茵陈蒿汤治疗肝纤维化的核心功能成分群以及潜在通路

doi:10.12122/j.issn.1673-4254.2024.08.09

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (8): 1508-1517.doi: 10.12122/j.issn.1673-4254.2024.08.09

• • 上一篇

茵陈蒿汤治疗肝纤维化的核心功能成分群以及潜在通路

陈星梅¹(), 刘琴文²^,³, 李镱²^,³, 钟晓宇¹, 樊奇灵²^,³, 马柯¹, 罗柳婷¹, 官道刚²^,³, 朱志博¹()

^1.南方医科大学中西医结合医院，广东广州 510220
^2.南方医科大学基础医学院生物化学与分子生物学系，广东广州 510410
^3.南方医科大学广东省单细胞技术与应用重点实验室，广东广州 510410

收稿日期:2024-03-21 出版日期:2024-08-20 发布日期:2024-09-06
通讯作者: 朱志博 E-mail:1842578607@qq.com;zhuzb676@smu.edu.cn
作者简介:陈星梅，在读硕士研究生，E-mail: 1842578607@qq.com
基金资助:
广东省基础与应用基础研究基金(2020A1515010412)

Analysis of core functional components in Yinchenhao Decoction and their pathways for treating liver fibrosis

Xingmei CHEN¹(), Qinwen LIU²^,³, Yi LI²^,³, Xiaoyu ZHONG¹, Qiling FAN²^,³, Ke MA¹, Liuting LUO¹, Daogang GUAN²^,³, Zhibo ZHU¹()

^1.Southern Medical University Hospital of Integrated Traditional Chinese and Western Medicine, Southern Medical University, Guangzhou 510220, China
^2.Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510410, China
^3.Guangdong Provincial Key Laboratory of Single Cell Technology and Application, Southern Medical University, Guangzhou 510410, China

Received:2024-03-21 Online:2024-08-20 Published:2024-09-06
Contact: Zhibo ZHU E-mail:1842578607@qq.com;zhuzb676@smu.edu.cn

摘要/Abstract

摘要：

目的基于网络药理学和体外实验分析验证茵陈蒿汤（YCHD）治疗肝纤维化（HF）的核心功能成分群（CFCG）以及潜在通路。方法在DisGeNET、Genecards、CMGRN和PTHGRN提取了HF的PPI数据，使用Cytoscape 3.9.1构建权重网络。从TCMSP中收集茵陈蒿汤所有化学成分，用PreADMET Web服务器和SwissTargetPrediction选择茵陈蒿汤潜在活性成分和靶点。构建融合模型获取功能效应空间并评估有效蛋白，得到CFCG，再进一步对所有目标进行GO和KEGG通路富集分析。细胞实验：体外培养人肝星状细胞（LX-2），分别设置空白组（不加TGF-β1刺激）、对照组NC（20 ng/mL TGF-β1刺激）和化合物组（0、50、100、200 μmol/L），CCK-8实验检测药物敏感性，qPCR实验检测化合物对LX-2中Ⅰ型胶原Α1（COL1A1）的影响，Western blotting实验分析评估化合物在TGF-β1刺激下对LX-2中潜在通路的影响，验证潜在治疗机制。结果分析得到1005个致病基因，茵陈蒿汤潜在活性成分和靶点分别有226个和1529个，核心功能成分群有52个。根据模型计算结果，选取得分最高的乙酸苄酯、香草酸、苯甲酸甲酯、虎杖苷、月桂酸、阿魏酸进行CCK-8验证发现在200 μmol/L内无细胞毒性；在qPCR实验中，与TGF-β1组相比苯甲酸甲酯、虎杖苷、月桂酸和阿魏酸能够抑制TGF-β1诱导的LX-2活化。GO和KEGG分析及Western blotting验证发现，这4种成分在200 μmol/L浓度时对PI3K、p-PI3K、AKT、p-AKT、ERK、p-ERK、P38 MAPK、p-P38 MAPK有不同程度的抑制。结论茵陈蒿汤抗肝纤维化可能是其中的乙酸苄酯、香草酸、苯甲酸甲酯、虎杖苷、月桂酸、阿魏酸等成分通过抑制PI3K-AKT和MAPK通路实现的。

