“白花蛇舌草-半枝莲”治疗原发性肝癌的机制研究：基于网络药理学、分子对接及体外实验验证

doi:10.12122/j.issn.1673-4254.2025.01.11

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (1): 80-89.doi: 10.12122/j.issn.1673-4254.2025.01.11

“白花蛇舌草-半枝莲”治疗原发性肝癌的机制研究：基于网络药理学、分子对接及体外实验验证

徐朦¹(), 陈丽娜², 吴金玉³, 刘丽丽⁴, 施美⁴, 周灏⁴, 张国梁⁴()

^1.广西中医药大学，研究生院，广西南宁 530000
^2.广西中医药大学，金沙洲医院肾病科，广西南宁 530000
^3.广西中医药大学，第一附属医院风湿科，广西南宁 530000
^4.安徽中医药大学附属医院感染科，安徽合肥 230038

收稿日期:2024-09-06 出版日期:2025-01-20 发布日期:2025-01-20
通讯作者: 张国梁 E-mail:1025473030@qq.com;zhangguoliang61@sina.com
作者简介:徐朦，在读博士研究生，E-mail: 1025473030@qq.com
基金资助:
国家自然科学基金(81874451);全国名中医肝胆系疾病学术思想传承及特色方药的临床开发研究(202204295107020040);安徽中医药大学临床科研项目(2021yfylc23)

Mechanism of Hedyotis diffusa-Scutellaria barbata D. Don for treatment of primary liver cancer: analysis with network pharmacology, molecular docking and in vitro validation

Meng XU¹(), Lina CHEN², Jinyu WU³, Lili LIU⁴, Mei SHI⁴, Hao ZHOU⁴, Guoliang ZHANG⁴()

^1.Graduate School, Guangxi University of Chinese Medicine, Nanning 530000, China
^2.Department of Nephrology of Jinshazhou Hospital, Guangxi University of Chinese Medicine, Nanning 530000, China
^3.Department of Rheumatology of First Affiliated Hospital, Guangxi University of Chinese Medicine, Nanning 530000, China
^4.Department of Infection, Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Hefei 230038, China

Received:2024-09-06 Online:2025-01-20 Published:2025-01-20
Contact: Guoliang ZHANG E-mail:1025473030@qq.com;zhangguoliang61@sina.com
Supported by:
National Natural Science Foundation of China(81874451)

摘要/Abstract

摘要：

目的通过网络药理学及分子对接探究“白花蛇舌草-半枝莲”的有效成分及其作用于原发性肝癌的主要生物过程及信号通路。方法通过TCMSP、Uniport、Genecards、String数据库以及Cytoscape软件得出最终核心基因；通过ClueGo对药物-疾病共有基因做GO、KEGG富集分析；通过Pubcham、RCSB、Autoduck把药物有效成分与最终核心基因进行分子对接，得出结合能最高药物有效成分；再通过CCK-8、细胞凋亡、Western blotting实验研究此药物有效成分对HepG2的作用。结果筛选得出最终核心基因为TP53、ESR1。GO分析显示主要生物过程为BP- regulation of apoptotic signaling pathway，negative regulation of cell population proliferation；CC-membrane raft； MF-protein kinase activity等。KEGG分析显示主要信号通路为Apoptosis，Proteoglycans in cancer，PI3K-Akt signaling pathway，Hepatitis B等。分子对接结果显示，药物有效成分与最终核心基因均可以在自然条件下进行对接，其中ESR1与ursolic acid结合能最高（-4.98 kcal/mol）。CCK-8、细胞凋亡、Western blotting实验显示ursolic acid对HepG2有明显抑制作用。结论 “白花蛇舌草-半枝莲” 通过多种有效成分与原发性肝癌紧密结合，继而对原发性肝癌起到治疗作用。

关键词: 白花蛇舌草, 半枝莲, 原发性肝癌, 网络药理学, 分子对接

Abstract:

