JNK通过增强神经元自噬流提高大鼠缺血性脑卒中后的神经保护

JNK-mediated Neuroprotective Mechanism after Ischemic Stroke in Rats

为了探究应激激活蛋白激酶JNK在缺血性脑卒中后对神经元细胞的保护机制,本文采用栓线法构建雄性SD大鼠大脑中动脉梗塞(model of middle cerebral artery occlusion, MCAO)模型。在自噬变化特征性时间点加入JNK激动剂茴香霉素(anisomycin, AN),随后采用Western印迹和免疫荧光检测缺血半影区自噬流通路蛋白质表达水平,分析JNK对Bcl-2-Beclin1复合物稳定性的影响和对自噬流通路的影响。结果显示:与MCAO+Veh组相比:在12 h, AN组LC3(**P<0.01)、Beclin1(**P<0.01)和Bcl-2(*P<0.05)的表达水平显著升高,而切割胱天蛋白酶(cleaved caspase-3)的表达水平显著降低(**P<0.01),以及组织蛋白酶B(cathepsin B)(***P<0.001)和LAMP1(***P<0.001)表达水平均显著升高;在第2天,AN组的组织蛋白酶B(**P<0.01)和溶酶体关联膜蛋白1(recombinant lysosomal associated membrane protein1,LAMP1)(*P<0.05)表达水平显著降低。同时,通过神经功能损伤评分、TTC染色分析JNK活性激活对MCAO大鼠神经功能损伤、脑梗死体积的影响,结果显示:与MCAO+Veh组相比,MCAO+AN组的SD大鼠脑梗死体积显著减少(12 h:***P<0.001,第2 d:*P<0.05)且神经功能评分显著降低(12 h:***P<0.001,第2 d:**P<0.01)。上述结果提示:在缺血性脑卒中的急性期,JNK活性激活促进Bcl-2-Beclin1复合物解离,使神经元凋亡向自噬转换,提高了神经元的存活;在亚急性期,JNK活性激活使自噬产物堆积导致了神经元的自噬性损伤。

In order to explore the protective mechanism of stress-activated protein kinase JNK on neurons after ischemic stroke, the model of middle cerebral artery occlusion(MCAO) in male SD rats was established by suture methods. Anisomycin(AN), a JNK agonist, was added at the characteristic time point of autophagy, and then Western blotting and immunofluorescence were used to detect the protein expression of autophagy flow pathways in ischemic penumbra, and the effects of JNK on the stability of Bcl-2-Beclin1 complex and autophagy flow pathway were analyzed.The results showed that compared with the MCAO+Veh group, the expression levels of LC3(**P<0.01), Beclin1(**P<0.01) and Bcl-2(*P<0.05) atHour 12 and theANgroup were significantly increased, while the expression level of cleaved caspase-3 was significantly decreased(**P<0.01), and the expression levels of cathepsin B(***P<0.001) and Lamp1(***P<0.001) were significantly increased;On Day2, the expression levels of cathepsin B(**P<0.01)and recombinant lysosomal associated membrane protein1(LAMP1)(*P<0.05)were significantly increased.Meanwhile, the effects of JNK activation on neurological function damage and cerebral infarction volumes of MCAO rats were analyzed by neurological injury score and TTC staining, the results showed that: compared with the MCAO+Veh group, the MCAO+AN group significantly reduced the cerebral infarction volume(Hour 12:***P<0.001,Day 2:*P<0.05) and neurological function score(Hour 12:***P<0.001,Day 2:**P<0.01).These results suggest that: in the acute stage of ischemic stroke, JNK activation promotes the dissociation of the Bcl-2-Beclin1 complex, convertsneuronal apoptosis to autophagy, and improves the survival of neurons;in the subacute stage, JNK activation inducesautophagy product accumulation, leading to autophagy injury of neurons.