大黄酚对局灶性脑缺血再灌注小鼠脑组织p-CREB、BDNF和p-STAT3的影响

doi:10.3969/j.issn.1006-7795.2018.03.010

摘要/Abstract

摘要： 目的研究大黄酚（chrysophanol，CHR）对短暂性局灶性脑缺血再灌注小鼠缺血半暗带区磷酸化环磷酸腺苷反应元件结合蛋白（phosphorylated cyclic AMP response element binding protein，p-CREB）、脑源性神经营养因子（brain derived neurotrophic factor，BDNF）和缺血侧脑组织磷酸化转录激活因子3（phosphorylated signal transducer and activator of transcription 3，p-STAT3）表达水平的影响，探究CHR保护脑缺血损伤的机制。方法将18只雄性健康C57BL小鼠按照数字表法随机分为3组：假手术（Sham）组、大脑中动脉梗死（middle cerebral artery occlusion，MCAO）组、CHR组（自造模当天至再灌注后14 d按0.1 mg/kg腹腔注射CHR），每组6只。采用线栓法制作右侧大脑中动脉缺血45 min再灌注模型。于再灌注后14 d处死小鼠，迅速断头、取脑，用免疫荧光染色方法检测脑组织冰冻切片缺血半暗带区p-CREB和BDNF的水平，应用免疫荧光双标染色方法对p-CREB和BDNF在缺血脑组织内的表达进行细胞定位，并应用Western blotting检测缺血侧脑组织p-STAT3蛋白水平。结果 1） Sham组小鼠脑组织可见p-CREB阳性染色细胞。MCAO组小鼠脑缺血半暗带区p-CREB表达水平比Sham组明显降低（P<0.05）。给予CHR治疗的脑缺血再灌注小鼠半暗带区p-CREB的表达水平比MCAO组明显增多（P<0.05）。在半暗带区，p-CREB与神经元标志物NeuN免疫荧光染色共定位。2） Sham组小鼠脑组织可见BDNF阳性细胞。MCAO组小鼠半暗带区BDNF水平比Sham组明显降低（P<0.05）。CHR组小鼠脑缺血半暗带区BDNF水平比MCAO组明显增多（P<0.05）。在半暗带区，BDNF与神经元标志物NeuN免疫荧光染色共定位。3） MCAO组小鼠缺血侧脑组织p-STAT3蛋白水平比Sham组明显升高（P<0.05）。CHR组小鼠缺血侧脑组织p-STAT3蛋白水平比MCAO组明显减少（P<0.05）。结论 CHR可能通过上调p-CREB和BDNF表达水平，抑制p-STAT3蛋白表达，对脑缺血再灌注损伤发挥长期神经保护作用。

关键词: 大黄酚, 脑缺血再灌注, 大脑中动脉梗死, 磷酸化环磷酸腺苷反应元件结合蛋白, 脑源性神经营养因子, 磷酸化转录激活因子3

Abstract: Objective To explore the effects of chrysophanol (CHR) on the levels of phosphorylated cyclic adenine monophosphate (AMP) response element binding protein (p-CREB), brain derived neurotrophic factor (BDNF), and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in middle cerebral artery occlusion (MCAO)/reperfusion mice and to investigate the protective efficacy of CHR and the relative mechanism.Methods Totally 18 male C57BL mice were randomly divided into three groups:Sham group (n=6), MCAO group (n=6), and CHR group (CHR was intraperitoneally injected at a dose of 0.1 mg/kg for 14 days after reperfusion, n=6). MCAO was made by using the suture method. The mice were reperfused after right MCAO for 45 minutes. The mice were sacrificed and rapidly decapitated and the brains were separated on 14 d after reperfusion. Immunofluorescent staining was used to detect the levels of p-CREB and BDNF. And double immunofluorescence labeling was used to explore the cellular location of p-CREB and BDNF. Western blotting analysis was used to investigate the level of p-STAT3.Results 1) Many p-CREB positive cells were observed in Sham group. In the penumbra of MCAO group, the expression of p-CREB decreased significantly compared with Sham group (P<0.05). Compared with MCAO group, the expression of p-CREB increased significantly in penumbra zone of cerebral ischemia/reperfusion mice treated with CHR on 14 d after reperfusion (P<0.05). The p-CREB-positive cells were co-localized with general neuronal marker, NeuN, in the penumbra of ischemia/reperfusion mice. 2) A large number of BDNF-positive cells were observed in Sham group. The level of BDNF decreased significantly in the penumbra of MCAO group compared with Sham group (P<0.05). Compared with MCAO group, the expression of BDNF increased significantly in penumbra zone of cerebral ischemia/reperfusion mice treated with CHR 14 d after reperfusion (P<0.05). And BDNF-positive cells were co-localized with NeuN in the penumbra of ischemia/reperfusion mice. 3) The level of p-STAT3 increased significantly in ischemic brain tissue of MCAO group compared with Sham group (P<0.05). Compared with MCAO group, the level of p-STAT3 decreased significantly in ischemic brain tissue of cerebral ischemia/reperfusion mice treated with CHR 14 d after reperfusion (P<0.05).Conclusion CHR may provide long-term neuroprotective effect against cerebral ischemia/reperfusion injury by up-regulating the expressions of p-CREB and BDNF, and inhibiting p-STAT3 protein expression.

Key words: chrysophanol, cerebral ischemia/reperfusion, middle cerebral artery occlusion, phosphorylated cyclic AMP response element binding protein, brain derived neurotrophic factor, phosphorylated signal transducer and activator of transcription 3

中图分类号:

R743.3

房亚兰, 黄语悠, 赵咏梅, 李锦程, 段云霞, 高利, 罗玉敏. 大黄酚对局灶性脑缺血再灌注小鼠脑组织p-CREB、BDNF和p-STAT3的影响[J]. 首都医科大学学报, 2018, 39(3): 360-365.

Fang Yalan, Huang Yuyou, Zhao Yongmei, Li Jincheng, Duan Yunxia, Gao Li, Luo Yumin. Effects of chrysophanol on the expressions of p-CREB, BDNF and p-STAT3 in the brain of focal cerebral ischemia/reperfusion mice[J]. Journal of Capital Medical University, 2018, 39(3): 360-365.

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