Objective To investigate the active ingredients in Hedyotis diffusa-Scutellaria barbata D. Don and the main biological processes and signaling pathways mediating their inhibitory effect on primary hepatocellular carcinoma (HCC). Methods The core intersecting genes of HCC and the two drugs were screened from TCMSP, Uniport, Genecards, and String databases using Cytoscape software, and GO and KEGG enrichment analyses of the intersecting genes were conducted. Molecular docking between the active ingredients of the drugs and the core genes was carried out using Pubcham, RCSB and Autoduckto to identify the active ingredients with the highest binding energy, whose inhibitory effect on HepG2 cells was verifies using CCK-8 assay, flow cytometry and Western blotting. Results TP53 and ESR1 were identified as the core genes of HCC and the two drugs. GO and KEGG analyses showed that the two genes were mainly involved in regulation of apoptotic signaling pathway, cell population proliferation, methane raft, and protein kinase activity, and participated in the signaling pathways of apoptosis, proteoglycans in cancer, PI3K Akt signaling pathway, and hepatitis B. Molecular docking studies showed that the active ingredients of the drugs could be docked with TP53 and ESR1 genes under natural conditions, and ursolic acid had the highest binding energy to ESR1 (-4.98 kcal/mol). The results of CCK-8 assay, flow cytometry and Western blotting all demonstrated significant inhibitory effect of ursolic acid on HepG2 cells. Conclusion The inhibitory effect of Hedyotis diffusa-scutellariae barbatae on HCC is mediated by multiple active ingredients in the two drugs.

Key words: Hedyotis diffusa, Scutellaria barbata D. Don, primary hepatocellular carcinoma, network pharmacology, molecular docking

徐朦, 陈丽娜, 吴金玉, 刘丽丽, 施美, 周灏, 张国梁. “白花蛇舌草-半枝莲”治疗原发性肝癌的机制研究：基于网络药理学、分子对接及体外实验验证[J]. 南方医科大学学报, 2025, 45(1): 80-89.

Meng XU, Lina CHEN, Jinyu WU, Lili LIU, Mei SHI, Hao ZHOU, Guoliang ZHANG. Mechanism of Hedyotis diffusa-Scutellaria barbata D. Don for treatment of primary liver cancer: analysis with network pharmacology, molecular docking and in vitro validation[J]. Journal of Southern Medical University, 2025, 45(1): 80-89.

图/表 15

表1 一抗稀释液比例

Tab.1 List of the primary antibodies used for Western blotting

Antibody name	Dilution ratio	Species and genus
JNK	1:5000	Mouse
p-JNK	1:1000	Rabbit
P38	1:2000	Rabbit
p-P38	1:1000	Mouse
ERK1/2	1:1000	Rabbit
P-ERK1/2	1:1000	Rabbit

图1 "药物-靶点-基因"网络图

Fig.1 "Drug target gene" network diagram. A, B, C: Drug common targets; SSC: Hedyotis diffusa; BZL: S. barbata.

表2 药物共有基因

Tab.2 Drug shared genes

MOL	MOL name	OB%	DL
MOL000098	quercetin	46.43	0.28
MOL000358	beta-sitosterol	36.91	0.75
MOL000449	Stigmasterol	43.83	0.76

图2 疾病-药物共有基因

Fig. 2 Intersecting genes of HCC and the two drugs.

图3 最终核心基因

Fig. 3 Final core genes.

图4 GO分析

Fig. 4 GO analysis.

图5 KEGG分析

Fig. 5 KEGG analysis.

图6 分子对接结果图

Fig.6 Molecular docking results (purple is the docking part) between ESR1 and ursolic acid.

