Mechanism of Honghua Oral Liquid in Alleviating Neuropathic Pain

GUO Qiuyan; ZHAO Minghong; LU Tianming; XIA Fei; ZHANG Ying; ZHANG Hongbing; ZHAI Xiaoru; YANG Qian; LI Yongdong; LI Jin; LI Xin; SHEN Shuo; GU Liwei; DU Maobo

doi:10.13422/j.cnki.syfjx.20222442

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Mechanism of Honghua Oral Liquid in Alleviating Neuropathic Pain

更新时间：2023-02-15

- Mechanism of Honghua Oral Liquid in Alleviating Neuropathic Pain
  增强出版
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 6, Pages: 222-230(2023)
- 作者机构：
  
  1.中国中医科学院中药研究所，北京 100700
  2.中国中医科学院青蒿素研究中心，北京 100700
  3.赣南医学院第一临床医学院，江西赣州341000
  4.山西华卫药业有限公司，山西晋中 030600
  5.青岛妇女儿童医院，山东青岛 266000
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20222442
  CLC： R2-0;R33;R289;R322.8
- Published：20 March 2023，
  
  Published Online：05 December 2022，
  
  Received：16 September 2022，
- 稿件说明：
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郭秋岩,赵明洪,卢天明等.红花口服液缓解神经病理性疼痛的分子机制[J].中国实验方剂学杂志,2023,29(06):222-230.

GUO Qiuyan,ZHAO Minghong,LU Tianming,et al.Mechanism of Honghua Oral Liquid in Alleviating Neuropathic Pain[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(06):222-230.
郭秋岩,赵明洪,卢天明等.红花口服液缓解神经病理性疼痛的分子机制[J].中国实验方剂学杂志,2023,29(06):222-230. DOI： 10.13422/j.cnki.syfjx.20222442.

GUO Qiuyan,ZHAO Minghong,LU Tianming,et al.Mechanism of Honghua Oral Liquid in Alleviating Neuropathic Pain[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(06):222-230. DOI： 10.13422/j.cnki.syfjx.20222442.

摘要

目的

考察红花口服液（HOL）缓解神经病理性疼痛（NP）的药效作用特点并探究其分子作用机制。

方法

基于脊神经结扎（SNL）大鼠模型，将健康雄性SD大鼠随机分为假手术组，模型组，HOL低、中、高剂量组（0.5、1.0、2.0 mL·kg

-1

·d

-1

）及阳性药普瑞巴林组（25 mg·kg

-1

·d

-1

），每组6只。在脊神经结扎手术恢复3 d后，连续灌胃给药14 d并采集样本。期间检测各组大鼠的机械痛阈值和冷痛阈值，考察HOL的镇痛作用特点。将假手术组、模型组、HOL高剂量组的海马组织样本进行转录组测序，获取不同组别之间的差异表达基因，并进行通路富集分析，进而，选择与NP密切相关的靶标进行验证，进一步通过分子对接寻找HOL关键活性成分与靶标分子的具体结合位点。此外，检测血清中肿瘤坏死因子-

（TNF-

）和白细胞介素-10（IL-10）的表达水平，评价HOL对NP大鼠的影响。

结果

与假手术组比较，模型组大鼠的机械痛阈值及冷痛阈值均明显降低（

0.05）。与模型组比较，HOL组可有效升高其机械痛阈值及冷痛阈值（

0.05）；转录组分析发现，模型组与假手术组比较共有376个差异表达基因，其中上调基因124个、下调基因252个，模型组与HOL组比较共有194个差异表达基因，其中上调基因33个、下调基因161个。其中，类胰岛素一号生长因子（IGF1）、基质金属蛋白酶-2（MMP-2）、基质金属蛋白酶-14 （MMP-14）、酪氨酸激酶受体2（ERBB2）和整合素A5（ITGA5）与NP密切相关。实时荧光定量聚合酶链式反应（Real-time PCR）结果显示，与假手术组比较，模型组大鼠海马中上述各分子的mRNA表达量均显著升高（

