活血疏风颗粒通过抑制TLR4/NF-κB通路改善慢性偏头痛小鼠的中枢敏化

doi:10.12122/j.issn.1673-4254.2025.05.11

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (5): 986-994.doi: 10.12122/j.issn.1673-4254.2025.05.11

活血疏风颗粒通过抑制TLR4/NF-κB通路改善慢性偏头痛小鼠的中枢敏化

梁晓涛¹(), 熊一凡¹, 刘雪琪⁵, 梁小珊⁴, 朱晓煜¹, 谢炜¹^,²^,³^,⁴()

^1.南方医科大学中医药学院
^2.广东省中药制剂重点实验室
^3.广东省中西医结合防治情志病基础研究卓越中心
^4.南方医科大学南方医院中医科，广东广州 515510
^5.珠海高新技术产业开发区人民医院，广东珠海 519000

收稿日期:2025-01-23 出版日期:2025-05-20 发布日期:2025-05-23
通讯作者: 谢炜 E-mail:1092296938@qq.com;xieweizn@126.com
作者简介:梁晓涛，医师，在读博士研究生，E-mail: 1092296938@qq.com
基金资助:
第二届全国名中医工作室(G724290126);中西医结合情志病防治重大成果培育(G624290511);2023年度省基础与应用基础研究基金企业联合基金（公共卫生与医药健康领域）(2023A1515220030);重大疑难疾病中西医临床协作项目

Huoxue Shufeng Granule alleviates central sensitization in chronic migraine mice via TLR4/NF-κB inflammatory pathway

Xiaotao LIANG¹(), Yifan XIONG¹, Xueqi LIU⁵, Xiaoshan LIANG⁴, Xiaoyu ZHU¹, Wei XIE¹^,²^,³^,⁴()

^1.School of Traditional Chinese Medicine, Southern Medical University
^2.Guangdong Provincial Key Laboratory of Chinese Medicine Pharmaceutics
^3.Guangdong Basic Research Center of Excellence for Integrated Traditional and Western Medicine for Qingzhi Diseases
^4.Department of Traditional Chinese Medicine, Nanfang Hospital of Southern Medical University, Guangzhou 510515, Guangdong
^5.People's Hospital of Zhuhai High-tech Industrial Development Zone, Zhuhai 519000, Guangdong

Received:2025-01-23 Online:2025-05-20 Published:2025-05-23
Contact: Wei XIE E-mail:1092296938@qq.com;xieweizn@126.com
About author:First author contact:LIANG Xiaotao1, XIONG Yifan1, LIU Xueqi5, LIANG Xiaoshan4, ZHU Xiaoyu1, XIE Wei1, 2, 3, 4

摘要/Abstract

摘要：

目的探讨活血疏风颗粒对慢性偏头痛（CM）小鼠中枢敏化的干预作用及机制。方法通过文献检索获取活血疏风颗粒的主要化学成分，使用多重数据库分析预测药理机制。动物实验验证：选用雄性SPF C57BL/6J小鼠，使用腹腔注射硝酸甘油给药（10 mg/kg），隔日注射，连续注射5次，制备偏头痛小鼠模型。以活血疏风颗粒灌胃或托吡酯腹腔注射进行干预，根据干预方式的不同将小鼠分为对照组、慢性偏头痛模型组、托吡酯阳性对照组、活血疏风颗粒低剂量组、活血疏风颗粒高剂量组（HXSFG-H），12只/组。利用Von Frey和热板仪装置评估小鼠中枢敏化程度，RT-qPCR检测炎症相关因子，Western blotting检测TLR4/NF-κB通路表达水平，免疫荧光实验检测c-Fos、CGRP的活化水平。结果网络药理学结果显示，活血疏风颗粒改善CM的主要活性成分为芒柄花素、芍药苷、槲皮素、丹参酮等，GO功能富集分析得到共得到492条信号通路，其中生物过程366条，细胞组分46条，分子功能80条；KEGG富集分析显示，活血疏风颗粒改善慢性偏头痛的通路主要涉及Toll样受体和NF-κB信号通路。动物实验成功构建CM小鼠模型，托吡酯阳性对照组和活血疏风颗粒治疗组小鼠中枢敏化减轻，表现为机械痛阈值和热痛阈值下降程度的减小。活血疏风颗粒可降低c-Fos和CGRP的表达（P<0.01），改善炎症相关指标（P<0.01）；下调TLR4、p-NF-κB、IL-1β、TNF-α蛋白表达（P<0.01）。结论活血疏风颗粒能有效调节炎症通路，抑制TLR4/NF-κB信号转导，减轻中枢敏化。

