目的:探讨洛霉素A(Bafilomycin A1, Baf A1)对脑缺血再灌注损伤(middle cerebral artery occlusion-reperfusion, MCAO/R)小鼠的保护作用及机制。方法:Atg7f/f-Mx1-Cre小鼠15只,随机分为:假手术组(C)、模型组(M)、氟西汀组(M + F)、Baf A1组(M + B)和ATG7敲除组(M + ATG7 KO)。M + B组术前24小时给与脑立体定位注射,M + F组给与氟西汀灌胃连续14 d;其他组均于术前14天开始给予等体积生理盐水。缺血2小时再灌注24 h后,对各组小鼠进行神经行为学评分、脑含水量测定和脑梗死面积检测,Western blotting检测自噬相关蛋白Beclin 1和LC3的表达。结果:与C组相比,M组神经行为评分、脑含水量、脑梗死面积、自噬相关蛋白Beclin 1和LC3的表达显著增加,M + F组、M + B组和M + ATG7 KO组逆转以上结果,差异具有统计学意义。结论:Baf A1通过降低前期和后期细胞自噬水平实现对脑缺血再灌注损伤小鼠的保护作用。 Objective: To investigate the protective effect and mechanism of Bafilomycin A1 on middle cerebral ischemia-reperfusion injury in mice. Methods: 15 Atg7f/f-Mx1-Cre mice were randomly divided into sham group (C), model group (M), fluoxetine group (M + F), Baf A1 group (M + B) and ATG7 knockout group (M + ATG7 KO). Group (M + B) was given stereotactic injection 24 hours before operation, Group (M + F) was given intragastric fluoxetine for 14 days, and other groups were given an equal volume of saline 14 days before the operation. After 2 hours of ischemia and 24 hours of reperfusion, the mice in each group were evaluated by neurobehavioral score, cerebral water content and cerebral infarction area. The expression of autophagic related proteins Beclin 1 and LC3 was detected by western blotting. Results: Compared with Group C, the neurobehavioral score, cerebral water content, cerebral infarct size, autophagy associated protein Beclin 1 and LC3 were signifi- cantly increased in group M, and reversed in Group (M + F), Group (M + B) and Group (M + ATG7 KO) and the difference was statistically significant. Conclusion: Baf A1 may protect mice from cerebral ischemia-reperfusion injury by decreasing autophagy levels in the early and late stages.
目的:探讨洛霉素A(Bafilomycin A1, Baf A1)对脑缺血再灌注损伤(middle cerebral artery occlusion-reperfusion, MCAO/R)小鼠的保护作用及机制。方法:Atg7f/f-Mx1-Cre小鼠15只,随机分为:假手术组(C)、模型组(M)、氟西汀组(M + F)、Baf A1组(M + B)和ATG7敲除组(M + ATG7 KO)。M + B组术前24小时给与脑立体定位注射,M + F组给与氟西汀灌胃连续14 d;其他组均于术前14天开始给予等体积生理盐水。缺血2小时再灌注24 h后,对各组小鼠进行神经行为学评分、脑含水量测定和脑梗死面积检测,Western blotting检测自噬相关蛋白Beclin 1和LC3的表达。结果:与C组相比,M组神经行为评分、脑含水量、脑梗死面积、自噬相关蛋白Beclin 1和LC3的表达显著增加,M + F组、M + B组和M + ATG7 KO组逆转以上结果,差异具有统计学意义。结论:Baf A1通过降低前期和后期细胞自噬水平实现对脑缺血再灌注损伤小鼠的保护作用。
洛霉素A1,脑立体定位注射,脑缺血再灌注损伤(MCAO/R),ATG7,细胞自噬
Leyi Yin, Zhijian Wang
Jiaxing University College of Medicine, Jiaxing Zhejiang
Received: Jan. 3rd, 2022; accepted: Mar. 21st, 2022; published: Mar. 29th, 2022
Objective: To investigate the protective effect and mechanism of Bafilomycin A1 on middle cerebral ischemia-reperfusion injury in mice. Methods: 15 Atg7f/f-Mx1-Cre mice were randomly divided into sham group (C), model group (M), fluoxetine group (M + F), Baf A1 group (M + B) and ATG7 knockout group (M + ATG7 KO). Group (M + B) was given stereotactic injection 24 hours before operation, Group (M + F) was given intragastric fluoxetine for 14 days, and other groups were given an equal volume of saline 14 days before the operation. After 2 hours of ischemia and 24 hours of reperfusion, the mice in each group were evaluated by neurobehavioral score, cerebral water content and cerebral infarction area. The expression of autophagic related proteins Beclin 1 and LC3 was detected by western blotting. Results: Compared with Group C, the neurobehavioral score, cerebral water content, cerebral infarct size, autophagy associated protein Beclin 1 and LC3 were significantly increased in group M, and reversed in Group (M + F), Group (M + B) and Group (M + ATG7 KO) and the difference was statistically significant. Conclusion: Baf A1 may protect mice from cerebral ischemia-reperfusion injury by decreasing autophagy levels in the early and late stages.
