临床和实验医学杂志2024，Vol.23Issue(12) ：1233-1237.DOI:10.3969/j.issn.1671-4695.2024.12.001

G-CSF介导的小胶质细胞自噬活性降低在大鼠酒精使用障碍脑卒中中的脑保护作用研究

Study of the cerebroprotective role of G-CSF-mediated reduction of microglia autophagic activity in alcohol use disorder stroke in rats

王力 高勇 胡晓璐 李丽 张杰
临床和实验医学杂志2024，Vol.23Issue(12) ：1233-1237.DOI:10.3969/j.issn.1671-4695.2024.12.001
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G-CSF介导的小胶质细胞自噬活性降低在大鼠酒精使用障碍脑卒中中的脑保护作用研究

Study of the cerebroprotective role of G-CSF-mediated reduction of microglia autophagic activity in alcohol use disorder stroke in rats

王力 1高勇 1胡晓璐 2李丽 3张杰3
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作者信息

  • 1. 山西省汾阳医院麻醉科 山西 汾阳 032200
  • 2. 北京市房山区妇幼保健院麻醉科 北京 102401
  • 3. 首都医科大学附属北京友谊医院麻醉科 北京 100050
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摘要

目的 探讨大鼠酒精使用障碍(AUD)脑卒中后粒细胞集落刺激因子(G-CSF)治疗对小胶质细胞自噬活性的影响及其神经保护作用.方法 将54只SD雄性大鼠按照随机数字表法分为AUD组、AUD+大脑中动脉栓塞(MCAO)组、AUD+MCAO+G-CSF组,每组各18只.AUD组用20％酒精双瓶法构建AUD大鼠模型,AUD+MCAO组、AUD+MCAO+G-CSF组均在AUD模型基础上制作MCAO局部脑缺血模型,AUD+MCAO+G-CSF组大鼠缺血再灌注损伤1 h后腹腔注射G-CSF 50μg/kg治疗.每天记录所有大鼠的酒精摄入量及偏好.观察各组大鼠脑缺血再灌注损伤24 h后检测脑梗死面积和神经功能评分.采用免疫荧光染色及蛋白质印迹法检测各组大鼠缺血侧大脑皮层小胶质细胞特异性标志物TMEM119、自噬标记物LC-3的表达水平.结果 到末次饮酒28 d时酒精摄入量已达到稳定水平,酒精偏好明显增加.与AUD组比较,AUD+MCAO组的脑梗死面积显著升高,神经功能评分显著减少,差异均有统计学意义(P＜0.05);与AUD+MCAO组比较,AUD+MCAO+G-CSF组的脑梗死面积明显减少,神经功能评分明显升高,差异均有统计学意义(P＜0.05.免疫荧光染色结果显示:与AUD组比较,AUD+MCAO组TMEM119和LC-3蛋白表达荧光强度明显增强,差异均有统计学意义(P＜0.05);与AUD+MCAO组比较,AUD+MCAO+G-CSF组TMEM119和LC-3蛋白表达荧光强度均明显减弱,差异均有统计学意义(P＜0.05).此外,免疫荧光显示TMEM119和LC-3二者有明显共定位.蛋白质印迹法结果显示,TMEM119和LC-3在AUD组表达微弱,与AUD组比较,AUD+MCAO组TMEM119和LC-3蛋白表达均明显升高,差异均有统计学意义(P＜0.05);与AUD+MCAO组比较,AUD+MCAO+G-CSF组TMEM119和LC-3蛋白表达明显降低,差异均有统计学意义(P＜0.05).结论 G-CSF通过降低小胶质细胞的自噬活性,在AUD脑损伤中发挥重要的脑保护作用.

Abstract

Objective To observe the effect of granulocyte colony-stimulating factor(G-CSF)on microglia autophagic activity and its neuroprotective effects after alcohol use disorder(AUD)stroke in rats.Methods Fifty-four SD male rats were divided into the AUD group,the AUD+Middle cerebral artery occlusion(MCAO)group and the AUD+MCAO+G-CSF group according to the random number table method,with 18 mice in each group.The AUD group constructed an AUD rat model using the 20％alcohol double bottle method,the AUD+MCAO group,AUD+MCAO+G-CSF group made MCAO local cerebral ischemia models based on the AUD model,and the AUD+MCAO+G-CSF group rats were treated with intraperitoneal injection of G-CSF 50 μg/kg 1 hour after ischemia-reperfusion injury.The daily alcohol intake and preferences of all rats were recorded.The cerebral infarction area and neurological function score were observed 24 hours after cerebral ischemia-reperfusion injury in each group of rats.Changes in the number of positive cells for microglia-specific marker TMEM119 and autophagy marker LC-3 in the cerebral cortex on the ischemic side were quantified using Western blotting analysis and the localizations of each was visualized via immunofluorescence staining.Results By the end of the last 28 days of drinking,alcohol intake had reached a stable level and alcohol preference had significantly increased.Compared to the AUD group,the stroke area of the AUD+MCAO group was significantly increased,and the neurolog-ical function score of the AUD+MCAO group was significantly decreased,the differences were statistically significant(P＜0.05);compared with the AUD+MCAO group,the cerebral infarction area of AUD+MCAO+G-CSF group was significantly reduced,and the neurological func-tion score of the AUD+MCAO group was significantly increased,the differences were statistically significant(P＜0.05).The immunofluores-cence staining results showed that compared with the AUD group,the fluorescence intensity of TMEM119 and LC-3 protein expression was signifi-cantly enhanced in the AUD+MCAO group,and the differences were statistically significant(P＜0.05);Compared with the AUD+MCAO group,the fluorescence intensity of TMEM119 and LC-3 protein expression was significantly reduced in the AUD+MCAO+G-CSF group,and the differences were statistically significant(P＜0.05).In addition,immunofluorescence showed significant co localization between TMEM119 and LC-3.The results of protein blotting showed that,TMEM119 and LC-3 were weakly expressed in the AUD group compared to the AUD group,the expressions of TMEM119 and LC-3 proteins were significantly increased in the AUD+MCAO group,and the differences were statisti-cally significant(P＜0.05);compared with the AUD+MCAO group,the expressions of TMEM119 and LC-3 proteins werr significantly re-duced in the AUD+MCAO+G-CSF group,and the differences were statistically significant(P＜0.05).Conclusion G-CSF plays an im-portant cerebroprotective role in MCAO brain injury following AUD by decreasing microglial autophagic activity.

关键词

大鼠/酒精使用障碍/脑卒中/小胶质细胞/自噬/粒细胞集落刺激因子

Key words

Rats/Alcohol use disorders/Stroke/Microglia/Autophagy/Granulocyte colony-stimulating factor

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基金项目

国家自然科学基金面上项目(82271307)

出版年

2024
临床和实验医学杂志
首都医科大学附属北京友谊医院

临床和实验医学杂志

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影响因子：1.504
ISSN：1671-4695
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