关键词: 活血疏风颗粒, 慢性偏头痛, TLR4/NF-κB, 网络药理学

Abstract:

Objective To investigate the therapeutic mechanism of Huoxue Shufeng Granules (HXSFG) for alleviating central sensitization in a mouse model of chronic migraine (CM). Methods We analyzed the main chemical components of HXSFG through literature review and explored their pharmacological mechanisms by bioinformatics analyses. In a male C57BL/6J mouse model of CM established by intraperitoneal injections of nitroglycerin (10 mg/kg) every other day (5 injections), the effects of gavage with low, and high doses of HXSFG or intraperitoneal injections of topiramate for ameliorating central sensitization were evaluated using Von Frey test and a hot plate apparatus; the changes in expressions of inflammatory factors, the proteins in the TLR4/NF‑κB signaling pathway, and activation of c-Fos and CGRP were detected using RT-qPCR, Western blotting and immunofluorescence staining. Results Network pharmacology analysis suggested that the main active components in HXSFG for alleviating CM included formononetin, paeoniflorin, quercetin, and tanshinone. Gene Ontology (GO) enrichment analysis identified 492 GO entries, comprising 366 biological processes, 46 cellular components, and 80 molecular functions. KEGG pathway enrichment analysis indicated that the Toll-like receptor and NF‑κB signaling pathways were crucial in mediating the therapeutic effects of HXSFG on CM. In the mouse models of CM, both topiramate and HXSFG treatments alleviated the symptoms of central sensitization, evidenced by improved mechanical and thermal pain thresholds in the mice. HXSFG significantly reduced the expression of c-Fos and CGRP, improved inflammatory markers, and downregulated the expressions of TLR4, p-NF‑κB, IL-1β, and TNF‑α proteins in the mouse models. Conclusion HXSFG effectively alleviates central sensitization in CM mice by modulating the inflammatory pathways and inhibiting the TLR4/ NF-κB signaling pathway, suggesting its potential as a therapeutic option for CM.

Key words: Huoxue Shufeng Granules, chronic migraine, TLR4/NF-κB, network pharmacology

梁晓涛, 熊一凡, 刘雪琪, 梁小珊, 朱晓煜, 谢炜. 活血疏风颗粒通过抑制TLR4/NF-κB通路改善慢性偏头痛小鼠的中枢敏化[J]. 南方医科大学学报, 2025, 45(5): 986-994.

Xiaotao LIANG, Yifan XIONG, Xueqi LIU, Xiaoshan LIANG, Xiaoyu ZHU, Wei XIE. Huoxue Shufeng Granule alleviates central sensitization in chronic migraine mice via TLR4/NF-κB inflammatory pathway[J]. Journal of Southern Medical University, 2025, 45(5): 986-994.

图/表 6

表1 本研究涉及的引物序列

Tab.1 Primer sequences used in this study

Primer name	Sequence
IFN-γ Forward	AGCAACAACATAAGCGTCATTGA
IFN-γ Reverse	TGACCTCAAACTTGGCAATACTC
TNF-α Forward	GCCTCCCTCTCATCAGTTCTATG
TNF-α Reverse	ACCTGGGAGTAGACAAGGTACAA
IL-1β Forward	GCAACTGTTCCTGAACTCAACT
IL-1β Reverse	ATCTTTTGGGGTCCGTCAACT
IL-6 Forward	ACAACCACGGCCTTCCCTACTT
IL-6 Reverse	CACGATTTCCCAGAGAACATGTG
GAPDH Forward	CAACTACATGGTCTACATGTTC
GAPDH Reverse	CTCGCTCCTGGAAGATG

