Neuroprotective Effect and Mechanism of Salidroside in Cerebral Ischemia-reperfusion Injury in Mouse Model

doi:10.3969/j.issn.1673-5765.2022.09.013

Abstract

Abstract: Objective To explore the neuroprotective effect and mechanism of salidroside (SAL) on cerebral ischemia-reperfusion injury through animal models and in vitro cell culture.
Methods A total of 36 male C57BL/6J mice were randomly divided into sham operation group (12 mice), vehicle group (12 mice) and SAL group (12 mice). The mice models of right middle cerebral artery occlusion were made by thread embolization method and were given reperfusion after 1 hour ischemia. At the onset of reperfusion, 150 μL SAL (10 mg/kg, 0.22 mg per mouse) was given to mice immediately by tail vein injection, the same volume of phosphate buffered saline was injected in vehicle group, while nothing was given in sham operation group. The neurological deficits of mice were evaluated 24 hours after ischemia. Cerebral infarction was calculated by neuronal nuclei (NeuN) staining. The autophagy marker microtubule associated protein 1 light chain 3 beta (LC3B) was co-stained with NeuN and glial fibrillary acidic protein (GFAP) to observe the colocalization of LC3B with neurons and astrocytes around infarction in vehicle group, so as to identify the main site of autophagy activity 24 hours after ischemia. Autophagy markers Beclin-1 and autophagy related protein 3 (Atg3) were co-stained with NeuN, respectively, thus to further observe the changes of autophagy level in neurons around infarction. The number of Beclin-1+/NeuN+ and Atg3+/NeuN+ cells per square millimeter was counted. Primary rat neurons were cultured for 10 days and subjected to oxygen glucose deprivation (OGD), and the cells were divided into six groups: normal control (control, CON) group, OGD 1 hour and reoxygenation 1 hour (OGD 1 h) group, OGD 1 hour and reoxygenation 4 hours (OGD 4 h) group, SAL group, OGD 1 hour and reoxygenation 1 hour with SAL (OGD+SAL 1 h) group, OGD 1 hour and reoxygenation 4 hours with SAL (OGD+SAL 4 h) group. CON group and SAL group were not subjected to OGD. The dosage of SAL was 50 μmol/L. The relative expression levels of LC3B, Beclin-1, Atg3, autophagy related protein 5 (Atg5) and Atg7 in each group were detected by immunoblotting. LC3B and NeuN in CON and OGD 4 h groups were co-stained to observe the expression changes of LC3B after OGD. The number of LC3B+/NeuN+ cells per square millimeter was counted.
Results At 24 hours after ischemia, the neurological deficit scores (5.8±1.4 vs. 7.1±1.4, P=0.0332) and cerebral infarction rate (28.7%±9.7% vs. 39.9%±9.4%, P=0.0038) of mice in SAL group were lower than that in vehicle group. LC3B colocalized with neurons around infarct, but not with astrocytes in vehicle group. The number of Beclin-1+/NeuN+ (312.4±45.6 vs. 471.2±50.3 per square millimeter, P=0.0121) and Atg3+/NeuN+ (322.5±26.5 vs. 491.3±42.1 per square millimeter, P=0.0013) cells in SAL group were less than that in vehicle group. Immunoblotting of primary neurons showed that LC3B relative expression in OGD 1 h group (1.096±0.004 vs. 1.000±0.000, P=0.0174) and OGD 4 h group (1.213±0.019 vs. 1.000±0.000, P<0.0001) was higher than that in CON group. The relative expression of LC3B, Beclin-1 and Atg3 in OGD+SAL 4 h group were significantly lower than that in OGD 4 h group (0.833±0.029 vs. 1.213±0.019, P<0.0001; 0.579±0.081 vs. 1.152±0.144, P<0.0001; 0.726±0.182 vs. 1.091±0.177, P=0.0211). The relative expression of LC3B and Beclin-1 in OGD+SAL 4 h group were significantly lower than that in OGD+SAL 1 h group (0.833±0.029 vs. 1.046±0.063, P<0.0001; 0.579±0.081 vs. 0.921±0.090, P=0.0030). The number of LC3B+/NeuN+ cells in CON group was less than that in OGD 4 h group (51.9±18.7 vs. 584.2±34.5 per square millimeter, P=0.0002).
Conclusions Salidroside may play a neuroprotective role in acute cerebral ischemia-reperfusion injury by inhibiting neuron autophagy.

