Heat-clearing and Toxin-removing Method Reduces Ischemic Stroke Injury by Protecting Endothelial-pericyte and Inhibiting Macrophage Migration

SUN Zijin; ZHANG Haojia; WANG Kai; WANG Zhaoyi; SONG Linjing; XU Wenxiu; JI Jing; LI Changxiang; WANG Qingguo; WANG Xueqian; CHENG Fafeng

doi:10.13422/j.cnki.syfjx.20260124

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Heat-clearing and Toxin-removing Method Reduces Ischemic Stroke Injury by Protecting Endothelial-pericyte and Inhibiting Macrophage Migration

更新时间：2026-04-30

- Heat-clearing and Toxin-removing Method Reduces Ischemic Stroke Injury by Protecting Endothelial-pericyte and Inhibiting Macrophage Migration
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 32, Issue 11, Pages: 56-67(2026)
- 作者机构：
  
  1.北京中医药大学中医学院，北京 102446
  2.北京中医药大学第一临床医学院，北京 100700
  3.康复大学生命科学与健康学院，山东青岛 266113
  4.安徽中医药大学中西医结合学院，合肥 230038
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20260124
  CLC： R256;R259;R285
- Received：20 September 2025，
  
  Revised：2025-12-09，
  
  Accepted：10 December 2025，
  
  Online First：12 December 2025，
  
  Published：05 June 2026
- 稿件说明：
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孙资金,张浩嘉,王凯等.清热解毒法通过保护内皮-周细胞并抑制巨噬细胞迁移协同减轻缺血性中风损伤[J].中国实验方剂学杂志,2026,32(11):56-67.

SUN Zijin,ZHANG Haojia,WANG Kai,et al.Heat-clearing and Toxin-removing Method Reduces Ischemic Stroke Injury by Protecting Endothelial-pericyte and Inhibiting Macrophage Migration[J].Chinese Journal of Experimental Traditional Medical Formulae,2026,32(11):56-67.
孙资金,张浩嘉,王凯等.清热解毒法通过保护内皮-周细胞并抑制巨噬细胞迁移协同减轻缺血性中风损伤[J].中国实验方剂学杂志,2026,32(11):56-67. DOI： 10.13422/j.cnki.syfjx.20260124.

SUN Zijin,ZHANG Haojia,WANG Kai,et al.Heat-clearing and Toxin-removing Method Reduces Ischemic Stroke Injury by Protecting Endothelial-pericyte and Inhibiting Macrophage Migration[J].Chinese Journal of Experimental Traditional Medical Formulae,2026,32(11):56-67. DOI： 10.13422/j.cnki.syfjx.20260124.

摘要

目的

该研究旨在探讨清热解毒法代表方剂黄连解毒汤对缺血性脑卒中模型中免疫细胞迁移、血-脑屏障保护及细胞功能恢复的调节作用。

方法

采用大脑中动脉瞬时缺血-再灌注（tMCAO）建立小鼠缺血性中风模型，并通过激光散斑成像和神经功能评分评估脑血流与神经功能。脑组织经苏木素-伊红（HE）与尼氏（Nissl）染色评价病理损伤。小鼠分为假手术组、模型组、黄连解毒汤组和银杏叶提取物组，适应1周后开始灌胃：假手术组和模型组给予生理盐水，黄连解毒汤组按1.82 g·kg

-1

给药，银杏叶提取物组按0.432 g·kg

-1

给药，连续5 d，第6天末次给药后进行tMCAO造模。对脑组织及外周免疫细胞进行单细胞RNA测序，利用均匀流形逼近与投影（UMAP）和比值比（OR）指标分析细胞分布，开展差异表达分析以评估黄连解毒汤对内皮细胞、周细胞和巨噬细胞的影响，并结合细胞通讯分析工具（CellChat）和解偶联调控分析工具（decoupler）分析细胞通讯和转录因子调控。最终使用实时荧光定量聚合酶链式反应（Real-time PCR）与酶联免疫吸附测定法（ELISA）验证相关mRNA和蛋白表达。

结果

与假手术组比较，模型组表现出神经功能评分显著升高（

0.01）及血流量水平显著下降（

0.01），且呈现皮质结构紊乱、胞体空泡化程度加剧及尼氏小体数量增多；与模型组比较，黄连解毒汤组与银杏叶提取物组则显示出神经功能评分显著降低（

0.01）及血流量水平显著回升（

0.01），同时皮质结构紊乱得以改善，胞体空泡化程度减轻，尼氏小体数量亦有所减少。单细胞数据显示，黄连解毒汤可保护内皮细胞与周细胞，防止其数量减少，同时可恢复内皮与周细胞中的功能基因［如血小板内皮细胞黏附分子1（PECAM1）、一氧化氮合酶3（NOS3）］表达，并降低其趋化因子与黏附因子表达水平［如C-C趋化因子配体2（CCL2）、C-X-C趋化因子配体2（CXCL2）］；对于巨噬细胞，黄连解毒汤可减少其向中枢的趋化数目，并可其趋化因子受体与炎症因子表达水平［如白细胞介素（IL）-6、C-C趋化因子受体2（CCR2）、C-X-C趋化因子受体2（CXCR2）］。同时，细胞通讯发现黄连解毒汤通过上述作用，降低周细胞与内皮细胞损害，减少其对巨噬细胞的吸引，降低周细胞-巨噬与内皮-巨噬之间的C-C趋化因子配体（CCL）、C-X-C趋化因子配体（CXCL）、粒细胞集落刺激因子3（CSF3）等趋化信号通路的配体-受体互作关系，避免造成二次损害。与假手术组比较，模型组小鼠IL-1