Keywords:Bafilomycin A1, Stereotactic Injection, MCAO/R, ATG7, Cell Autophagy
Copyright © 2022 by author(s) and Hans Publishers Inc.
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缺血性脑损伤的发病率逐年升高,目前已成为威胁人类生命的最主要疾病之一 [
Atg7f/f-Mx1-Cre小鼠15只,将含Atg7基因打靶序列的纯合转基因小鼠,与诱导型的Mx1-Cre转基因小鼠(由美国Jackson实验室引进)交配,进而筛选获得,可以用pIpC诱导敲除Atg7基因的转基因小鼠模型(Atg7f/f-Mx1-Cre)。
B组术前24小时给与脑立体定位注射,F组给与氟西汀灌胃连续14 d,其他组均于术前14天开始给予等体积生理盐水。
采用改良longa. Zea氏线栓法 [
采用改良Bederson 评分法进行神经功能缺损评分 [
小鼠麻醉后颈椎脱臼,冰上快速断头取脑。把脑组织迅速放入−20℃冰箱冷冻10 min,切成2 mm的冠状切片放入六孔板,加入1% TTC溶液,避光,37℃烘箱30 min后取出按顺序摆好位置,拍照分析梗死面积,正常组织被染成鲜红色,梗死组织不染色为白色,计算出梗死体积占大脑体积的百分比。
麻醉小鼠后开颅取新鲜大脑,用滤纸吸干表面水分,电子秤称量脑湿重,然后将其置于105℃的烤箱烘烤48 h至恒重,称量干重,计算脑含水量。脑组织含水量 = (脑湿重 − 脑干重)/脑湿重 × 100%。
收集大鼠左侧海马组织,提取组织总蛋白用BCA试剂盒进行蛋白定量和变性后,进行SDS-PAGE电泳(凝胶浓度为10%和5%,80 V,30 min后换110 V,70 min),转膜后于5%脱脂奶粉室温封闭1 h,加入一抗(Beclin 1、LC3) 4℃冰箱过夜,加入辣根过氧化物标记的兔源二抗,显影定影后用ImageJ分析蛋白相对表达量。
所有实验均重复3次以上,数据采用GraphPad Prism8.0处理。结果以均数±标准差(mean ± SD)表示,各组间计量资料的比较采用单因素方差分析(One-Way ANOVA),以P < 0.05为差异有统计学意义。
图1显示,与C组相比,M组神经行为评分、脑梗死面积和脑含水量显著增加,而M + F组、M + B组和M + ATG7 KO组神经行为评分、脑梗死面积和脑含水量均显著降低,差异具有统计学意义。
提取损伤侧海马组织总蛋白,图2结果显示,与C组相比,M组细胞自噬信号通路蛋白Beclin1和LC3表达显著增加,而M + F组、M + B组和M + ATG7 KO组细胞自噬信号通路蛋白Beclin1、LC3表达显著降低,差异具有统计学意义。
图1. 5组小鼠神经行为评分、脑梗死面积和脑含水量的变化。(a) 实验过程;(b) 神经行为学评分;(c) 脑梗死面积;(d) 脑含水量
图2. 5组小鼠海马自噬相关蛋白的变化。(a) 免疫印迹;(b) Beclin1统计学结果;(c) LC3统计学结果
我国已经进入老龄化社会,缺血性脑损伤的发病率逐年升高,目前已成为威胁人类生命的最主要疾病之一,由于发病率、致死率或致残率高给社会和家庭造成严重危害,缺血组织恢复血流后会引起更严重的组织损伤和功能障碍,并且治疗效果非常差,临床称为缺血再灌注损伤 [
研究表明,在脑缺血、炎症反应等应激状态下,神经元内Beclin1、LC3、ATG5、LAMP1等自噬相关蛋白表达增加,自噬溶酶体途径被激活且参与了脑缺血再灌注损伤 [
本实验采用改良longa. Zea氏线栓法 [
感谢浙江省实验动物科技计划项目(No. LGD19C090002)支持。
殷乐依,王志坚. Bafilomycin A1对脑缺血再灌注损伤小鼠的保护作用及其机制The Protective Effect and Mechanism of Bafilomycin A1 on Middle Cerebral Ischemia-Reperfusion Injury in Mice[J]. 生物医学, 2022, 12(02): 97-102. https://doi.org/10.12677/HJBM.2022.122012