Exploring the active ingredients of Banzhilian and its mechanism of action on diabetic Gastric cancer based on network pharmacology

Abstract

The incidence of Gastric cancer (GC) has shown a sharp upward trend, and patients with GC complicated by diabetes exhibit significantly worse clinical outcomes and prognosis compared to those without diabetes. Traditional Chinese medicine has played a crucial role in the treatment of both GC and diabetes. Currently, Banzhilian(Scutellaria barbata D. Don) is utilized in the treatment of GC; however, the specific small-molecule monomers it contains and their mechanisms of action have not yet been fully elucidated. This study aims to explore the mechanism of quercetin, a key component of Banzhilian, through network pharmacology, molecular docking, molecular dynamics (MD) simulation, bioinformatics, and in vitro and in vivo experiments. Initially, core targets and key pathways involved in the treatment of diabetes-associated GC (GC-diabetes) were identified using public databases. Subsequently, molecular docking, MD simulation, and survival analysis were performed. Experimental validation included CCK-8 assays, colony formation assays, apoptosis detection, cell cycle analysis, wound healing assays, Transwell migration assays, Western blotting, and mouse subcutaneous tumor formation experiments to evaluate the effects of quercetin, as an active monomer in Banzhilian, on Gastric cancer cells (HGC-27-HG cells) under high-glucose conditions. In this study, quercetin was identified as the key active component, with AKT1, TP53, JUN, MYC, and CCND1 recognized as the target genes, and the PI3K/AKT signaling pathway as the primary regulatory pathway. The results of the study indicate that the proliferation, migration, and invasion capabilities of HGC-27-HG cells are significantly higher than those of HGC-27 cells. However, quercetin inhibited the growth of HGC-27-HG cells, promoted apoptosis, induced cell cycle arrest at the G0/G1 phase, and reduced the cells' migration and invasion abilities. Furthermore, it downregulated the expression of target genes and their phosphorylation levels. The experimental findings confirmed that quercetin, as an active monomer in Banzhilian, suppresses the proliferation of HGC-27-HG cells by inhibiting the PI3K/AKT/MYC pathway, promoting apoptosis, blocking cell cycle progression, and inhibiting cell migration and invasion.

Keywords: Banzhilian; Chinese medicine; Diabetes; Gastric cancer; Quercetin.

Fig. 1

Flowchart illustrating the target and mechanism of Banzhilian in managing GC-diabetes.

Fig. 2

Prediction of Banzhilian’s components and targets in anti-GC-diabetes, along with GO and KEGG analysis. (A) Diabetes-related genes. (B) GC-related genes. (C) Identification of intersection targets between Banzhilian and GC-diabetes. (D) Interaction between Banzhilian’s active components and GC-diabetes pathogenic genes. (E,F) Key targets were identified using the CytoNCA plugin. (G) GO analysis. (H) KEGG enrichment analysis.

Fig. 3

Molecular docking and molecular dynamics simulation. (A) Molecular docking results were obtained using Autodock Vina software and visualized using Open-Source PyMOL(Schrodinger, LLC). (B) 2D representation of molecular docking, the 2D diagrams of molecular docking were visualized using LigPlot+ (v2.2). (C,D) Root Mean Square Deviation (RMSD) variation over time. (E) Hydrogen bond analysis.

Fig. 4

Survival analysis and immunohistochemistry. (A) Impact of elevated and reduced expression patterns of five key genes on overall survival (OS) and post-progression survival (PPS) in individuals with Gastric cancer. (B) Expression levels of P53, AKT, JUN, MYC, and CCND1 in normal gastric tissues and Gastric cancer tissues. scale bar, 25 μm.

Fig. 5

Investigation of the anti-GC-diabetes effect of quercetin on cell proliferation. (A) IC50 value of quercetin on HGC-27-HG cells. (B) CCK-8 assay for HGC-27-HG cells. (C,D) Clonal formation rate of HGC-27-HG cells. (E,F) Cell proliferation activity was assessed by EDU staining. scale bar, 200 μm. (G) Cell cycle alterations in HGC-27-HG cells. (H) Cell activity was measured by AMPI staining. scale bar, 50 μm. (I) Apoptosis rate in HGC-27 cells. (J–M) Expression of apoptosis-related proteins. HGC-27, HGC-27-HG, and the low, medium, and high dose groups of HGC-27-HG were labeled as Group 1, Group 2, Group 3, Group 4, and Group 5. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 6

Investigation of quercetin’s anti-GC-diabetes effect on cell transfer. (A,B) Scratch healing rate in HGC-27-HG cells. scale bar, 100 μm. (C,D) Transwell migration capacity of HGC-27-HG cells. scale bar, 50 μm. (E,F) invasiveness of HGC-27-HG cells. (G–K) Expression of EMT-related proteins. (L,M) Analysis of PI3K, p-PI3K, AKT, p-AKT, and MYC protein abundance through Western blot technique. HGC-27, HGC-27-HG, and the low, medium, and high dose groups of HGC-27-HG were labeled as Group 1, Group 2, Group 3, Group 4, and Group 5. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 7

In vivo validation of quercetin’s effects on diabetic Gastric cancer. (A) Blood glucose change curve in mice. (B) Weight change curve in mice. (C) Tumor volume change curve in mice. (D) Representative images of individual tumors from all experimental groups. (E,F) Tumor measurements and weight determination following euthanasia. (G) H&E staining of tumor tissues. scale bar, 100 μm. (H) Immunohistochemical analysis of Ki67 expression in tumor specimens. scale bar, 100 μm. (I) TUNEL assay to detect tissue apoptosis. scale bar, 100 μm. (J) Schematic diagram illustrating quercetin’s function in treating GC-diabetes generated by Figdraw. NG, HG, and HG + Q represent the normal blood glucose group, the high blood glucose group, and the high blood glucose group treated with quercetin, respectively. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.