0.01）。与模型组比较，HOL组可有效降低其mRNA表达量（

0.01）。分子对接结果显示，红花主要活性成分羟基红花黄色素A、山奈酚和槲皮素与上述靶标分子IGF1、MMP-2、MMP-14、ERBB2、ITGA5的氨基酸残基形成稳定的氢键作用力。酶联免疫吸附测定法（ELISA）检测结果显示，与假手术组比较，模型组大鼠血清中TNF-

/IL-10表达失衡（

0.01），与模型组比较，HOL组可显著减少促炎细胞因子TNF-

的含量（

0.01），增加抗炎细胞因子IL-10的含量（

0.05）。

结论

HOL对SNL大鼠具有镇痛作用，其机制与抑制神经炎症密切相关。

Abstract

Objective

To investigate the pharmacodynamic characteristics and explore the molecular mechanism of Honghua oral liquid （HOL） in relieving neuropathic pain （NP）.

Method

Healthy male SD rats were randomly assigned into sham group， model group， low-， medium-， high-dose （0.5， 1.0， 2.0 mL·kg

-1

·d

-1

， respectively） HOL groups， and a positive drug （pregabalin， 25 mg·kg

-1

·d

-1

） group， with 6 rats in each group. Spinal nerve ligation （SNL） of L5 was conducted in other groups except the sham group. Drug administration was performed 3 days after the SNL surgery for 2 consecutive weeks， and samples were collected after the end of the administration. During the treatment period， the mechanical pain threshold and cold pain threshold were determined to measure the pain-relieving effect of HOL. Transcriptome sequencing was performed on hippocampal tissue samples from the sham， model， and high-dose HOL groups， and differentially expressed genes between the sham group and the model group as well as the model group and HOL high-dose group were obtained. After pathway enrichment analysis， we selected the targets which were closely related to neuroinflammation for validation， and predicted the specific binding sites of the major active components in HOL with the targets through molecular docking. In addition， the serum levels of tumor necrosis factor-

（TNF-

） and interleukin-10 （IL-10） were determined by enzyme-linked immunosorbent assay （ELISA） to evaluate the effect of HOL on neuroinflammation in NP rats.

Result

Compared with the sham group， SNL decreased the mechanical pain threshold and cold pain threshold （

0.05）. Compared with the model group， HOL recovered the mechanical pain threshold and cold pain threshold （

0.05）. The transcriptome data showed that 376 differentially expressed genes （DEGs） were identified between the model group and the sham group， including 124 upregulated genes and 252 downregulated genes， and 194 DEGs between the model group and the high-dose HOL group， including 33 upregulated genes and 161 downregulated genes. Among them， insulin-like growth factor 1（IGF1）， matrix metallopeptidase-2 （MMP-2）， matrix metallopeptidase-14 （MMP-14）， erb-B2 receptor tyrosine kinase 2 （ERBB2）， and integrin subunit alpha 5 （ITGA5） associated with NP were selected for further validation. The Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） results showed that compared with the sham group， the modeling up-gurelated the mRNA levels of the above five molecules in the hippocampus （

0.01）. Compared with model group， HOL down-regulated the mRNA levels of these molecules （

0.01）. The molecular docking results showed that the main active components of safflower， hydroxysafflor yellow A， kaempferol， and quercetin， formed stable hydrogen bonds with the amino acid residues of IGF1， MMP-2， MMP-14， ERBB2， and ITGA5. The enzyme-linked immunosorbent assay（ELISA） results showed that compared with those in the sham group， the serum levels of TNF-

and IL-10 were out of balance in the model rats （

0.01）. Compared with the model group， HOL lowered the level of the pro-inflammatory cytokine TNF-

（

0.01） and elevated that of the anti-inflammatory cytokine IL-10 （

0.05）.

Conclusion

HOL exerts analgesic effect on SNL rats by inhibiting neuroinflammation.

关键词

红花口服液神经病理性疼痛胰岛素样生长因子基质金属蛋白酶酪氨酸激酶受体2整合素A5

Keywords

Honghua oral liquidneuropathic paininsulin-like growth factormatrix metalloproteinaseerb-B2 receptor tyrosine kinase 2integrin subunit alpha 5

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