益气活血通络方对脊髓型颈椎病模型大鼠miR-126a-5p及VEGF信号通路的影响

doi:10.11958/20230607

天津医药 ›› 2024, Vol. 52 ›› Issue (3): 273-277.doi: 10.11958/20230607

益气活血通络方对脊髓型颈椎病模型大鼠miR-126a-5p及VEGF信号通路的影响

刘丹(), 唐占英, 李攀, 袁薇娜, 李芳芳, 陈倩, 胡志俊^△()

上海中医药大学附属龙华医院康复医学科（邮编200032）

收稿日期:2023-04-23 修回日期:2023-06-08 出版日期:2024-03-15 发布日期:2024-03-13
通讯作者: ^△E-mail：hzjz1062@163.com
作者简介:刘丹（1988），女，主治医师，主要从事骨与关节慢性疾病的中西医结合康复治疗方面研究。E-mail：danliu0212@163.com
基金资助:
国家重点研发计划项目(2019YFC1709905);上海市进一步加快中医药传承创新发展三年行动计划项目[ZY(2021-2023)-0201-01];上海市浦东新区卫生系统浦东名中医培养计划项目(PWRzm2020-15)

Effect of Yiqi Huoxue Tongluo Decoction on miR-126a-5p and VEGF signaling pathway in cervical spondylotic myelopathy model rats

LIU Dan(), TANG Zhanying, LI Pan, YUAN Weina, LI Fangfang, CHEN Qian, HU Zhijun^△()

Department of Rehabilitation Medicine, Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China

Received:2023-04-23 Revised:2023-06-08 Published:2024-03-15 Online:2024-03-13
Contact: ^△E-mail: hzjz1062@163.com

刘丹, 唐占英, 李攀, 袁薇娜, 李芳芳, 陈倩, 胡志俊. 益气活血通络方对脊髓型颈椎病模型大鼠miR-126a-5p及VEGF信号通路的影响[J]. 天津医药, 2024, 52(3): 273-277.

LIU Dan, TANG Zhanying, LI Pan, YUAN Weina, LI Fangfang, CHEN Qian, HU Zhijun. Effect of Yiqi Huoxue Tongluo Decoction on miR-126a-5p and VEGF signaling pathway in cervical spondylotic myelopathy model rats[J]. Tianjin Medical Journal, 2024, 52(3): 273-277.

摘要/Abstract

摘要：

目的探讨益气活血通络方对脊髓型颈椎病模型大鼠微小RNA-126a-5p（miR-126a-5p）及血管内皮生长因子（VEGF）信号通路的影响。方法 30只健康雄性SD大鼠按照随机数字表法均分为假手术组、模型组和中药组。模型组、中药组均制备脊髓型颈椎病模型。中药组给予益气活血通络方灌胃，假手术组、模型组给予生理盐水灌胃，均为12周。各组干预结束后进行大鼠行为学测试，测定机械性刺激阈值和热刺激回缩时间。处死大鼠，HE染色观察各组椎间盘软骨终板结构的差异。TUNEL染色观察大鼠椎间盘纤维环细胞变化。实时荧光定量聚合酶链式反应检测大鼠椎间盘纤维环组织miR-126a-5p和VEGF mRNA。采用Western blot法检测大鼠椎间盘纤维环组织VEGF蛋白表达。结果与假手术组比较，模型组大鼠椎间盘血管芽数目减少，大鼠椎间盘纤维环细胞破坏明显、大量凋亡且细胞密度降低；模型组和中药组机械性刺激阈值降低，热刺激回缩时间缩短，miR-126a-5p水平降低，VEGF mRNA和蛋白表达水平升高（P<0.05）。与模型组比较，中药组大鼠椎间盘血管芽数目增加，大鼠椎间盘纤维环细胞破坏减轻，大鼠椎间盘纤维环细胞凋亡减少且细胞密度增加，机械性刺激阈值升高，热刺激回缩时间延长，miR-126a-5p水平增加，VEGF mRNA和蛋白表达水平降低（P<0.05）。结论益气活血通络方治疗脊髓型颈椎病的机制可能与上调miR-126a-5p、下调VEGF表达有关。

关键词: 颈椎病, 血管内皮生长因子类, 椎间盘, 脊髓, 益气活血通络方, miR-126a-5p

Abstract:

