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目的:比较研究热休克蛋白A5(HSPA5)和3-甲基腺嘌呤(3-MA)介导小鼠脑缺血再灌注(I/R)损伤性自噬的保护作用。方法:成年雄性BALB/c小鼠30只,分为6组:sham组、缺血再灌注组(I/R组)、vehicle+I/R组、3-甲基腺嘌呤(3-MA)+I/R组、scramble siRNA+I/R组和HSPA5 siRNA+I/R组,每组5只。采用大脑中动脉阻塞(MCAO)60 min后再灌注24 h制作小鼠I/R模型。Vehicle+I/R组和3-MA+I/R将5μl 0.9%Na Cl或3-MA(30 mg/ml)在MCAO前30 min侧脑室注射;scramble siRNA+I/R group组和HSPA5 siRNA+I/R组将5μl scramble siRNA或HSPA5 siRNA(2μg/μl)在MCAO前24 h侧脑室注射。通过小鼠I/R后神经功能评分确定运动功能障碍程度。小鼠神经细胞内自噬体形成用透射电子显微镜测定,再灌注24 h缺血大脑皮层(LC3)-II/LC3-I表达用Western Blot方法检测。小鼠I/R后缺血大脑神经细胞损伤用Nissl染色进行观察。结果:HSPA5和3-MA可以影响I/R小鼠行为学改变并使脑缺血性损伤和神经症状加重(P<0.05)。透射电子显微镜检测可见,sham组小鼠大脑皮层神经细胞形态正常,I/R组小鼠缺血大脑皮层神经元胞质中细胞器减少,形成自噬体。与sham组小鼠比较,I/R组小鼠缺血大脑皮层LC3-II蛋白表达水平显著增高(P<0.05);3-MA+I/R或HSPA5 siRNA+I/R小鼠缺血大脑皮层LC3-II蛋白表达水平明显低于I/R小鼠(P<0.05)。结论:HSPA5和3-MA在介导小鼠脑缺血再灌注损伤导致的自噬可发挥相同的保护作用,并且促进神经细胞凋亡。
AIM: To compare the protective effects of heat shock protein A5 (HSPA5) and 3-methyladenine (3-MA) on autophagy induced by cerebral ischemia-reperfusion (I / R) in mice. Methods: Thirty male BALB / c mice were randomly divided into 6 groups: sham group, ischemia / reperfusion group (I / R group), vehicle + I / R group, 3- methyladenine (3-MA) + I / R group, scramble siRNA + I / R group and HSPA5 siRNA + I / R group with 5 in each group. The mouse I / R model was established by reperfusion for 60 min after middle cerebral artery occlusion (MCAO) for 60 min. 5 μL of 0.9% NaCl or 3-MA (30 mg / ml) was intracerebroventricularly injected into Vehicle + I / R group and 3-MA + I / R 30 min before MCAO; scramble siRNA + I / R group and HSPA5 siRNA + I / R group Five μl of scramble siRNA or HSPA5 siRNA (2 μg / μl) were injected into the lateral ventricle 24 hours before MCAO. The degree of motor dysfunction was determined by the score of neurological function after I / R in mice. Autophagosome formation in mouse neurons was measured by transmission electron microscopy and expression of LC3-II / LC3-I at 24 h after reperfusion was measured by Western Blot. Nissl staining was used to observe the damage of neurons in ischemic brain after I / R in mice. Results: HSPA5 and 3-MA can affect the behavioral changes of I / R mice and aggravate cerebral ischemic injury and neurological symptoms (P <0.05). Transmission electron microscopy showed that the morphology of neurons in the cerebral cortex of sham-treated mice was normal. The organelles in neuronal cytoplasm of ischemic cortex decreased in I / R mice and formed autophagosomes. The expression of LC3-II protein in ischemic cortex of I / R group was significantly higher than that of sham group (P <0.05). The expression of LC3-II protein in ischemic cortex of I / R group was significantly higher than that of sham group The level of cortical LC3-II protein expression was significantly lower than that of I / R mice (P <0.05). CONCLUSIONS: HSPA5 and 3-MA can play the same protective role in mediating autophagy induced by cerebral ischemia-reperfusion injury in mice, and promote neuronal apoptosis.