沉默髓样细胞白血病-1基因对大鼠脑缺血再灌注后自噬和梗死体积的影响

Effects of knock-down myeloid cell leukemia-1 on the autophagy and infarction volume after cerebral ischemia/reperfusion in rats

目的研究沉默髓样细胞白血病-1(Mcl-1)基因对大鼠脑缺血再灌注后自噬和梗死体积的影响。方法以线栓法制作SD大鼠大脑中动脉阻塞再灌注(MCAO)模型,脑缺血1 h再灌注72 h。SD大鼠按照体重随机分为正常组、模型组和实验组,每组10只。分别在MCAO术前1 h,模型组和实验组以立体定位并脑内注射慢病毒载体Mcl-1短发夹RNA(shRNA)及阴性对照shRNA液体。以免疫荧光染色检测Mcl-1表达,以免疫印迹法分析Mcl-1、自噬相关蛋白微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)和Beclin-1蛋白表达,以噻唑蓝(TTC)染色法测定并计算脑梗死体积。结果脑缺血再灌注后,在梗死灶周边的皮质和纹状体区,散在大量Mcl-1荧光阳性细胞。梗死灶周边的皮质和纹状体区Mcl-1蛋白水平,模型组为1.17±0.03,约为正常组0.21±0.04的5倍,组间比较差异有统计学意义(P<0.001)。注射慢病毒Mcl-1-shRNA载体显著下调脑缺血再灌注后Mcl-1表达,实验组Mcl-1蛋白水平为0.43±0.06,约为模型组的36%,组间差异有统计学意义(P<0.001)。梗死灶周边的皮质和纹状体区Beclin-1蛋白水平,模型组为0.86±0.03,约为正常组0.21±0.05的4倍,组间比较差异有统计学意义(P<0.001)。梗死灶周边的皮质和纹状体区LC3-Ⅱ蛋白水平,模型组为0.83±0.04,约为正常组0.19±0.05的4.3倍,组间比较差异有统计学意义(P<0.001)。注射慢病毒Mcl-1-shRNA载体显著上调脑缺血再灌注后LC3-Ⅱ表达,实验组LC3-Ⅱ蛋白水平为2.17±0.06,约为模型组的2.6倍,组间比较差异有统计学意义(P<0.001)。注射慢病毒Mcl-1-shRNA载体后,实验组的Beclin-1蛋白水平为1.94±0.05,约为模型组的2.3倍,组间比较差异有统计学意义(P<0.01)。实验组大鼠脑梗死体积为(0.38±0.02)mm3,显著高于模型组脑梗死体积的(0.29±0.01)mm3,组间比较差异有统计学意义(P<0.001)。结论沉默Mcl-1基因可显著上调脑缺血后自噬反应和增加脑梗死体积,推测Mcl-1在脑梗死中发挥神经保护作用。

Objective To investigate effects of RNA interference targeting myeloid cell leukemia - 1 （ Mcl - 1 ） on the autophagy reaction and infarction volume after cerebral ischemia/reperfusion in rats. Methods The middle cerebral artery occlusion （MCAO）was induced using the intraluminal suture occlusion technique in SD rats. Reperfusion of cerebral blood flow was allowed by gently removing the monofilament after 1 hour ischemia, followed by 72 h reperfusion. Rats were randomly assigned into 3 groups： normal group, model group and test group （n = 10）. The preparations of lentivirus vector targeting Mcl -1 short hairpin RNA （shRNA） and carrying scrambled shRNA were infused stereotactically into the ipsilateral hemispheric region at 1 h before MCAO in model group and test group, respectively. The Mcl - 1 expression was detected by immunofluorescence staining. The expressions of Mcl - 1, microtubule - associated protein 1 light chain 5 - Ⅱ （ LC3 - Ⅱ ） and Beclin - 1 protein in the brain were detected by Western blot. The cerebral infarct volume was measured by use of 2,3,5 -triphenyltetrazolium chloride（TFC） stained. Results The number of cells expressing strong Mcl - 1 immunoreactivity was significantly higher in the cortex and striatum in model group than that in normal group. Western blot confirmed that Mcl - 1 in model group with 1.17 ±0. 03 and in normal group with 0.21 ± 0. 04, Beclin - 1 in model group with 0. 86 ± 0.03 and in normal group with 0. 21 ±0. 05 and LC3 - Ⅱ in model group with 0. 83 ±0.04 and in normal group with 0. 19 ±0. 05, all increased significantly in the cortex and striatum surrounding the infarct core in model group than that in normal group （all P 〈0. 001 ）. Compared to model group, quantification analysis confirmed that the level of Mcl- 1 protein decreased, Beclin - 1 1.94 ± 0. 05 and LC3 - Ⅱ 2. 17 ± 0. 06 in test group upregulated, more importantly （ P 〈 0. 001 ）. The shRNA - mediated inhibition of Mcl - 1 efficiently increased infarction volume of test group with （0. 38 ± 0. 02 ） mm3 and in model group with （0.29 ±0.01） mm3, the difference was significantly （ all P〈0.001）. Conclusion shRNA - mediated inhibition of Mcl - 1 significantly up -regulated the autophagy reaction and infarction volume, and Mcl - 1 maybe play neuroprotective effect after cerebral ischemia/reperfusion.