主 办:北 京 中 医 药 大 学
ISSN 1006-2157 CN 11-3574/R

北京中医药大学学报 ›› 2020, Vol. 43 ›› Issue (8): 653-660.doi: 10.3969/j.issn.1006-2157.2020.08.007

• 中药药理 • 上一篇    下一篇

化痰祛瘀法通过调控miR-181影响输入蛋白α3/NF-κB通路的抗动脉粥样硬化的研究*

秦合伟1,2, 李彦杰1, 张志鑫1#, 姬令山1, 郭宁2   

  1. 1 河南省中医院 河南中医药大学第二附属医院 河南 450002;
    2 河南中医药大学
  • 收稿日期:2020-01-29 出版日期:2020-08-30 发布日期:2020-08-27
  • 通讯作者: # 张志鑫,男,硕士,副主任医师,主要研究方向:中西医结合治疗心脑血管疾病,E-mail:qinheweii@126.com
  • 作者简介:秦合伟,男,博士,副主任医师
  • 基金资助:
    * 国家自然科学基金资助项目(No. 81704030),河南省中医药科学研究专项重点课题(No. 2019ZY1015),河南省高等学校重点科研项目计划资助(No. 20B360014),河南省中医药拔尖人才培养项目,河南省中医药文化与管理研究项目(No. TCM2020016)

Regulation of input protein α3/NF-κB pathway by adjusting miR-181 with dissipating phlegm and removing blood stasis method and its anti-atherosclerosis mechanism*

Qin Hewei1,2, Li Yanjie1, Zhang Zhixin1#, Ji Lingshan1, Guo Ning2   

  1. 1 Henan Province Hospital of & TCM Second Affiliated Hospital of Henan University of Chinese Medicine, Henan 450002, China;
    2 Henan University of Chinese Medicine, Henan 450002, China
  • Received:2020-01-29 Online:2020-08-30 Published:2020-08-27
  • Contact: Zhang Zhixin, M.D., Deputy Chief Physician. No. 6 Dongfeng Road, Zhengzhou, Henan Province Hospital of TCM, Henan 450002. E-mail: qinheweii@126.com
  • Supported by:
    National Natural Science Foundation of China (No.81704030)

摘要: 目的 观察化痰祛瘀法抗动脉粥样硬化的效应及作用机制。方法 ①体外实验:通过加入氧化型低密度脂蛋白(ox-LDL)构建血管内皮细胞损伤模型。随机将细胞分为4组:模型组、微小核糖核酸181(miR-181)模拟物组、miR-181抑制物组、化痰祛瘀法药物血清组。检测各组细胞活性,miR-181、输入蛋白α3和核因子κB(NF-κB)的基因和蛋白表达水平。②动物实验:60只ApoE-/-小鼠随机分为5组:模型组,miR-181抑制物组,miR-181 模拟物组,化痰祛瘀法高、低剂量组,每组12只,连续给药8周后检测外周血浆白细胞介素-6(IL-6)、血管细胞黏附因子-1(VCAM-1)和E-选择素水平,各组主动脉壁miR-181、输入蛋白α3和NF-κB的基因表达水平,并进行病理学观察。结果 ①体外实验:与模型组相比,化痰祛瘀法药物血清组和miR-181模拟物组的细胞活性升高(P<0.05),输入蛋白α3和NF-κB的基因和蛋白表达下调(P<0.05)。②动物实验:与模型组相比,化痰祛瘀法高、低剂量组和miR-181模拟物组外周血浆IL-6、VCAM-1和E-选择素水平降低(P<0.05);与miR-181抑制物组相比,化痰祛瘀法各组外周血浆IL-6、VCAM-1和E-选择素水平降低(P<0.05)。与模型组相比,化痰祛瘀法各组和miR-181模拟物组主动脉miR-181 mRNA表达上调(P<0.05),输入蛋白α3和NF-κB mRNA表达下调(P<0.05);与miR-181 模拟物组相比,化痰祛瘀法高、中剂量组主动脉miR-181 mRNA表达下调,而输入蛋白α3和NF-κB mRNA表达上调(P<0.05)。结论 化痰祛瘀法抗动脉粥样硬化的作用机制可能与其调控miR-181从而靶向影响输入蛋白α3/NF-κB信号通路,降低IL-6、VCAM-1和E-选择素等炎性介质水平,减轻血管内皮细胞损伤有关。

关键词: 动脉粥样硬化, 化痰祛瘀法, 内皮细胞, 微小核糖核酸181, 输入蛋白α3, 核因子κB, 小鼠

Abstract: Objective To observe the correlation between the anti-atherosclerosis mechanism of dissipating phlegm and removing blood stasis method and the effect on the input protein α3/NF-κB signaling pathway through the regulation of microribonucleic acid 181 (miR-181). Methods In vitro experiments, vascular endothelial cell injury model was established with oxidized low-density lipoprotein (ox-LDL). According to different intervention methods, cells were randomly divided into four groups: model group (blank serum), miR-181 mimic group, miR -181 inhibitor group, phlegm-resolving and blood stasis-eliminating TCM serum group (TCM serum group). After intervention, the cell activity of each group was detected. RT-PCR and Western blot were used to detect the mRNA and protein expression levels of miR-181, input protein α3 and NF-κB. In vivo experiments, 60 ApoE-/- mice were randomly divided into 5 groups: model group, miR-181 inhibitor group, miR-181 mimic group, high-dose and low-dose phlegm-resolving and blood stasis-eliminating TCM serum group (TCM high-dose serum group, and TCM low-dose serum group respectively) (n=12). After continuous intervention for 8 weeks, the plasma and aorta were collected for testing. The levels of IL-6, VCAM-1 and E-selectin in the peripheral plasma were tested by ELISA. The expression of microRNA-181, input protein α3 and NF-κB in the aortic wall of each group was tested by RT-PCR, and the pathological changes were observed. Results In vitro experiments, the cell activity of the TCM serum group and the miR-181 mimic group was significantly increased (P<0.05), and the expression of α3 and NF-κB mRNA were significantly down-regulated (P<0.05). In vivo experiments, compared with the model group, the plasma levels of IL-6, VCAM-1 and E-selectin in the TCM high-dose and low-dose serum groups and miR-181 mimic group were significantly lower, compared with the model group (P<0.05); compared with the inhibitor group, the levels of peripheral plasma IL-6, VCAM-1 and E-selectin in the TCM serum group were lower (P<0.05). Compared with the model group, the mRNA expression of aortic miR-181 in the TCM high-dose and low-dose serum groups and miR-181 mimic group was significantly increased (P<0.05), and the expression level of α3 and NF-κB significantly down-regulated (P<0.05). There was no significant difference in the indexes between the TCM high-dose and low-dose serum groups (P>0.05). Conclusion The anti-atherosclerosis mechanism of resolving phlegm and eliminating blood stasis may be related to the targeted regulation of miR-181 to affect the input protein α3/NF-κB signaling pathway, which could possibly reduce the levels of inflammatory mediators such as IL-6, VCAM-1 and E-selectin, thus reducing the damage of vascular endothelial cells.

Key words: atherosclerosis, phlegm-resolving and blood stasis-eliminating method, endothelial cells, miR-181, input protein α3, NF-κB, mice

中图分类号: 

  • R285.5