关键词: 茵陈蒿汤, 核心功能成分群, 网络药理学, 成分贡献率, 肝纤维化, LX-2细胞

Abstract:

Objective To analyze the core functional component groups (CFCG) in Yinchenhao Decoction (YCHD) and their possible pathways for treating hepatic fibrosis based on network pharmacology. Methods PPI data were extracted from DisGeNET, Genecards, CMGRN and PTHGRN to construct a weighted network using Cytoscape 3.9.1. The data of the chemical components in YCHD were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and the potential active components and targets were selected using PreADMET Web server and SwissTargetPrediction. A fusion model was constructed to obtain the functional effect space and evaluate the effective proteins to identify the CFCG followed by GO and KEGG pathway enrichment analyses for all the targets. In cultured human hepatic stellate cells (LX-2 cells), the cytotoxicity of different compounds in YCHD was tested using CCK-8 assay; the effects of these compounds on collagen α1 (Col1a1) mRNA expression and the pathways in 20 ng/mL TGF-β1-stimulated cells were analyzed using RT-qPCR and Western blotting. Results A total of 1005 pathogenic genes, 226 potential active components and 1529 potential targets in YCHD and 52 potential targets of CFCG were obtained. Benzyl acetate, vanillic acid, clorius, polydatin, lauric acid and ferulic acid were selected for CCK-8 verification, and they all showed minimal cytotoxicity below the concentration of 200 μmol/L. Clorius, polydatin, lauric acid and ferulic acid all effectively inhibited TGF-β1-induced LX-2 cell activation. At the concentration of 200 μmol/L, all these 4 components inhibited PI3K, p-PI3K, AKT, p-AKT, ERK, p-ERK, P38 MAPK and p-P38 MAPK expressions in TGF-β1-induced LX-2 cells. Conclusion The therapeutic effect of YCHD on hepatic fibrosis is probably mediated by its core functional components including benzyl acetate, vanillic acid, clorius, polydatin, lauric acid and ferulic acid, which inhibit the PI3K-AKT and MAPK pathways in hepatic stellate cells.

Key words: Yinchenhao Decoction, core functional component groups, network pharmacology, CDR, hepatic fibrosis, LX-2 cells

陈星梅, 刘琴文, 李镱, 钟晓宇, 樊奇灵, 马柯, 罗柳婷, 官道刚, 朱志博. 茵陈蒿汤治疗肝纤维化的核心功能成分群以及潜在通路[J]. 南方医科大学学报, 2024, 44(8): 1508-1517.

Xingmei CHEN, Qinwen LIU, Yi LI, Xiaoyu ZHONG, Qiling FAN, Ke MA, Liuting LUO, Daogang GUAN, Zhibo ZHU. Analysis of core functional components in Yinchenhao Decoction and their pathways for treating liver fibrosis[J]. Journal of Southern Medical University, 2024, 44(8): 1508-1517.

图/表 9

图1 致病基因的权重网络和功能分析

Fig.1 Weighted network and functional analysis of the pathogenic genes. A: Gene selection and PPI network for hepatic fibrosis (HF) based on Genescards and Disease databases. B: KEGG enrichment analysis of the pathogenic genes. C: GO enrichment analysis of the pathogenic genes.

表1 实验验证的方剂关键成分的收集

Tab.1 Collection of experimentally validated key components of the formula by HPLC

