Molecular targets of Yangyin Fuzheng Jiedu Prescription in the treatment of hepatocellular carcinoma based on network pharmacology analysis

Abstract

Background: Yangyin Fuzheng Jiedu Prescription (YFJP) is a traditional Chinese medicine (TCM) indicated for the treatment of hepatocellular carcinoma (HCC). Its potential targets and molecular mechanisms are not clear. Therefore, this study intends to explore the molecular mechanism of YFJP based on network pharmacology analysis and in vitro validation.

Methods and results: Through univariate and multivariate analyses and survival analysis in HCC patients with or without YFJP treatment we found that drinking alcohol, alfafeto protein ≥ 400 ng/l, baseline portal vein tumor thrombus and total bilirubin level ≥ 18.8 μM) were independent risk factors for poor prognosis, while red blood cell count ≥ 4 × 10⁹/l and TCM treatment were independent protective factors. Besides, YFJP prolonged the cumulative survival of HCC patients. Using online pharmacological methods, we obtained 58 relevant compounds and molecular 53 targets. By using scratch test, Transwell assay, EdU assay, and TUNEL staining, we found that YFJP-containing serum repressed the migration, invasion and proliferation of HCC cells in vitro, and induced cell apoptosis. Moreover, YFJP diminished the gene expression of TP53, CCND1, p-EGFR, EGF, VEGFA, JUN, IL6, COX-2, AKT1, and MAPK1 in HCC cells, but elevated the expression of ESR1 and CASP3.

Conclusions: Taken together, results showed that YFJP attenuated HCC progression through mediating effects on HCC-related genes.

Keywords: Apoptosis; Cumulative survival; Hepatocellular carcinoma; Invasion; Migration; Network pharmacology; Proliferation; Yangyin fuzheng jiedu prescription.

Fig. 1

Kaplan–Meier analysis. Kaplan–Meier analysis for the 5-year cumulative survival rate between patients in the YFJP group (n = 159) and non-YFJP group (n = 318)

Fig. 2

The HCC-related target network

Fig. 3

Cluster analysis for HCC-related targets

Fig. 4

The network for KEGG enrichment analysis

Fig. 5

The YFJP-TCM-compound-target network and cluster analysis. The YFJP-TCM-compound-target network contains 1 TCM prescription, 8 TCMs, 120 compounds and 411 targets (The blue diamond node represents the TCM prescription, the red V-shaped nodes represent TCMs, the pink hexagons represent specific compounds, and the purple circles represent targets)

Fig. 6

The potential target-compound-HCC-YFJP network and cluster analysis. a The potential target-compound-HCC-YFJP network contains 1 TCM prescription, 8 TCMs, 58 compounds, and 53 targets (The blue diamond node represents TCM prescription, the red V-shaped nodes represent TCM, the pink hexagons represent compounds, and the purple circles represent targets). b, c Cluster analysis for topological modules. d KEGG analysis network for Cluster 1

Fig. 7

The key target-compound-HCC-YFJP network and cluster analysis. a Network for putative targets for YFJP treatment of HCC. b The key target-compound-HCC-YFJP network includes 1 TCM prescription, 7 Chinese medicines, 51 compounds and 12 targets (The blue diamond node represents TCM prescription, the red V-shaped nodes represent TCM, the pink hexagons represent compounds, and the purple circles represent targets). c GO analysis for key targets. d KEGG analysis for key targets

Fig. 8

YFJP inhibits the migration, invasion, and proliferation, while inducing the apoptosis of HepG2 cells. a The migration of HepG2 cells as detected by scratch test (× 200). b The invasion of HepG2 cells as detected by Transwell assay (×200). c The proliferation of HepG2 cells as detected by EdU assay. d The apoptosis of HepG2 cells as detected by TUNEL staining (×200). e, f The mRNA expression as detected by qRT-PCR. * p < 0.05 vs. the blank control group. ^# p < 0.05 vs. the YFJP-L group. These data were measurement data, expressed as mean ± standard deviation. Data among multiple groups were compared using one-way ANOVA, followed by Tukey’s post hoc test. The experiments were repeated three times