Anti-inflammatory Effect and Mechanism of Lianggesan and Its Component Glycyrrhiza Radix et Rhizoma Based on Zebrafish Inflammation Model

YU Jingtao; ZHOU Hongling; LIU Junshan; XU Nishan; QUAN Jingyu; LU Zibin; LIU Dongyi; CAO Huihui; YU Linzhong

doi:10.13422/j.cnki.syfjx.20221938

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Anti-inflammatory Effect and Mechanism of Lianggesan and Its Component Glycyrrhiza Radix et Rhizoma Based on Zebrafish Inflammation Model

更新时间：2022-11-17

- Anti-inflammatory Effect and Mechanism of Lianggesan and Its Component Glycyrrhiza Radix et Rhizoma Based on Zebrafish Inflammation Model
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 28, Issue 20, Pages: 17-26(2022)
- 作者机构：
  
  南方医科大学中医药学院，国家中医药管理局科研三级实验室中药药理实验室，中药制剂实验室，广州 510515
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20221938
  CLC： R2-0;R33;R289
- Received：16 April 2022，
  
  Published Online：22 July 2022，
  
  Published：20 October 2022
- 稿件说明：
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余景滔,周红玲,刘俊珊等.基于斑马鱼炎症模型的凉膈散及其组分甘草不同极性部位的抗炎活性评价及作用机制[J].中国实验方剂学杂志,2022,28(20):17-26.

YU Jingtao,ZHOU Hongling,LIU Junshan,et al.Anti-inflammatory Effect and Mechanism of Lianggesan and Its Component Glycyrrhiza Radix et Rhizoma Based on Zebrafish Inflammation Model[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(20):17-26.
余景滔,周红玲,刘俊珊等.基于斑马鱼炎症模型的凉膈散及其组分甘草不同极性部位的抗炎活性评价及作用机制[J].中国实验方剂学杂志,2022,28(20):17-26. DOI： 10.13422/j.cnki.syfjx.20221938.

YU Jingtao,ZHOU Hongling,LIU Junshan,et al.Anti-inflammatory Effect and Mechanism of Lianggesan and Its Component Glycyrrhiza Radix et Rhizoma Based on Zebrafish Inflammation Model[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(20):17-26. DOI： 10.13422/j.cnki.syfjx.20221938.

摘要

目的

采用脂多糖（LPS）诱导斑马鱼炎症模型，通过浸泡给药方式，评价凉膈散及其组分甘草不同极性部位的抗LPS活性。

方法

利用相似相溶原理分离得到凉膈散及其组分甘草的不同极性部位，随机选择受精后3 d的斑马鱼，暴露于不同浓度的药物提取物中，进行为期24、48、72 h的毒性观察；斑马鱼卵黄囊显微注射0.5 g·L

-1

LPS造模，浸泡给予不同浓度的凉膈散及甘草提取物，统计72 h内斑马鱼的生存数目并在显微镜下观察12 h时斑马鱼卵黄囊中性粒细胞的聚集情况；苏木素-伊红（HE）染色对LPS显微注射斑马鱼卵黄囊进行组织病理学分析；实时荧光定量聚合酶链式反应（Real-time PCR）进一步探讨凉膈散及甘草体内抗炎作用及其可能的机制。

结果

凉膈散及甘草提取物毒性随着极性升高而降低，药物的毒性大小依次为石油醚部位

乙酸乙酯部位

正丁醇部位

水部位；与LPS模型组比较，各提取物均能提高LPS斑马鱼炎症模型的动物生存率，抑制斑马鱼卵黄囊中性粒细胞的聚集及炎症细胞浸润，其中凉膈散水部位与甘草乙酸乙酯部位的效果最为显著（

0.01）；此外，与LPS模型组比较，凉膈散水部位与甘草乙酸乙酯部位降低了斑马鱼炎症模型中促炎细胞因子白细胞介素-6（IL-6）和肿瘤坏死因子-

（TNF-

）的mRNA表达，抑制了LPS诱导的Toll样受体4（TLR4）和核转录因子-

B（NF-

B）的上调（

0.01）。

结论

凉膈散及其组分甘草的不同提取部位对LPS炎症均有保护作用，其机制可能与抑制TLR4和NF-

B信号通路相关；在斑马鱼模型中，浸泡给药方式适用于中药的抗LPS活性筛选，可用于单味中药、复方及其不同提取部位的抗内毒素活性评价。

Abstract

Objective

Lipopolysaccharide （LPS）-induced zebrafish inflammation model was used to evaluate the anti-inflammatory activity of different extracts from Lianggesan （LGS） and its component Glycyrrhiza Radix et Rhizoma.

Method

Different polar fractions of LGS and Glycyrrhiza Radix et Rhizoma were obtained by the principle of similar miscibility. For toxicity observation， the zebrafish （3 day-post-fertilization） was exposed to different concentrations of extracts for 24， 48 and 72 h. The yolk sac of the zebrafish was microinjected with 0.5 g·L

-1

LPS to establish the inflammation model， and then the embryos were soaked with different concentrations of extracts to observe their survival status at 72 h and the aggregation of neutrophils in yolk sac at 12 h after treatment. Hematoxylin-eosin staining was used to analyze the yolk sac of the zebrafish microinjected with LPS. Quantitative Real-time polymerase chain reaction （Real-time PCR） was performed to further investigate the anti-inflammatory effects and mechanisms of LGS and Glycyrrhiza Radix et Rhizoma.

Result

The toxicity of LGS and Glycyrrhiza Radix et Rhizoma was decreased with the increase of polarity， and the descending order was petroleum ether

ethyl acetate

-butanol

water. Compared with model group， the extracts from different fractions of LGS

and

Glycyrrhiza Radix et Rhizoma prolonged the survival time of the zebrafish， and inhibited the recruitment and aggregation of neutrophils and decreased the infiltration of inflammatory cells in the yolk sac， among which the water fraction of LGS and the ethyl acetate fraction of Glycyrrhiza Radix et Rhizoma had the most significant effect （

0.01）. In addition， compared with model group， the water fraction of LGS and the ethyl acetate fraction of Glycyrrhiza Radix et Rhizoma down-regulated the mRNA expression of interleukin-6 （IL-6） and tumor necrosis factor-

（TNF-

）， and suppressed the expression of toll like receptor 4 （TLR4） and nuclear factor kappa-B （NF-

B） in LPS-stimulated zebrafish （

0.01）.

Conclusion

The extracts from different fractions of LGS and Glycyrrhiza Radix et Rhizoma

exerted protective effects in LPS-induced zebrafish by inhibiting the TLR4 and NF-

B signaling pathways. Moreover， in zebrafish model， the method of administration by soaking was applicable to the high-throughput screening of anti-inflammatory Chinese medicine， which was suitable for the evaluation of anti-LPS activity of Chinese medicine and the different extracts.

关键词

Keywords

references

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