3-methyladenine下调自噬对小鼠脑缺血再灌注损伤的影响

Effect of Autophagy Down-regulation by 3-methyladenine on Focal Ischemia-Reperfusion Injury in Mice

目的探讨自噬对脑缺血再灌注损伤的影响。方法 54只CD-1小鼠随机分为假手术对照组6只,缺血再灌注组24只,盐水对照组9只和3-MA组15只。缺血再灌注组再分为6、12、24和48h组,每组6只。采用线拴法建立t-MCAO模型。假手术组和缺血再灌注组小鼠不给药,盐水对照组小鼠给予5μL生理盐水;3-MA组小鼠麻醉后置于立体定位架,术前30min用Hamilton注射器于左侧侧脑室定位后分别注射5μL浓度为2、10、50mM的3-MA。采用Western blot半定量检测再灌注后缺血侧/左侧皮层、海马和纹状体LC3Ⅱ蛋白表达变化,TTC染色观察3-MA(10mM)组和对照组缺血再灌注后24h脑梗死体积及水肿率。结果缺血再灌注组6、12、24、48h皮层、海马和纹状体LC3Ⅱ/LC3Ⅰ蛋白表达均有升高趋势,其中皮层和海马区LC3Ⅱ/LC3Ⅰ蛋白表达于24h升高最为明显,与假手术组比较,差异有统计学意义(P<0.05);而缺血再灌注组各个时间点纹状体区LC3Ⅱ/LC3Ⅰ蛋白表达量与假手术组比较,差异均无统计学意义(P>0.05);与盐水对照组比较,3-MA(10mM)组小鼠脑梗死体积明显减少[(26.72±1.53)mm^3对(51.80±2.85)mm^3,P<0.001],水肿率明显降低[(7.82±0.75)%对(16.55±4.47)%,P<0.01]。结论自噬的下调对脑缺血再灌注损伤有神经保护作用。

Objective To investigate the effect of autophagy on the brain of mice with focal ischemia-reperfusion（IR） injury.Methods Fifty-four CD-1 mice were randomly divided into sham operation group（n=6）,IR group（n=24）,saline control group（n=9）,and 3-methyladenine group（n=15）.Every 6 mice in IR group were sacrificed at 6h,12 h,24h,and 48 h after reperfusion.T-MCAO model in mice was established with left middle cerebral suture occlusion.Saline control group was given 5｜xL saline.Mice from 3-methyladenine group was put on anesthesia and was injected 5｜xL 3-methyladenine at the concentrations of 2,10,and 50 mM to left venticle with Hamilton syringe 30 min before operation.The expression of LC3 Ⅱ protein in the cortex,hippocampus and striatum were detected by western blot.TTC staining method was used to observe cerebral infarction area and edema in the brain of mice at 24 h after reperfusion in 10 mM 3-methyladenine group and saline control group.Results Comparing to sham operation group,the expression of LC3Ⅱ/LC3 Ⅰ protein in the cortex,hippocampus and striatum at 6,12,24,and 48 h after reperfusion increased;the level of LC3Ⅱ/LC3 Ⅰ protein in the cortex and hippocampus at 24 h after reperfusion was the highest and significantly different（P〈0.05）,but no significant difference in he level of LC3Ⅱ/LC3 Ⅰ protein in the striatum was found at any time point between IR group and sham operation group（P〈0.05）.3-methyladenine（10mM） group had smaller infarction area and lighter swelling than saline control group did（26.72±1.53mm^3 vs 51.80±2.85mm^3,P〈0.001;7.82%±0.75%vs 16.55%±4.47%,P〈0.01）.Conclusion Down-regulation of autophagy plays an neuroprotective role in cerebral ischemia reperfusion injury.