机分为假手术组、对照组、预处理组、治疗组。应用线栓法建立MCAO模型，经左颈外-颈内动脉单剂量注射NRG-1β干预治疗。Longa法进行神经功能学评分，干湿重法测量脑组织含水量，氯化三苯基四氮唑(TTC)染色法测定脑梗塞体积，TUNEL法检测细胞凋亡，免疫组化、免疫荧光双标法、免疫印迹及RT-PCR等检测AQP-4、Erg-1、STAT-3、GFAP、MMP-9、NSE、XIAP和Smac等蛋白及其mRNA表达水平的变化。
结果①模型建立后，对照组大鼠均出现神经功能缺失症状，脑组织含水量和脑梗塞体积增大，NRG-1β预处理及治疗能明显改善大鼠神经功能缺损症状，减少脑组织含水量及脑梗死体积。②缺血再灌注能诱导神经细胞凋亡，减少抗凋亡蛋白XIAP的水平，增加凋亡蛋白Smac的表达；NRG-1β预处理能减少大鼠缺血脑组织细胞凋亡数，明显增加抗凋亡蛋白、减少促凋亡蛋白的表达水平、均衡抗凋亡蛋白(XIAP)／促凋亡蛋白(Smac)的表达水平、促进某些及早基因(Erg-1)的表达、有效抑制脑损伤后炎症反应的发生发展；与对照组相比存在显著差别(P＜0．05)。③缺血后24 h内给NRG-1β干预均能减少大鼠缺血脑组织细胞凋亡数，抑制脑损伤后炎症反应，尤其以缺血1h给药效果最好。NRG-1β的神经保护作用至少持续至给药后24 h。④随着缺血时间的延长，对照组脑组织AQP-4、STAT-3、GFAP、MMP-9、NSE等蛋白的表达水平有不同程度的增加；NRG-1β治疗在一定时间内能增加AQP-4、STAT-3、GFAP和NSE的表达水平，减少MMP-9蛋白的表达量。
结论①NRG-1β预处理能诱发机体产生缺血性耐受，有效保护缺血脑组织继发性损伤；②NRG-1β对大鼠脑缺血再灌注损伤有积极的保护作用，缺血24 h内给药均可减轻脑损伤程度，缺血1 h给药可能是其最佳治疗时间窗，这种神经保护作用至少持续至给药后24 h。③NRG-1β的神经保护作用可能是通过激活某些及早基因、结合并活化其特异性受体、抑制凋亡途径中的多个环节、减少脑缺血再灌注后炎症反应的发生发展、诱导胶质细胞反应，改善神经元生存环境而实现的。④NRG-1β作为一种新型神经保护剂，可能对缺血性卒中患者有积极的治疗作用。
Objective The aim is to investigate the precaution and the treatment effects ofNeuregulin-1β(NRG-1β)on the middle cerebral artery occlusion (MCAO)injury in rats,toexplore the pathophysiology mechanism of brain damage,the perfect therapeutic time andthe neuroprotection of NRG-1βin rats after MCAO.
Methods Four hundred and seventy-five MCAO animal models were establishedwith a suture and randomly designed into a sham-operation group,a control group,apretreatment group and a treatment group.NRG-1βwas administrated through theexternal-internal carotid artery (ICA)for intervention.Neurology score was evaluated withLonga's.Brain water content (BWC)was detected with dry-humid weight method.Tetrazolium chloride (TTC)stain was used to count the volume percentage of the cerebralinfarction.Cell apoptosis was observed by TUNEL.And the alternation of aquaporin-4(AQP-4),Erg-1,STAT-3,GFAP,MMP-9,NSE,XIAP and Smac and their mRNA weredetermined through immunohistochemistry,double immunofluorescence labeling,immunoblotting and RT-PCR.
Results①After the animal models were accomplished,all rats in the control groupshowed the neurology deficit symptom.Their BWC and the volume percentage of cerebralinfarction increased while the pretreatment and the treatment of NRG-1βobviouslyimproved the neurology deficit and decreased BWC and the infaction size.②NRG-1βpretreatment could attenuate the amount of cell apotosis,balanced the expression levels ofanti-apoptosis protein (XIAP)/pro-apoptosis protein (Smac),promote the presence of earlygrowth response gene-1 (Egr-1),actively suppress the development of inflammatoryresponse after ischemia cerebral damage.There were significant differences compared withthat in the control group (P＜0.05).③The administration of NRG-1βwithin 24 h aftercerebral ischemia could decrease the number of cell apoptosis in brain tissue and inhibit theinflammatory response,especially within 1.0 h,suggesting that the perfect therapeutic timewindow was at ischemic 1.0 h after MCAO.Its neuro-protective effects kept at least 24 h after the administration.④In the control group,ischemia and reperfusion could induceneurocyte apoptosis,reduce the expression levels of anti-apoptosis protein XIAP,andelevated the presence of pro-apoptosis protein Smac.NRG-1βtreatment significantlyincreased the anti-apoptosis and decreased the pro-apoptosis protein expressions in giventime after MCAO.⑤With the duration of brain ischemia,the increased expressions ofAQP-4,STAT-3,GFAP,MMP-9 and NSE proteins were shown in brain tissues to someextent;NRG-1βinjection could elevate AQP-4,STAT-3,GFAP and NSE and decreaseMMP-9 expression levels in some certain periods.
Conclusion①NRG-1βpretreatment can stimulate the protective tolerance and efficientlykeep brain tissue from the second ischemic damage;②NRG-1βsignificantly attenueatesthe degree of brain damage,plays an active neuro-protective effects inischemia-reperfusion rats,which kept at least 24 h after the administration.The bestadministration time may be ischemic 1.0 h.③The neuroprotective effect of NRG-1βmay be achieved through activating early response gene,combining with its specialreceptors,inhibiting different links in the apoptosis pathway,reducing the development ofinflammatory response after cerebral ischemia and reperfusion,promoting the gliocyteproliferation and improving the survival environment of neurons.④As a newneuroprotective regent,NRG-1βis potent to apply for the clinical treatment of ischemicstroke.
引文
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