Analysis of the Anti-Tumour Effect of Xuefu Zhuyu Decoction Based on Network Pharmacology and Experimental Verification in Drosophila

Abstract

Background: Tumours are among the most lethal diseases that heavily endanger human health globally. Xuefu Zhuyu Decoction (XFZYD) is a prescription used to treat blood-activating stasis. Although XFZYD has been shown to suppress migration and invasion of tumour cells, the active ingredients, potential targets, and underlying mechanism remain largely elusive. Purpose: To identify the prospective ingredients and major targets of XFZYD against tumours, and evaluate the efficacy and potential molecular mechanisms of XFZYD extract on tumour growth and invasion. Methods: We predicted that XFZYD might act on 80 targets through 128 active components using the network pharmacology analysis method. In addition, we prepared an XFZYD aqueous extract and employed the Ras^V12/lgl ^-/- -induced Drosophila tumour model to carry out experimental verification. Results: XFZYD did not exhibit any side effects on development, viability, and fertility. Furthermore, XFZYD significantly impeded tumour size and invasion at moderate concentrations and suppressed the increased phosphorylation of JNK but strongly enhanced the expression of Caspase 3 in the Ras^V12/lgl ^-/- model. Finally, the mRNA level of the transcription complex AP-1 component c-FOS was remarkably reduced. In contrast, the transcription of three pro-apoptotic genes was significantly increased when XFZYD was used to treat the tumour model. Conclusion: The study findings suggest that XFZYD may promote tumour cell apoptosis by activating caspase signalling to control primary growth and hinder tumour cell invasion by suppressing JNK/AP-1 signalling activity, thus providing a potential therapeutic strategy for XFZYD in the clinical treatment of cancer and other related diseases.

Keywords: Xuefu Zhuyu decoction; anti-tumour; cancer treatment; drosophila; network pharmacology; traditional Chinese medicine.

FIGURE 1

Bioinformatic analysis of XFZYD ingredients. XFZYD anti-tumour related targets network comprises 11 herbs, 128 ingredients, and 80 targets. The herbs are presented as triangles, the ingredients as hexagons, and the targets as rounded rectangles. Node size and transparency were determined by the degree and value depicted as differences in size and colour shade.

FIGURE 2

PPI analysis. (A) The PPI was constructed by STRING databases. Node size and depth of gene targets are proportional to the degrees; (B) PPI analysis for the top 30 targets.

FIGURE 3

GO and KEGG enrichment analysis of 80 overlapped genes. (A) Top 20 GO enrichment analysis for the functional processes, molecular mechanism, and function; (B) Top 20 KEGG enrichment pathway; (C) Enrichment functional classification for top 20 KEGG pathways. (D) Crosstalk of key targets and pathways.

FIGURE 4

The HPLC chromatogram of XFZYD. HPLC analysis of five chemical constituents, quercetin (a, 360 nm), kaempferol [(A), 360 nm], isorhamnetin [(A), 360 nm], luteolin [(B), 350 nm), and baicalein [(C), 335 nm], in XFZYD. The tested peaks are indicated by green, blue, red, purple, and pink arrows.

FIGURE 5

Effect of XFZYD on the development, viability, and fertility of Drosophila. Statistical analysis of the white pupae rate [(A), n = 3] and time to pupariation [(B), n = 3] of w ¹¹¹⁸ larvae fed control food, or XFZYD-added food are shown. For time to pupariation statistics, a one-way ANOVA with Bonferroni’s multiple comparison test was applied, and ns indicates no significant difference. For the histogram of viability [(C), n = 30] and fertility [(D), n = 30] in w ¹¹¹⁸ adults, the chi-squared test was applied (n = 30).

FIGURE 6

XFZYD inhibits tumour invasion in Drosophila VNC. Fluorescent images showing Drosophila larval whole body (A–G), cephalic complexes (H–N), and ventral nerve cords [VNC, (O–U)]; the anterior is up in all panels. Statistical analysis of the invasion percentage (V) is shown in figures (O–U) [(O), 0%, n = 50; (P), 75%, n = 44; (Q), 63%, n = 46; (R) 43.9%, n = 57; (S), 46.5%, n = 43; (T), 66.7%, n = 45; (U), 5.5%, n = 55]. The histogram of tumour size (W) is shown in figures (I–N), and is measured in pixels by Image-Pro Plus 6.0 (n = 10). Stacked bar graph of the pupariation percentage (X) is shown in figures (A–G) [(A), 100%, n = 50; (B), 0%, n = 51; (C), 0%, n = 52; (D) 19.67%, n = 61; (E), 6.25%, n = 43; (F), 0%, n = 45; (G), 96.6%, n = 58]. (Y) Measurement of 20 min food intake by early third-instar larvae as calculated by blue food ingestion (6–8 larvae per pool, n = 3). For invasion and pupariation statistics, the chi-square test was applied. For tumour size and food intake statistics, one-way ANOVA with Bonferroni’s multiple comparison test was applied. Compared to the Ras^V12/lgl ^−/− group, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05; ns, no significant difference. Scale bar: 500 µm (A–G), 200 µm (H–N) and 100 µm (O–U).

FIGURE 7

XFZYD alters pJNK and Caspase3 expression. Merged fluorescence micrographs of 3^rd instar larval cephalic complexes (A–F) are shown. The individual channels detect only pJNK [red, (A’–C’)] or only cleaved-Caspase3 signal [red, (D’–F’)]. Tumour cells are marked with a green fluorescent protein (GFP), and nuclei (DNA) are labelled with DAPI (blue). Scale bar: 200 µm (A–F).

FIGURE 8

XFZYD regulates the transcription of c-FOS, rpr, hid, and grim. mRNA levels of c-FOS (A), rpr, hid, and grim (B), as measured by qRT-PCR (n = 3). Error bars represent the standard deviation. One-way ANOVA with Bonferroni’s multiple comparison test was used to compute p-values. ****p < 0.0001, ***p < 0.001, **p < 0.01.