Key words: Cerebral ischemia-reperfusion; Neuron; Autophagy; Salidroside; Neuroprotection

摘要： 目的通过动物模型和体外细胞培养探索毛柳苷（salidroside，SAL）对脑缺血再灌注损伤的神经保护作用及其可能机制。
方法 36只雄性C57BL/6J小鼠随机分为假手术组（12只）、溶剂组（12只）和SAL组（12只）。线栓法制作小鼠右侧大脑中动脉闭塞模型，缺血1 h后再灌注，而后即刻尾静脉注射150 μL SAL（剂量10 mg/kg，每只实际用量0.22 mg），溶剂组注射同等体积磷酸盐缓冲液，假手术组不给药。缺血再灌注后24 h进行小鼠神经功能缺损评分，神经元特异核蛋白（neuronal nuclei，NeuN）染色统计脑梗死率。取溶剂组小鼠全脑冰冻切片，自噬标记物微管相关蛋白1轻链3B（microtubule associated protein 1 light chain 3 beta，LC3B）分别与NeuN、胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）免疫荧光共染，观察LC3B与梗死周边区神经元、星形胶质细胞共定位情况，以明确缺血再灌注后24 h产生自噬活动的主要部位。自噬标记物苄氯素1（Beclin-1）、自噬相关蛋白3（autophagy related protein 3，Atg3）分别与NeuN免疫荧光共染，进一步观察梗死周边区神经元自噬水平变化情况，并统计Beclin-1+/NeuN+、Atg3+/NeuN+细胞在单位面积（1 mm2）内的个数。原代大鼠神经元培养10 d后进行氧糖剥夺（oxygen glucose deprivation，OGD），将细胞分为以下6组：对照（control，CON）组、OGD 1 h后复氧1 h（OGD 1 h）组、OGD 1 h后复氧4 h（OGD 4 h）组、SAL组、OGD 1 h后复氧1 h/全程SAL治疗（OGD+SAL 1 h）组、OGD 1 h后复氧4 h/全程SAL治疗（OGD+SAL 4 h）组，CON组和SAL组不进行OGD，SAL剂量50 μmol/L。采用免疫印迹法检测各组细胞中自噬标记物LC3B、Beclin-1、Atg3、Atg5和Atg7的相对表达量；CON组、OGD 4 h组细胞进行LC3B、NeuN免疫荧光共染，观察OGD后神经元内LC3B表达变化情况，并统计LC3B+/NeuN+细胞在单位面积（1 mm2）内的个数。
结果缺血再灌注后24 h，SAL组小鼠神经功能缺损评分（5.8±1.4分 vs. 7.1±1.4分，P=0.0332）和脑梗死率（28.7%±9.7% vs. 39.9%±9.4%，P=0.0038）均低于溶剂组。溶剂组LC3B与梗死周边区神经元有共定位，与星形胶质细胞无共定位。SAL组Beclin-1+/NeuN+（312.4±45.6个/平方毫米 vs. 471.2±50.3个/平方毫米，P=0.0121）、Atg3+/NeuN+（322.5±26.5个/平方毫米 vs. 491.3±42.1个/平方毫米，P=0.0013）细胞数量少于溶剂组。大鼠原代神经元免疫印迹结果显示，OGD 1 h组（1.096±0.004 vs. 1.000±0.000，P=0.0174）、OGD 4 h组（1.213±0.019 vs. 1.000±0.000，P<0.0001）LC3B相对表达量高于CON组；OGD+SAL 4 h组LC3B、Beclin-1、Atg3相对表达量低于OGD 4 h组（0.833±0.029 vs. 1.213±0.019，P<0.0001，0.579±0.081 vs. 1.152±0.144，P<0.0001，0.726±0.182 vs. 1.091±0.177，P=0.0211）；OGD+SAL 4 h组LC3B、Beclin-1相对表达量低于OGD+SAL 1 h组（0.833±0.029 vs. 1.046±0.063，P<0.0001；0.579±0.081 vs. 0.921±0.09，P=0.0030）。CON组LC3B+/NeuN+细胞数少于OGD 4 h组（51.9±18.7个/平方毫米 vs. 584.2±34.5个/平方毫米，P=0.0002）。
结论 SAL可能通过抑制神经元自噬在急性脑缺血再灌注损伤中发挥神经保护作用。

关键词: 脑缺血再灌注; 神经元; 自噬; 毛柳苷; 神经保护

WEN Shaohong, LIU Kuan, ZHAO Shunying, DONG Wen, CHEN Qingfang, CHEN Wentao, YE Weizhen, JIANG Mingyu, LIU Xiangrong. Neuroprotective Effect and Mechanism of Salidroside in Cerebral Ischemia-reperfusion Injury in Mouse Model[J]. Chinese Journal of Stroke, 2022, 17(09): 991-1001.

温少红, 刘宽, 赵顺英, 董雯, 陈青芳, 陈文涛, 叶维贞, 姜鸣钰, 刘向荣. 毛柳苷在脑缺血再灌注后抑制神经元自噬及神经保护作用研究[J]. 中国卒中杂志, 2022, 17(09): 991-1001.

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