The Therapeutic Potential of Bombyx Batryticatus for Chronic Atrophic Gastritis Precancerous Lesions via the PI3K/AKT/mTOR Pathway Based on Network Pharmacology of Blood-Entering Components

Abstract

Background: Chronic atrophic gastritis precancerous lesions (PL-CAG) are characterized by the atrophy of gastric mucosal glands, often accompanied by intestinal metaplasia or dysplasia. Timely intervention and treatment can effectively reverse its malignant progression and prevent the onset of gastric cancer. Bombyx Batryticatus (BB) exhibits a range of pharmacological effects, including anticoagulation, antiepileptic properties, anticancer activity, and antibacterial effects. However, the pharmacological basis and mechanisms underlying BB's efficacy in treating PL-CAG remain unclear. Methods: A three-factor modeling approach was implemented to develop a rat PL-CAG model, while the MNNG-induced PLGC (precancerous lesions of gastric cancer) cell model was served as a cell PL-CAG model. UPLC-QE-Orbitrap-MS/MS (Ultra performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry) was utilized to perform an in-depth analysis of the components in the plasma extract of BB. Leveraging network pharmacology, molecular docking analyses, and experimental validation, we initially elucidated the potential mechanisms through which BB mediates its therapeutic effects on PL-CAG at both in vivo and in vitro levels. Results: Prototype compounds of 42 blood-entering components were identified by UPLC-QE-Orbitrap-MS/MS analysis. Network pharmacology analysis and molecular docking studies indicate that the core targets are primarily enriched in the PI3K-Akt signaling pathway, and the key components, including Nepitrin, Quercetin 3-O-neohesperidoside, Rutin, and others, exhibited stable docking conformations with the first eleven pivotal targets. Both in vivo and in vitro experiments validated that BB may effectively treat PL-CAG via modulation of the PI3K-Akt signaling pathway. Conclusions: The therapeutic efficacy of BB in the management of PL-CAG may be achieved through the synergistic interaction of multiple components and targets, which may be more closely related to the inhibition of the PI3K/AKT signaling pathway. This approach will establish a solid experimental foundation and provide essential data for the clinical application of BB in treating PL-CAG, while also facilitating further research initiatives.

Keywords: Bombyx Batryticatus; PI3K/AKT/mTOR pathway; UPLC-QE-Orbitrap-MS/MS; chronic atrophic gastritis precancerous lesions; network pharmacology.

Figure 1

Effects of BB on CAG rats. (A) Macroscopic examination of the stomachs of different groups of rats. (B) Macroscopic examination of the gastric mucosa of different groups of rats. (C) Typical representative sections of HE staining of gastric histopathology in rats (the yellow arrow indicates capillary congestion). Effect of BB on serum levels of GAS (D), PG I (E), PG II (F), PG I /PG II (G). Results were represented as mean ± SD. n = 6. Compared to control, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001. Compared to model, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Expression of inflammatory factors in rats. Effect of BB on serum levels of IL-1β (A), IL-6 (B), and TNF-α (C). (D–G) Representative Western blot images and bar graphs of the relative expressions of IL-1β, IL-6, and TNF-α in each group. Results were represented as mean ± SD. n = 6. Compared to control, ^# p < 0.05, ^### p < 0.001, ^#### p < 0.0001. Compared to model, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Impact of BB on the proliferation, migration, and invasion of PLGC cells in vitro. (A) The IC50 value of MNNG was determined by CCK-8 assay for PLGC cells. (B) Morphological observation of normal gastric mucosal epithelial cells (GES-1) and MNNG-induced PLGC cells. Scale bar: 200 μm. (C) The effect of BB serum-containing on the viability of PLGC cells was assessed by CCK-8 assay. (D,E) Colony formation assay of PLGC cells that were treated with the BB drug serum. Representative images and quantification of cell colonies are shown. (F,G) The effect of BB drug serum on the migration of PLGC cells was evaluated by scratch healing assays. Representative images and quantification of the wound healing rate are shown. Scale bar: 500 μm. (H,I) The effect of BB drug serum on the invasion of PLGC cells was evaluated by Transwell assays. Representative images and quantification of invading PLGC cells are shown. Scale bar: 50 μm. Data are presented as mean ± SD. n = 3. Compared to control, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001. Compared to model, ** p < 0.01, **** p < 0.0001.

Figure 4

BB chromatogram and prototype compounds of blood-entering components. (A) Total ion chromatograms (TICs) recorded in the positive ion mode of BB. (B) TICs recorded in the negative ion mode of BB. (C) Structures of compounds 1–42.

Figure 5

Network analysis of BB treating PL-CAG. (A) Venn diagram of 293 common targets of BB and CAG. (B) Acquirement of the 48 key targets from the 293 common targets via STRING network topological analysis. (C) The top 10 significantly enriched terms in biological processes, molecular functions, and cellular components. (D) The top 20 significantly enriched terms in KEGG pathways. (E) Drug–components–diseases–targets–pathway network. Illustration of the relevance among components of BB, the four stages of CAG progression (CAG, IM, DYS, PLGC), the key targets, and the top corresponding pathways. Orange nodes refer to the 42 components of BB, purple nodes refer to the four stages of CAG progression, green nodes refer to the key targets, and yellow nodes refer to the signaling pathways.

Figure 6

Molecular docking results. (A–O) The representative docking complex of key targets and compounds. (P) The heatmap of docking scores of key targets combining the top 5 active compounds in BB.

Figure 7

BB suppresses the activation of the PI3K/AKT/mTOR signaling pathway in vivo and in vitro. (A) The impact of BB on the expression of proteins associated with the PI3K-AKT signaling pathway in vivo. (B–J) Representative Western blot images and bar graphs of the relative expressions of PI3K, p-PI3K, Akt, p-Akt, mTOR, and p-mTOR in rats. (K) The impact of BB on the expression of proteins associated with the PI3K-AKT signaling pathway in vitro. (H–V) Representative Western blot images and bar graphs of the relative expressions of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, E-cadherin, and N-cadherin in PLGC cell. Results were represented as mean ± SD. n = 6. Compared to control, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001. Compared to model, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 8

Schematic diagram of the regulatory mechanism of BB in the treatment of CAG. BB can restrain uncontrolled proliferation, migration, and invasion by regulating the PI3K-Akt pathway to inhibit CAG progression.