Effect of Gupi Xiaoji Decoction on Pyroptosis of HepG2.2.15 Cells Based on Network Pharmacology and Molecular Docking

Hui-ying JIAN; Wen-hui GAO; Xiao-ning TAN; Zhuo LIU; Zhen ZHANG; Ke-xin LI; Pu-hua ZENG

doi:10.13422/j.cnki.syfjx.20220318

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Effect of Gupi Xiaoji Decoction on Pyroptosis of HepG2.2.15 Cells Based on Network Pharmacology and Molecular Docking

更新时间：2022-02-15

- Effect of Gupi Xiaoji Decoction on Pyroptosis of HepG2.2.15 Cells Based on Network Pharmacology and Molecular Docking
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 28, Issue 6, Pages: 159-166(2022)
- 作者机构：
  
  1.湖南中医药大学,长沙 410208
  2.湖南省中医药研究院附属医院,长沙 410006
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20220318
  CLC： R285;R289;R22;R2-031;R33
- Received：25 October 2021，
  
  Published Online：14 January 2022，
  
  Published：20 March 2022
- 稿件说明：
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翦慧颖,郜文辉,谭小宁等.基于网络药理学与分子对接技术探讨固脾消积饮对肝癌HepG2.2.15细胞焦亡的影响[J].中国实验方剂学杂志,2022,28(06):159-166.

JIAN Hui-ying,GAO Wen-hui,TAN Xiao-ning,et al.Effect of Gupi Xiaoji Decoction on Pyroptosis of HepG2.2.15 Cells Based on Network Pharmacology and Molecular Docking[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(06):159-166.
翦慧颖,郜文辉,谭小宁等.基于网络药理学与分子对接技术探讨固脾消积饮对肝癌HepG2.2.15细胞焦亡的影响[J].中国实验方剂学杂志,2022,28(06):159-166. DOI： 10.13422/j.cnki.syfjx.20220318.

JIAN Hui-ying,GAO Wen-hui,TAN Xiao-ning,et al.Effect of Gupi Xiaoji Decoction on Pyroptosis of HepG2.2.15 Cells Based on Network Pharmacology and Molecular Docking[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(06):159-166. DOI： 10.13422/j.cnki.syfjx.20220318.

摘要

目的

以半胱氨酸天冬氨酸蛋白水解酶-1（Caspase-1）介导的细胞焦亡为基础，采用网络药理学及分子对接技术筛选中药复方固脾消积饮中抗肿瘤的活性成分，通过体外实验探讨该方干预肝癌HepG2.2.15细胞焦亡的分子机制。

方法

利用中药系统药理学分析平台（TCMSP）筛选中药复方固脾消积饮的化合物及靶点，并获取对应的基因Symbol；从GeneCards数据库、在线人类孟德尔遗传（OMIM）数据库、PharmGKB数据库、TTD数据库搜集Caspase-1的靶点，运用Cytoscape构建化合物-基因靶点调控网络；采用STRING数据库进行蛋白质-蛋白质相互作用（PPI）网络分析，采用基因本体（GO）功能富集和京都基因与基因组百科全书（KEGG）通路富集分析预测该方有效成分对Caspase-1的作用机制，应用AutoDock Vina进行分子对接验证。制备固脾消积饮含药血浆，体外培养肝癌HepG2.2.15细胞；将HepG2.2.15细胞分为空白血浆组、VX-765组、VX-765+含药血浆组、含药血浆组，使用15%含药血浆干预48 h后，免疫荧光染色法检测细胞膜表面GSDMD-N的表达及分布情况，检测HepG2.2.15细胞上清乳酸脱氢酶（LDH）、白细胞介素-1

（IL-1

）、白细胞介素-18（IL-18）的释放情况，蛋白免疫印迹法（Western blot）检测细胞中特征蛋白Caspase-1、消皮素D-N端（GSDMD-N）的表达水平。

结果

网络药理学筛选得到丝裂原活化蛋白激酶14（MAPK14）、丝裂原活化蛋白激酶1（MAPK1）、蛋白激酶B1（Akt1）、丝裂原活化蛋白激酶8（MAPK8）、V-Jun肉瘤病毒癌基因同源物（JUN）、TP53为主要作用靶点，化合物7-hydroxy-5，8-dimethoxy-2-phenyl-chromone、黄芩素、鼠李素、荠苎黄酮、异鼠李黄素、7-