表3 药物有效成分与最终核心基因对接的结合能（部分）

Tab.3 Binding energy of drug active ingredients and final core gene docking (part)

Active ingredient	Coregene	Bindingenergy (kcal/mol)
2-hydroxy-3-methylanthraquinone	TP53	-4.41
	ESR1	-4.34
2-methoxy-3-methyl-9,10-anthraquinone	TP53	-4.73
	ESR1	-4.01
Quercetin	TP53	-3.05
Beta-sitosterol	TP53	-3.79
	ESR1	-4.18
Ursolic acid	TP53	-4.49
	ESR1	-4.98
Poriferasterol	TP53	-4.7
	ESR1	-4.52
Stigmasterol	TP53	-3.97
	ESR1	-4.7
Rivularin	TP53	-3.2
Chrysin-5-methylether	TP53	-4.26
7-hydroxy-5,8-dimethoxy-2-phenyl-chromone	TP53	-3.48
	ESR1	-3.06
5-hydroxy-7,8-dimethoxy-2-(4-methoxyphenyl)chromone	TP53	-3.38
5,7,4'-trihydroxy-6-methoxyflavanone	TP53	-3.53
Moslosooflavone	TP53	-3.18
eriodictyol	TP53	-3.68
Salvigenin	TP53	-3.56
	ESR1	-3.18
Baicalin	TP53	-3.14
	ESR1	-3.51
Baicalein	TP53	-3.69
Sitosteryl acetate	TP53	-4.71
	ESR1	-4.34
24-Ethylcholest-4-en-3-one	TP53	-4.26
	ESR1	-3.89
Dinatin	TP53	-3.39
(2R)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one	TP53	-3.38
	ESR1	-3.21
CLR	TP53	-4.45
	ESR1	-4.63
Sitosterol	TP53	-3.83
	ESR1	-4.25
Wogonin	TP53	-3.61

图7 不同浓度、培养时间的Ursolic acid对HepG2活力的影响

Fig.7 Effect of different concentrations and treatment time of ursolic acid on viability of HepG2 cells.

图8 ROS流式检测图

Fig.8 ROS flow cytometry analysis. A: HepG2. B: 0 µmol/L ursolic acid. C: 20 µmol/L ursolic acid. D: 40 µmol/L ursolic acid. E: 80 µmol/L ursolic acid.

图9 不同浓度的JNK抑制剂和不同处理时间对HepG2活力的影响

Fig.9 Effect of different concentrations and treatment time of JNK inhibitor on viability of HepG2 cells.

图10 Ursolic acid及索拉非尼对HepG2细胞活力的影响

Fig.10 Effects of usolic acid and sorafenib on viability of HepG2 cells. A: LO-2. B: HepG2+blank serum. C: HepG2+40 µmol/L ursolic acid. D: HepG2+1.5 µmol/L JNK inhibitor. E: HepG2+sorafenib (10 µmol/L, cultured for 24 hours). F: HepG2+40 µmol/L ursolic acid+1.5 µmol/L JNK inhibitor. G: HepG2+sorafenib (10 µmol/L, cultured for 24 hours)+1.5 µmol/L JNK inhibitor. **P<0.01 vs A.

图11 HepG2细胞凋亡率

Fig.11 Apoptosis rate of HepG2 cells with different treatments. A: LO-2. B: HepG2. C: HepG2+optimal concentration of ursolic acid. D: HepG2+optimal concentration of JNK inhibitor. E: HepG2+sorafenib (10 µmol/L, cultured for 24 hours). F: HepG2+optimal concentration of ursolic acid+optimal concentration of JNK inhibitor. G: HepG2+sorafenib (10 µmol/L, cultured for 24 hours)+optimal concentration of JNK inhibitor.

图12 各组HepG2中蛋白的相对表达量

Fig.12 Relative expression levels of proteins in HepG2 cells with different treatments. A: LO-2. B: HepG2. C: HepG2+optimal concentration of ursolic acid. D: HepG2+optimal concentration of JNK inhibitor. E: HepG2+sorafenib (10 µmol/L, cultured for 24 hours). F:HepG2+optimal concentration of ursolic acid+optimal concentration of JNK inhibitor. G: HepG2+sorafenib (10 µmol/L, cultured for 24hours)+optimal concentration of JNK inhibitor *P<0.05, **P<0.01vs A.

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“白花蛇舌草-半枝莲”治疗原发性肝癌的机制研究：基于网络药理学、分子对接及体外实验验证

Mechanism of Hedyotis diffusa-Scutellaria barbata D. Don for treatment of primary liver cancer: analysis with network pharmacology, molecular docking and in vitro validation

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