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

doi:10.12122/j.issn.1673-4254.2025.01.11

Abstract

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

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.

Figures/Tables 15

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

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

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

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

Fig. 3 Final core genes.

Fig. 4 GO analysis.

Fig. 5 KEGG analysis.

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

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

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

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.

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

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.

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.

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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