、IL-6、肿瘤坏死因子-

（TNF-

）、CCL2、CXCL2、CSF3分子的mRNA表达水平显著上调（

0.01），且G蛋白信号调节因子5（RGS5）、PECAM1、血管内皮生长因子B（VEGFB）与NOS3等内皮与周细胞功能相关mRNA表达水平显著下调（

0.01）；而与模型组比较，在黄连解毒汤组与银杏叶提取物组中，IL-1

、IL-6、TNF-

、CCL2、CXCL2、CSF3分子的mRNA表达水平显著下调（

0.01），且RGS5、PECAM1、VEGFB与NOS3等内皮与周细胞功能相关mRNA表达水平亦显著上调（

0.01）。与假手术组比较，模型组小鼠IL-1

、IL-6、TNF-

蛋白表达水平显著

上调（

0.01）；与模型组比较，黄连解毒汤与银杏叶提取物组的IL-1

、IL-6、TNF-

蛋白表达水平显著下调（

0.01）。

结论

黄连解毒汤通过保护缺血性中风的内皮细胞与周细胞，降低神经损害，同时抑制其与巨噬细胞的互作，减少对中枢内的二次损伤。

Abstract

Objective

To investigate the regulatory effects of Huanglian Jiedutang （HLJDT） on immune cell migration， blood-brain barrier protection， and cellular functional recovery in a model of ischemic stroke.

Methods

A transient middle cerebral artery occlusion （tMCAO） model was established in mice to induce ischemic stroke. Cerebral blood flow and neurological function were evaluated using laser speckle imaging and neurological deficit scoring. Histopathological damage in brain tissues was assessed by hematoxylin-eosin （HE） and Nissl staining. Mice were divided into a sham group， a model group， an HLJDT group， and a

Ginkgo biloba

extract （GBE） group. After one week of acclimatization， intragastric administration was initiated. The sham and model groups received normal saline， the HLJDT group received HLJDT at 1.82 g·kg

¹， and the GBE group received GBE at 0.432 g·kg

¹. Administration was continued for 5 consecutive days， and the tMCAO model was established after the final dose on day 6. Single-cell RNA sequencing was performed on brain tissues and peripheral immune cells. UMAP and odds ratio （OR） indices were used to analyze cell distribution. Differential expression analysis was conducted to evaluate the effects of HLJDT on endothelial cells， pericytes， and macrophages， combined with CellChat and decoupler to analyze cell-cell communication and transcription factor regulation. Finally， PCR and ELISA were used to validate the mRNA and protein expression of relevant genes.

Results

Compared with the sham group， the model group showed significantly increased neurological deficit scores （

0.01） and significantly decreased cerebral blood flow （

0.01）， accompanied by cortical structural disorder， aggravated cytoplasmic vacuolization， and increased numbers of Nissl bodies. Compared with the model group， both the HLJDT and GBE groups exhibited significantly reduced neurological deficit scores （

0.01） and markedly improved cerebral blood flow （

0.01）， along with amelioration of cortical structural disorder， alleviated cytoplasmic vacuolization， and reduced numbers of Nissl bodies. Single-cell analysis showed that HLJDT protected endothelial cells and pericytes by preventing their reduction， restored the expression of functional genes in these cells （

e.g

.， PECAM1 and NOS3）， and downregulated the expression of chemokines and adhesion-related factors （

e.g

.， CCL2 and CXCL2）. In macrophages， HLJDT reduced their recruitment to the central nervous system and downregulated the expression of chemokine receptors and inflammatory factors （

e.g

.， IL-6， CCR2， and CXCR2）. Cell-cell communication analysis further indicated that HLJDT， through the above mechanisms， alleviated damage to pericytes and endothelial cells， reduced their recruitment of macrophages， and decreased ligand-receptor interactions in chemokine signaling pathways （including CCL， CXCL， and CSF3） between pericytes/endothelial cells and macrophages， thereby preventing secondary injury. Compared with the sham group， the model group showed significantly upregulated mRNA expression levels of IL-1

， IL-6， TNF-

， CCL2， CXCL2， and CSF3 （

0.01）， while mRNA expression levels of endothelial- and pericyte function-related genes （RGS5， PECAM1， VEGFB， and NOS3） were significantly downregulated （

0.01）. In contrast， compared with the model group， the HLJDT and GBE groups exhibited significantly decreased mRNA expression levels of IL-1

， IL-6， TNF-

， CCL2， CXCL2， and CSF3 （

0.01）， and significantly increased expression of RGS5， PECAM1， VEGFB， and NOS3 （

0.01）. At the protein level， compared with the sham group， the model group showed significantly increased expression of IL-1

， IL-6， and TNF-

（

0.01）， whereas these protein levels were significantly reduced in the HLJDT and GBE groups compared with the model group （

0.01）.

Conclusion

HLJDT reduces neuronal damage in ischemic stroke by protecting endothelial cells and pericytes， while inhibiting their interaction with macrophages， thereby mitigating secondary injury in the central nervous system.

关键词

Keywords

references

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