粒细胞集落刺激因子在酒精使用障碍后卒中中的脑保护作用研究

Study on the Brain Protection of Granulocyte Colony-Stimulating Factor in Stroke after Alcohol Use Disorder

目的探讨大鼠酒精使用障碍(alcohol use disorder,AUD)后卒中应用粒细胞集落刺激因子(granulocyte colony-stimulating factor,G-CSF)治疗对小胶质细胞中转化生长因子α(transforming growth factor-α,TGF-α)表达的影响及其神经保护作用。方法选择雄性Sprague-Dawley大鼠,应用双瓶法构建AUD模型,在AUD模型基础上应用线栓法制作大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)局部脑缺血模型。MCAO前24 h侧脑室注射TGF-α小干扰RNA(small interfering RNA,siRNA)或阴性对照siRNA。缺血再灌注损伤1 h后腹腔注射G-CSF。大鼠随机分为AUD组(18只)、AUD+MCAO组(18只)、AUD+MCAO+G-CSF组(18只)、AUD+MCAO+G-CSF+TGF-αsiRNA组(18只)及AUD+MCAO+G-CSF+阴性对照siRNA组(6只)。记录大鼠酒精摄入量、酒精偏好变化及神经功能评分。采用2,3,5-氯化三苯基四氮唑染色法测定MCAO后24 h脑梗死体积。采用免疫荧光染色观察缺血侧大脑皮质小胶质细胞特异性标志物跨膜蛋白119(transmembrane protein 119,TMEM119)阳性细胞数量、G-CSF受体(G-CSF receptor,G-CSFR)及TGF-α蛋白表达的变化。采用免疫印迹法检测缺血侧脑组织TMEM119及TGF-α蛋白表达水平。结果大鼠酒精摄入量和酒精偏好随饮酒时间的增加而逐渐增加。G-CSFR在小胶质细胞中有明显的表达。与AUD组相比,AUD+MCAO组TMEM119和TGF-a蛋白荧光强度明显增强,阳性细胞数量增加。与AUD+MCAO组相比,AUD+MCAO+G-CSF组治疗后梗死体积明显缩小,大鼠神经功能缺损症状改善,TMEM119蛋白荧光强度明显减弱,阳性细胞数量减少;TGF-α蛋白荧光强度明显增强,阳性细胞数量增加。侧脑室注射TGF-αsiRNA后大鼠脑梗死体积增加,神经功能缺损加重。结论G-CSF通过增加小胶质细胞中TGF-α的表达,在AUD后卒中中发挥重要的脑保护作用。

Objective To examine t he effects of g ranulocyte colony-stimulating factor(G-CSF)t reatment on the expression of transforming growth factor-α(TGF-α)in microglia and its neuroprotective effects in stroke after alcohol use disorder(AUD)in rats.Methods M ale Sprague-Dawley r ats were u sed t o e stablish a n AUD m odel u sing t he d ouble bottle method.Based on the AUD model,the focal cerebral ischemia model of middle cerebral artery occlusion(MCAO)was established using the thread embolism method.TGF-αsmall interfering RNA(siRNA)or negative control siRNA was injected into the lateral ventricle 24 h before MCAO.G-CSF was injected intraperitoneally l h after ischemia-reperfusion injury.Rats were randomly divided into the AUD group(18 rats),the AUD+MCAO group(18 rats),the AUD+MCAO+G-CSF group(18 rats),the AUD+MCAO+G-CSF+TGF-αsiRNA group(18 rats),and the AUD+MCAO+G-CSF+control siRNA group(6 rats).Alcohol intake,changes in alcohol preference,and neurological function scores of the rats were recorded.The volume of cerebral infarction 24 h after MCAO was measured by 2,3,5-triphenyltetrazolium chloride staining.The number of positive cells of transmembrane protein 119(TMEM119),a specific marker of microglia in the cerebral cortex of the ischemic side,and the G-CSF receptor(G-CSFR),and the protein expression of TGF-αwere observed by immunofluorescence staining.The expression levels of TMEM119 and TGF-αprotein in brain tissue of the ischemic side were detected by Western blot.Results Alcohol intake and alcohol preference of rats gradually increased with the increase of drinking time.G-CSFR was significantly expressed in microglia.Compared with the AUD group,the fluorescence intensity of TMEM119 protein and TGF-αprotein in the AUD+MCAO group was significantly enhanced,and the number of positive cells increased.Compared with the AUD+MCAO group,the volume of infarction in the AUD+MCAO+G-CSF group was significantly reduced,and the symptoms of neurological impairment improved.The fluorescence intensity of TMEM119 protein was significantly weakened,and the number of positive cells decreased.The fluorescence intensity of TGF-αprotein was significantly enhanced,and the number of positive cells increased.Volume of cerebral infarction increased and neurological deficits aggravated in the rats after lateral ventricle injection of TGF-αsiRNA.Conclusions G-CSF plays an important brain protective effect in stroke after AUD by increasing the expression of TGF-αin microglia.