图2 活血疏风颗粒显著减轻 NTG 诱导的CM小鼠后爪基础及急性机械痛觉和热痛觉过敏

Fig.2 HXSFG alleviates baseline and acute mechanical and thermal hyperalgesia in mice with NTG-induced CM. A: Changes in baseline mechanical pain thresholds of the hind paws of the mice. B: Changes in acute mechanical pain thresholds of the hind paws of the mice. C: Changes in baseline thermal pain thresholds of the hind paws. D: Changes in acute thermal pain thresholds of the hind paws. Data are presented as Mean±SE (n=12). Statistical analysis was performed using two-way ANOVA followed by Tukey's post hoc test. **P<0.01 Model group vs Control group; #P<0.05, ##P<0.01 Topiramate group vs Model group; &P<0.05, &&P<0.01 HXSFG-L group vs Model group; ^^P<0.01 HXSFG-H group vs Model group.

图3 活血疏风颗粒对NTG 诱导的CM小鼠TNC部位c-Fos表达水平的影响

Fig.3 Effects of HXSFG on c-Fos expression levels in the TNC region of mice with NTG-induced CM. A: Immunofluorescence images of c-Fos expression (Scale bar: 100 μm). B: Quantitative analysis of c-Fos immunoreactivity (Each group included 4 mice, and 3 slices were analyzed for each mouse). C: Protein immunoblot images of c-Fos expression. D: Quantitative analysis of c-Fos protein immunoblotting (n=4). Data are presented as Mean±SE. Statistical analysis was performed using two-way ANOVA followed by Tukey's post hoc test. **P<0.01 vs Control group; #P<0.05, ##P<0.01 vs Model group; &P<0.05, &&P<0.01 vs Model group; ^^P<0.01 vs Model group.

图4 活血疏风颗粒对NTG 诱导的CM小鼠TNC部位CGRP表达水平的影响

Fig.4 Effects of HXSFG on CGRP expression levels in the TNC region of mice with NTG-induced CM. A: Immunofluorescence images of CGRP expression (×40, scale bar: 500 μm). B: Quantitative analysis of CGRP immunoreactivity (Each group included 4 mice, and 3 slices were analyzed for each mouse). C: Protein immunoblot images of CGRP expression. D: Quantitative analysis of CGRP protein immunoblotting (n=4). Data are presented as Mean±SE. Statistical analysis was performed using two-way ANOVA followed by Tukey's post hoc test. **P<0.01 vs Control group; ##P<0.01 vs Model group; &&P<0.01 vs Model group; ^^P<0.01 vs Model group.

图5 活血疏风颗粒对NTG 诱导的CM小鼠TNC部位炎症因子表达水平的影响

Fig.5 Effects of HXSFG on mRNA levels of inflammatory factors in the TNC region of mice with NTG-induced CM. A: Relative mRNA level of IL-1β. B: Relative mRNA level of IL-6. C: Relative mRNA level of TNF‑α. D: Relative mRNA level of IFN-γ. **P<0.01 vs Control group; ##P<0.01 vs Model group; &&P<0.01 vs Model group; ^^P<0.01 vs Model group.

图6 活血疏风颗粒对NTG 诱导的CM小鼠TNC部位TLR4/NF-κB信号通路的影响

Fig. 6 Effects of HXSFG on the TLR4/NF-κB signaling pathway in the TNC region of mice with NTG-induced CM. A: Protein immunoblot images of TLR4, p-NF-κB, NF-κB, IL-1β, TNF-α expression. B: Quantitative analysis of TLR4 protein immunoblotting. C: Quantitative analysis of p-NF-κB/NF-κB protein immunoblotting. D: Quantitative analysis of IL-1β protein immunoblotting. E: Quantitative analysis of TNF-α protein immunoblotting (n=4). Data are presented as Mean±SE. Statistical analysis was performed using two-way ANOVA followed by Tukey's post hoc test. **P<0.01 vs Control group; ##P<0.01 vs Model group; &&P<0.01 vs Model group; ^^P<0.01 vs Model group.

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活血疏风颗粒通过抑制TLR4/NF-κB通路改善慢性偏头痛小鼠的中枢敏化

Huoxue Shufeng Granule alleviates central sensitization in chronic migraine mice via TLR4/NF-κB inflammatory pathway

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