Mechanism underlying the effect of Pulsatilla decoction in hepatocellular carcinoma treatment: a network pharmacology and in vitro analysis

Abstract

Background: Currently, hepatocellular carcinoma (HCC) is associated with a poor prognosis. Moreover, there exist limited strategies for treating HCC. Pulsatilla decoction (PD), a traditional Chinese medicine formula, has been used to treat inflammatory bowel disease and several cancer types. Accordingly, we explored the mechanism of PD in HCC treatment via network pharmacology and in vitro experiments.

Methods: Online databases were searched for gene data, active components, and potential target genes associated with HCC development. Subsequently, bioinformatics analysis was performed using protein-protein interaction and Network Construction and Kyoto Encyclopedia of Genes and Genomes (KEGG) to screen for potential anticancer components and therapeutic targets of PD. Finally, the effect of PD on HCC was further verified by in vitro experiments.

Results: Network pharmacological analysis revealed that 65 compounds and 180 possible target genes were associated with the effect of PD on HCC. These included PI3K, AKT, NF-κB, FOS, and NFKBIA. KEGG analysis demonstrated that PD exerted its effect on HCC mainly via the PI3K-AKT, IL-17, and TNF signaling pathways. Cell viability and cell cycle experiments revealed that PD could significantly inhibit cancer cell proliferation and kill HCC cells by inducing apoptosis. Furthermore, western blotting confirmed that apoptosis was mediated primarily via the PI3K-AKT, IL-17, and TNF signaling pathways.

Conclusion: To the best of our knowledge, this is the first study to elucidate the molecular mechanism and potential targets of PD in the treatment of HCC using network pharmacology.

Keywords: Apoptosis.; Hepatocellular carcinoma; Network pharmacology; Proliferation; Pulsatilla decoction; Traditional Chinese medicine.

Fig. 1

The technical strategy used in this study. The active components and potential target genes of PD were analyzed by network pharmacology. Network structure, PPI and enrichment analysis were further analyzed. Finally, in vitro experiments were conducted to verify the therapeutic effect of PD on HCC and explore its potential molecular mechanism

Fig. 2

Prediction targets and C-D-T network analysis. (A) 6310 HCC-related genes. (B) 152 overlapping genes of compounds and HCC. (C) The C-D-T network

Fig. 3

PPI network, GO and KEGG enrichment analysis. (A) PPI network for PD treating HCC. Red nodes stand for ranking top ten with degree for PD anti-HCC targets. (B) BP, CC, and MF analysis. The lower q-value and redder color indicated greater enrichment of the GO terms. (C) Top 20 KEGG enrichment pathways. The x-axis represents the counts target genes in each pathway and the ordinate represents pathway. Redder means lower p-value

Fig. 4

C-P-T network of top 20 pathway. The C-P-T network contains 153 nodes and 807 edges

Fig. 5

The docking model of quercetin with JUN and AKT1, respectively. Binding model of quercetin on the molecular surface of JUN (A), AKT1 (B). The interaction model of quercetin with JUN (C) and AKT1 (D). The ligands in binding model and interaction model are colored in green

Fig. 6

PD inhibits the proliferation of HCC cells. (A) Cell viability assay showed that PD could decrease the viability of HCC cells. The HCC cells were treated with PD at different concentrations for 24, 48 and 72 h. (B) Representative images showing colonies formed by HCC cells treated with various concentrations of PD for 2 weeks. (C) Representative images and statistical graphs of HCC-LM3 and MHCC-97 H cell cycle analysis. *P < 0.05, **P < 0.01 versus the untreated group

Fig. 7

PD promotes the apoptosis of HCC cells and inhibits the proliferation of HCC orthotopic tumors in mice. HCC cells were treated with different concentrations of PD for 48 h, and the apoptosis of HCC cells was detected by flow cytometry. The percentage of apoptotic cells was expressed as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus the control group

Fig. 8

Changes in protein expression of HCC cells treated with PD at different concentrations. (A) PI3K/AKT signaling pathway. (B) TNF signaling pathway. (C) IL-17 signaling pathway. *P < 0.05, **P < 0.01 versus the control group