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J Zhejiang Univ (Med Sci)  2020, Vol. 49 Issue (6): 687-696    DOI: 10.3785/j.issn.1008-9292.2020.12.03
    
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 Lisheng2,*()
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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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 wordsmicroRNA      miR-199a-5p      Cerebral ischemia      Buyang Huanwu decoction      Neurogenesis      Angiogenesis      Rats, Sprague-Dawley     
Received: 24 October 2020      Published: 14 January 2021
CLC:  R285.5  
Corresponding Authors: CHU Lisheng     E-mail: 554192941@qq.com;cls2004@zcmu.edu.cn
Cite this article:

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.

URL:

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


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

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 
评分项目 得分
运动实验
  提尾实验
    前肢屈曲 1
    后肢屈曲 1
    30 s内头转动偏离垂直轴的角度>10° 1
  把大鼠放置地板上
    正常运动 0
    不能直线行走 1
    向偏瘫侧方向转圈 2
    向偏瘫侧方向倾倒 3
  感觉实验
    放置实验(视觉和触觉测试) 1
    本体感觉实验(深感觉,向桌子边缘按压鼠爪刺激肢体肌肉) 1
  平衡木实验
    稳定平衡姿势 0
    紧抓平衡木一侧 1
    紧抱平衡木且一肢体从平衡木滑落 2
    紧抱平衡木且两肢体从平衡木滑落,或在平衡木上旋转(>60 s) 3
    试图在平衡木保持平衡但跌落(>40 s) 4
    试图在平衡木保持平衡但跌落(>20 s) 5
    跌落, 未尝试在平衡木上平衡或悬吊(<20 s) 6
  反射消失和异常运动
    耳郭反射(触碰耳道时摇头) 1
    角膜反射(用棉签轻触角膜时眨眼) 1
    惊吓反射(对快弹硬纸板的噪音有运动反应) 1
    癫痫发作、肌阵挛,肌张力障碍 1
Tab 1 Modified neurological severity score
Fig 1 Neurological function of rats in five groups (n=14)
Fig 2 Toluidine blue staining showed the infarct volume in each group
Fig 3 Immunofluorescence double staining of BrdU+/DCX+、BrdU+/NeuN+、BrdU+/ GFAP+、BrdU+/vWF+ cells in each group
组别 n BrdU+/DCX+细胞数 BrdU+/NeuN+细胞数 BrdU+/GFAP+细胞数 BrdU+/vWF+细胞数
与手术对照组比较,**P<0.01;与模型对照组比较,##P<0.01;与补阳还五汤组比较,ΔΔP<0.01.BrdU:5-溴脱氧尿嘧啶核苷;DCX:双肾上腺皮质激素;NeuN:抗神经元核抗原;GFAP:胶质纤维酸性蛋白;vWF:血管性假血友病因子.
手术对照组 8 0.75±0.50 0.50±0.58 1.00±0.82 0.50±0.58
模型对照组 8 24.00±2.71** 17.75±1.71** 13.00±0.82** 23.00±3.16**
补阳还五汤组 8 36.00±1.41## 27.50±1.29## 15.25±2.38 35.75±0.96##
拮抗剂组 8 19.75±2.22ΔΔ 13.00±0.82ΔΔ 16.50±1.73 14.00±1.41ΔΔ
拮抗剂对照组 8 38.25±1.71 27.00±1.41 16.00±2.58 38.75±1.26
Tab 2 BrdU+/DCX+、BrdU+/NeuN+、BrdU+/GFAP+and BrdU+/vWF+ cells in each group  (${\bar x}$±s, 每视野)
Fig 4 Expression of vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) in ischemic peripheral brain tissues in each group
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