Objective To investigate the effect of Yiqi Huoxue Tongluo Decoction on microRNA-126a-5p (miR-126a-5p) and vascular endothelial growth factor (VEGF) signaling pathway in cervical spondylotic myelopathy model rats. Methods Thirty healthy male SD rats were divided into the sham operation group, the model group and the traditional Chinese medicine (TCM) group by random number table method. Cervical spondylotic myelopathy models were prepared in the model group and the TCM group. The TCM group was given intragastric administration of Yiqi Huoxue Tongluo Decoction, while the sham operation group and the model group were given intragastric administration of normal saline for 12 weeks. After intervention, the threshold of mechanical stimulation and retraction time of thermal stimulation in each group were measured by behavior tests. Rats were sacrificed to collect intervertebral disc tissue for hematoxylin-eosin (HE) staining and observe the number of vascular buds in intervertebral disc. Rat intervertebral disc annulus fibrosus cells were subjected to terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. The miR-126a-5p and VEGF mRNA of rat intervertebral disc tissue were detected by real-time fluorescence quantitative polymerase chain reaction (RT-PCR). The expression of VEGF protein of rat intervertebral disc tissue was detected by Western blot assay. Results Compared with the sham operation group, the number of vascular buds in intervertebral disc was decreased in the model group and the TCM group. The cell destruction of intervertebral disc annulus was obvious in rats, and apoptosis was high and cell density decreased. Mechanical stimulation threshold decreased, and mechanical stimulation threshold decreased. The level of miR-126a-5p was decreased, and the expression levels of VEGF mRNA and protein were increased. Compared with the model group, the number of vascular buds in intervertebral disc was increased in the TCM group. The destruction of intervertebral disc annulus cells was alleviated in rats. The apoptosis of annulus fibrosus cells in intervertebral disc decreased and cell density increased. The threshold of mechanical stimulation increased, and the retraction time of thermal stimulation was prolonged. The level of miR-126a-5p increased, and the expression levels of VEGF mRNA and protein decreased (P<0.05). Conclusion The mechanism of Yiqi Huoxue Tongluo Decoction in the treatment of cervical spondylotic myelopathy may be related to the up-regulation of miR-126a-5p expression and the down-regulation of VEGF expression.

Key words: cervical spondylosis, vascular endothelial growth factors, intervertebral disc, spinal cord, Yiqi Huoxue Tongluo Decoction, miR-126a-5p

中图分类号:

R681.531.1

图/表 4

表1 各组大鼠机械性刺激阈值、热刺激回缩时间比较（n=10，$\bar{x} \pm s$）

Tab.1 Comparison of the threshold of mechanical stimulation and retraction time of thermal stimulation between the three groups

组别	机械性刺激阈值/N	热刺激回缩时间/s
假手术组	17.67±1.32	2.84±0.20
模型组	8.34±0.95^a	1.56±0.19^a
中药组	14.07±1.54^ab	2.40±0.31^ab
F	132.238^＊＊	76.859^＊＊

图1 各组大鼠椎间盘纤维环组织HE染色及TUNEL染色结果 A：椎间盘纤维环组织HE染色（×400，标尺=50 μm）；B：椎间盘纤维环细胞TUNEL染色（×200，标尺=100 μm）。

Fig.1 HE staining and TUNEL staining results of rat intervertebral disc annulus fibrosus tissue in each group

表2 各组大鼠miR-126a-5p、VEGF mRNA及蛋白表达水平比较（n=10，$\bar{x} \pm s$）

Tab.2 Comparison of miR-126a-5p, VEGF mRNA and protein expression level between the three groups

组别	miR-126a-5p	VEGF mRNA	VEGF蛋白
假手术组	1.07±0.21	1.03±0.12	0.36±0.05
模型组	0.16±0.05^a	4.27±1.15^a	0.76±0.10^a
中药组	0.56±0.11^ab	2.26±0.61^ab	0.53±0.09^ab
F	110.156^＊＊	47.260^＊＊	57.921^＊＊

图2 各组大鼠VEGF Western blot结果

Fig.2 Results of Western blot assay of VEGF protein in three groups

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益气活血通络方对脊髓型颈椎病模型大鼠miR-126a-5p及VEGF信号通路的影响

Effect of Yiqi Huoxue Tongluo Decoction on miR-126a-5p and VEGF signaling pathway in cervical spondylotic myelopathy model rats

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