补阳还五汤上调miR-199a-5p表达促进脑缺血大鼠神经发生和血管生成

doi:10.3785/j.issn.1008-9292.2020.12.03

浙江大学学报（医学版）

2020, Vol. 49

Issue (6): 687-696 DOI: 10.3785/j.issn.1008-9292.2020.12.03

专题报道

补阳还五汤上调miR-199a-5p表达促进脑缺血大鼠神经发生和血管生成

诸葛陆杰¹(

),方燕²,金华倩¹,李琳²,杨琰²,胡小伟²,储利胜^2,*(

)

1. 浙江中医药大学药学院, 浙江杭州 310053
2. 浙江中医药大学基础医学院, 浙江杭州 310053

Chinese medicine Buyang Huanwu decoction promotes neurogenesis and angiogenesis in ischemic stroke rats by upregulating miR-199a-5p expression

ZHUGE Lujie¹(

),FANG Yan²,JIN Huaqian¹,LI Lin²,YANG Yan²,HU Xiaowei²,CHU Lisheng^2,*(

)

1. College of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou 310053, China
2. College of Basic Medical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China

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摘要：

目的: 探讨补阳还五汤促进脑缺血大鼠神经发生和血管生成的机制。方法: 实验大鼠随机分为手术对照组、模型对照组、补阳还五汤组、拮抗剂组、拮抗剂对照组，各14只。除手术对照组外，其余组采用线栓法诱导大脑中动脉阻塞模型，缺血90 min后再灌注14 d；补阳还五汤组、拮抗剂组和拮抗剂对照组在缺血后24 h灌胃补阳还五汤（13 g/kg）；所有大鼠腹腔注射5-溴脱氧尿苷（BrdU，50 mg/kg），1次/d，连续14 d；缺血后第5天，拮抗剂组和拮抗剂对照组分别于侧脑室注射miR-199a-5p拮抗剂和miR-199a-5p拮抗剂对照10 nmol。采用改良神经损伤严重程度评分（mNSS）和角试验评价神经功能缺损，甲苯胺蓝染色检测脑梗死体积，免疫荧光双标记法检测脑缺血大鼠神经发生和血管生成，实时逆转录PCR检测miR-199a-5p表达，蛋白质印迹法检测血管内皮生长因子（VEGF）和脑源性神经营养因子（BDNF）蛋白表达。结果: 补阳还五汤可促进脑缺血大鼠神经功能恢复（P < 0.05或P < 0.01），减小梗死体积（P < 0.05），增加BrdU⁺/DCX⁺、BrdU⁺/NeuN⁺、BrdU⁺/vWF⁺细胞数（均P < 0.01），上调miR-199a-5p表达（P < 0.01），促进VEGF和BDNF蛋白表达（均P < 0.05）；侧脑室注射miR-199a-5p拮抗剂后则逆转上述效应。结论: 补阳还五汤可促进脑缺血大鼠神经发生和血管生成，其机制可能与上调miR-199a-5p增加VEGF和BDNF表达有关。

关键词： 微RNA; miR-199a-5p; 脑缺血; 补阳还五汤; 神经发生; 血管生成; 大鼠, Sprague-Dawley

Abstract:

Objective: To investigate the mechanism of Chinese medicine Buyang Huanwu decoction (BYHWD) promoting neurogenesis and angiogenesis in ischemic stroke rats. Methods: Male SD rats were randomly divided into sham operation group, model group, BYHWD group, antagonist group and antagonist control group with 14 rats in each. Focal cerebral ischemia was induced by occlusion of the right middle cerebral artery for 90 min with intraluminal filament and reperfusion for 14 d in all groups except sham operation group. BYHWD (13 g/kg) was administrated by gastrogavage in BYHWD group, antagonist group and antagonist control group at 24 h after modeling respectively, and BrdU (50 mg/kg) was injected intraperitoneally in all groups once a day for 14 consecutive days. miR-199a-5p antagomir or NC (10 nmol) was injected into the lateral ventricle at d5 after ischemia in antagonist and antagonist control groups, respectively. The neurological deficits were evaluated by the modified neurological severity score (mNSS) and the corner test, and the infract volume was measured by toluidine blue staining. Neurogenesis and angiogenesis were detected by immunofluorescence double labeling method. The expression level of miR-199a-5p was tested by real-time RT-PCR, and the protein expressions of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) were determined by Western blotting. Results: BYHWD treatment significantly promoted the recovery of neurological function (P < 0.05 or P < 0.01), reduced the infarct volume (P < 0.05), increased the number of BrdU⁺/DCX⁺, BrdU⁺/NeuN⁺ and BrdU⁺/vWF⁺ cells (all P < 0.01), upregulated the expression of miR-199a-5p (P < 0.01), and increased the protein expression of VEGF and BDNF at d14 after cerebral ischemia (all P < 0.05). The above effects were reversed by intracerebroventricular injection of miR-199a-5p antagomir. Conclusion: Buyang Huanwu decoction promotes neurogenesis and angiogenesis in rats with cerebral ischemia, which may be related to increased protein expression of VEGF and BDNF through upregulating miR-199a-5p.

Key words: microRNA miR-199a-5p Cerebral ischemia Buyang Huanwu decoction Neurogenesis Angiogenesis Rats, Sprague-Dawley

收稿日期: 2020-10-24 出版日期: 2021-01-14

CLC:

R285.5

基金资助: 国家自然科学基金(81073075, 81873028);浙江省自然科学基金(Y207771)

通讯作者: 储利胜 E-mail: 554192941@qq.com;cls2004@zcmu.edu.cn

作者简介: 诸葛陆杰(1995-), 男, 硕士研究生, 主要从事中药促进脑缺血损伤修复的机制研究; E-mail:554192941@qq.com; https://orcid.org/0000-0003-3147-552X

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引用本文:

诸葛陆杰,方燕,金华倩,李琳,杨琰,胡小伟,储利胜. 补阳还五汤上调miR-199a-5p表达促进脑缺血大鼠神经发生和血管生成[J]. 浙江大学学报（医学版）, 2020, 49(6): 687-696.

ZHUGE Lujie,FANG Yan,JIN Huaqian,LI Lin,YANG Yan,HU Xiaowei,CHU Lisheng. Chinese medicine Buyang Huanwu decoction promotes neurogenesis and angiogenesis in ischemic stroke rats by upregulating miR-199a-5p expression. J Zhejiang Univ (Med Sci), 2020, 49(6): 687-696.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2020.12.03 或 http://www.zjujournals.com/med/CN/Y2020/V49/I6/687

表 1 改良的神经功能损伤严重程度评分表

图 1 各组神经功能检测结果比较(n=14)

图 2 各组缺血后甲苯胺蓝染色示脑梗死情况

图 3 各组BrdU+/DCX+、BrdU+/NeuN+、BrdU+/GFAP+、BrdU+/vWF+细胞免疫荧光双标染色图

表 2 各组BrdU+/DCX+、BrdU+/NeuN+、BrdU+/GFAP+和BrdU+/vWF+细胞数比较

图 4 各组缺血周边脑组织中抗血管内皮生长因子(VEGF)和脑源性神经营养因子(BDNF)蛋白表达

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