-methylisomucronulatol、芒柄花黄素、毛蕊异黄酮、木犀草素、槲皮素、山柰酚、

-谷甾醇、黄芩苷为主要活性成分，GO富集分析涉及氧化应激反应、对金属离子的反应、与泛素-类蛋白连接酶结合、磷酸酶结合等多个生物过程，KEGG通路富集分析涉及MAPK、核转录因子（NF）-

B、p53、低氧诱导因子-1（HIF-1）等信号通路，分子对接表明靶点与成分具有较好结合性。体外实验表明，与空白血浆组相比，VX-765组的GSDMD-N荧光信号减少减弱，LDH、IL-1

、IL-18的释放量均减少，Caspase-1、GSDMD-N的表达显著下调（

0.01）；固脾消积饮含药血浆组的GSDMD-N荧光信号增加增强，LDH、IL-1

、IL-18的释放量显著增加，Caspase-1、GSDMD-N的表达显著增高（

0.01）。

结论

固脾消积饮通过多靶点多通路调控Caspase-1以诱导肝癌HepG2.2.15细胞发生焦亡。

Abstract

Objective

To screen the active antitumor components of Gupi Xiaoji decoction by network pharmacology and molecular docking based on the pyroptosis mediated by cysteinyl aspartate-specific protease 1 （Caspase-1） and explore its molecular mechanism in intervening in the pyroptosis of HepG2.2.15 cells through

in vitro

experiments.

Method

The compounds and targets of Gupi Xiaoji decoction were screened out by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP） to obtain the corresponding gene symbols. The targets of Caspase-1 were collected from GeneCards，online mendelian inheritance in man（OMIM），PharmGKB，and TTD，and the compound-gene target regulatory network was constructed by Cytoscape. The protein-protein interaction（PPI） network was established and analyzed by STRING. The mechanism of the effective components of Gupi Xiaoji decoction on Caspase-1 was predicted by gene ontology（GO） functional enrichment and Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analyses. The molecular docking was verified with AutoDock Vina. The plasma medicated with Gupi Xiaoji Decoction was prepared and HepG2.2.15 cells were cultured

in vitro

. HepG2.2.15 cells were divided into a blank plasma group，a VX-765 group，a VX-765+medicated plasma group， and a medicated plasma group. After 48 hours of intervention with 15% medicated plasma， the expression and distribution of gasdermin D-N （GSDMD-N） on the surface of the cell membrane were detected by immunofluorescence staining. The release of lactic dehydrogenase （LDH）， interleukin（IL）-1

，and IL-18 in the cell supernatant was measured by enzyme-linked immunosorbent assay（ELISA） kits. The expression of Caspase-1 and GSDMD-N was measured by Western blot.

Result

The mitogen-activated protein kinase 14 （MAPK14），MAPK1，protein kinase B1 （Akt1）， MAPK8， V-Jun sarcoma virus oncogene homolog （JUN）， and TP53 screened by network pharmacology were the main targets. The compounds 7-hydroxy-5，8-dimethoxy-2-phenyl-chromone，wogonin，rhamnazin，moslosooflavone，isorhamnetin，7-

-methylisomucronulatol，formononetin，calycosin，luteolin，quercetin，kaempferol，

-sitosterol，and baicalein screened by network pharmacology were the main active components of Gupi Xiaoji decoction. Go enrichment analysis showed that multiple biological processes were involved， including responses to oxidative stress and metal ions，ubiquitin-like protein ligase binding，and phosphatase binding. KEGG pathway enrichment analysis showed MAPK pathway，nuclear factor（NF）-

B pathway，p53 pathway， and hypoxia-inducible factor-1（HIF-1） pathway were involved. Molecular docking showed that the targets had good binding with the components.

In vitro

experiments displayed that compared with the blank plasma group，the VX-765 group showed weakened GSDMD-N fluorescence signal，reduced release of LDH，IL-1

，and IL-18，and declining expression of Caspase-1 and GSDMD-N（

0.01）， and the medicated plasma group showed increased GSDMD-N fluorescence signal， increased release of LDH，IL-1

，and IL-18，and up-regulated expression of Caspase-1 and GSDMD-N（

0.01）.

Conclusion

Gupi Xiaoji Decoction can induce the pyroptosis of HepG2.2.15 cells by regulating Caspase-1 through multiple targets and multiple pathways.

关键词

Keywords

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