Formula/Herbs	Component	Content (mg/g)
Rheum palmatumL.	Catechin	0.015-11.66
	Chrysophanol	0.17-4.36
	(-)-epicatechin	0.98-6.33
	Gallic acid	0.041-4.21
	Isolindleyin	0.056-4.37
	Lindleyin	0.40-4.02
	Phenylbutanone-glucoside	0.36
	Physcion	0.09-0.15
	4,3,5'-trihydroxystilbene-4-(6"-galloyl-glucoside)	0.14-0.20
	Emodin	0.058-4.15
	Rhein	0.08-4.97
	Physcione	0.056-1.66
	Sennoside B	0.47-1.59
	3,4,3',5'-tetrahydroxystilbene-3-glucoside	0.05
	(-)-Epicatechin gallate	0.37-0.045
	(+)-catechin	0.07-0.79
	1,6-digalloyl-2-cinnamoyi-glucose	0.07-0.30
	1-galloyl-2-cinnamoyl-glucose	0.63-1.48
	2-cinnamoyl-glucose	0.13-0.42
	4-4-HydroxyphenylButan-2-One	0.48
	Aloe-emodin-8-glucoside	0.31-0.46
	Chrysophanol-8-glucoside	0.52-1.08
	Emodin-8-glucoside	0.10-1.16
	Epicatechin	0.28-0.70
	Musizin-glucoside	0.06-0.82
	Physcion-8-glucoside	0.20-0.78
	Sennosides	0.40-2.20
	Torachrysone-8-glucoside	0.12-0.25
	Trans-cinnamic acid	1.05
Artemisia capillaris Thunb.	Caffeic acid	0.31
	Chlorogenic acid	0.49-25.09
	P-hydroxyacetophenone	0.03
	Oleanolic acid	4.03
	Rutin	0.045
	Isoquercitrin	0.0094
	4,5-Dicaffeoylquinic acid	0.92
	Hyperin	0.0129
	Isorhamnetin-3-o-glucoside	0.0039
	3, 5-Dicaffeoylquinic acid	4.21
	4,5-O-dicaffeoyl quinic acid	4.55
	Hyperoside	0.52-7.81
Yinchenhao Decoction	Catechin	0.09-1.29
	Chlorogenic acid	0.43-18.12
	Gallic acid	0.58
	Genipin	0.0053
	Geniposide	0.033-23.07
	p-Hydroxyacetophenone	0.69
	Physcion	0.004-1.20
	Quercetin	0.041
	Scoparone	0.15
	Scopoletin	0.0087
	Emodin	0.0083-1.39
	Crocin I	1.38
	Crocin II	0.15
	Rhein	0.15-13.04
	Esculetin	0.024
	Aloe-emodin	0.041-6.26
	Aloe-emodin-8-O-β-D-glucoside	0.20
	Chrysophanol-1-O-β-D-glucoside	0.17
	deacetyl asperulosidic acid methylester	0.67
	Hyperoside	0.065
Gardenia jasminoides Ellis	Chlorogenic acid	0.12-0.27
	Geniposide	2.13-74.65
	Geniposidic acid	1.04-1.99
	Neochlorogenic acid	0.43
	Picrocrocin	1.30
	Crocin I	1.27-19.16
	Crocin II	0.96-1.18
	4-Dicaffeoylquinic Acid	2.14
	Crocin III	0.44
	Deacetyl asperulosidic acid methylester	0.44-3.36
	Genipin 1-gentiobioside	0.56-17.94
	Rutinum	1.03

表1 实验验证的方剂关键成分的收集

Tab.1 Collection of experimentally validated key components of the formula by HPLC

Formula/Herbs	Component	Content (mg/g)
Rheum palmatumL.	Catechin	0.015-11.66
	Chrysophanol	0.17-4.36
	(-)-epicatechin	0.98-6.33
	Gallic acid	0.041-4.21
	Isolindleyin	0.056-4.37
	Lindleyin	0.40-4.02
	Phenylbutanone-glucoside	0.36
	Physcion	0.09-0.15
	4,3,5'-trihydroxystilbene-4-(6"-galloyl-glucoside)	0.14-0.20
	Emodin	0.058-4.15
	Rhein	0.08-4.97
	Physcione	0.056-1.66
	Sennoside B	0.47-1.59
	3,4,3',5'-tetrahydroxystilbene-3-glucoside	0.05
	(-)-Epicatechin gallate	0.37-0.045
	(+)-catechin	0.07-0.79
	1,6-digalloyl-2-cinnamoyi-glucose	0.07-0.30
	1-galloyl-2-cinnamoyl-glucose	0.63-1.48
	2-cinnamoyl-glucose	0.13-0.42
	4-4-HydroxyphenylButan-2-One	0.48
	Aloe-emodin-8-glucoside	0.31-0.46
	Chrysophanol-8-glucoside	0.52-1.08
	Emodin-8-glucoside	0.10-1.16
	Epicatechin	0.28-0.70
	Musizin-glucoside	0.06-0.82
	Physcion-8-glucoside	0.20-0.78
	Sennosides	0.40-2.20
	Torachrysone-8-glucoside	0.12-0.25
	Trans-cinnamic acid	1.05
Artemisia capillaris Thunb.	Caffeic acid	0.31
	Chlorogenic acid	0.49-25.09
	P-hydroxyacetophenone	0.03
	Oleanolic acid	4.03
	Rutin	0.045
	Isoquercitrin	0.0094
	4,5-Dicaffeoylquinic acid	0.92
	Hyperin	0.0129
	Isorhamnetin-3-o-glucoside	0.0039
	3, 5-Dicaffeoylquinic acid	4.21
	4,5-O-dicaffeoyl quinic acid	4.55
	Hyperoside	0.52-7.81
Yinchenhao Decoction	Catechin	0.09-1.29
	Chlorogenic acid	0.43-18.12
	Gallic acid	0.58
	Genipin	0.0053
	Geniposide	0.033-23.07
	p-Hydroxyacetophenone	0.69
	Physcion	0.004-1.20
	Quercetin	0.041
	Scoparone	0.15
	Scopoletin	0.0087
	Emodin	0.0083-1.39
	Crocin I	1.38
	Crocin II	0.15
	Rhein	0.15-13.04
	Esculetin	0.024
	Aloe-emodin	0.041-6.26
	Aloe-emodin-8-O-β-D-glucoside	0.20
	Chrysophanol-1-O-β-D-glucoside	0.17
	deacetyl asperulosidic acid methylester	0.67
	Hyperoside	0.065
Gardenia jasminoides Ellis	Chlorogenic acid	0.12-0.27
	Geniposide	2.13-74.65
	Geniposidic acid	1.04-1.99
	Neochlorogenic acid	0.43
	Picrocrocin	1.30
	Crocin I	1.27-19.16
	Crocin II	0.96-1.18
	4-Dicaffeoylquinic Acid	2.14
	Crocin III	0.44
	Deacetyl asperulosidic acid methylester	0.44-3.36
	Genipin 1-gentiobioside	0.56-17.94
	Rutinum	1.03

图2 茵陈蒿汤中3种药用植物和方剂中化学成分的交集情况

Fig.2 Distribution of sepecific and common chemical components in 3 medicinal plants and prescriptions in YCHD (ZZ, DH and YCH refer to Zhizi, Dahuang and Yinchenhao).

图3 茵陈蒿汤中核心功能成分群及其贡献率

Fig.3 Core functional component groups (CFCG) in YCHD and its distribution rate. MID and NEWID represent the ID of the active ingredients; the red line represents the coverage of active ingredients from 0 to 100%; the blue line represents 90% coverage; the contribution rate of 52 components of labeled red was less than 90%, which was defined as the CFCG of YCHD.

图4 核心功能成分群与致病基因相同的富集途径

Fig.4 Common enriched pathways of the CFCG and the pathogenic genes. Blue represents CFCG, and red represents pathogenic gene.

图5 核心功能成分群靶点在综合途径中的分布

Fig.5 Distribution of CFCG targets and pathogenic genes in the integrated pathway. The blue, orange and pink frames represent pathogenic genes, CFCG targets and their common genes, respectively.

图6 CCK-8实验结果

Fig.6 CCK-8 assay of LX-2 cells treated with benzyl acetate, vanillic acid, clorius, polydatin, lauric acid or ferulic acid (drug concentration: 0-200 μmol/L).

图7 qPCR实验结果

图8 核心功能成分群的Western blotting结果

Fig.8 Western blotting of PI3K, AKT and MAPK expressions in TGF‑β1-induced LX-2 cells treated with clorius, polydatin, lauric acid and ferulic acid (Mean±SD, n=3).

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茵陈蒿汤治疗肝纤维化的核心功能成分群以及潜在通路

Analysis of core functional components in Yinchenhao Decoction and their pathways for treating liver fibrosis

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