Gleditsiae Sinensis Fructus ingredients and mechanism in anti-asthmatic bronchitis research

Gleditsiae Sinensis Fructus (GSF) is commonly used in traditional medicine to treat respiratory diseases such as bronchial asthma. However, there is a lack of research on the chemical composition of GSF and the pharmacological substance and mechanism of action for GSF in treating bronchial asthma.

The chemical constituents of GSF were analyzed using ultrahigh-performance liquid chromatography-quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). In this study, we combined network pharmacology, molecular docking techniques, and experimental validation to explore the therapeutic efficacy and underlying mechanism of GSF in the treatment of bronchial asthma.

Characterization of the chemical constituents of GSF was conducted using UHPLC-Q-Orbitrap HRMS. The identified chemical components were subjected to screening for active ingredients in the Swiss Absorption, Distribution, Metabolism, and Excretion (ADME) database. Relevant databases were utilized to retrieve target proteins for the active ingredients and targets associated with bronchial asthma disease, and the common targets between the two were selected. Subsequently, the protein-protein interaction (PPI) network was constructed using the String database and Cytoscape software to identify key targets. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the Metascape database. The "component-common target" network was constructed using Cytoscape to identify the primary active ingredients. Molecular docking validation was conducted using AutoDock software. The bronchial asthma mouse model was established using ovalbumin (OVA), and the lung organ index of the mice was measured. Lung tissue pathological changes were observed using hematoxylin and eosin (HE), Periodic Acid-Schiff (PAS), and Masson staining. The respiratory resistance (Penh) of the mice was assessed using a pulmonary function test instrument. An enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of IgE, IL-4, IL-5, and IL-13 in the mouse serum. Immunofluorescence staining was performed to detect the protein expression levels of AKT and PI3K in the lung tissues. An in vitro experiment was performed to observe the effects of echinocystic acid (EA) on IL-4 stimulated Human ASMCs (hASMCs). Cell viability was measured using a CCK-8 assay to calculate the IC50 value of the EA. A wound healing test was conducted to observe the effect of EA on degree of healing. RT-qPCR was performed to detect the influence of EA on the mRNA expression levels of ALB, SRC, TNF-α, AKT1, and IL6 in the cells.

A total of 95 chemical constituents were identified from the GSF. Of these, 37 were identified as active ingredients. There were 169 overlapping targets between the active ingredients and the disease targets. A topological analysis of the protein-protein interaction (PPI) network identified the core targets as IL6, TNF, ALB, AKT1, and SRC. An enrichment analysis revealed that the treatment of bronchial asthma with GSF primarily involved the AGE-RAGE signaling pathway and the PI3K-Akt signaling pathway, among others. The primary active ingredients included 13(s)-HOTRE, linolenic acid, and acacetin. The molecular docking results demonstrated a favorable binding activity between the critical components of GSF and the core targets. Animal experimental studies indicated that GSF effectively improved symptoms, lung function, and lung tissue pathological changes in the OVA-induced asthmatic mice, while alleviating inflammatory responses. GSF decreased the fluorescent intensity of the AKT and PI3K proteins. The IC50 value of EA was 30.02μg/ml. EA (30) significantly promoted the proliferation of IL4-stimulated hASMCs cells. EA (30) significantly increased the expression of ALB and SRC mRNA and decreased the expressions of TNF-α, AKT, and IL6 mRNA.

The multiple active ingredients found in GSF exerted their anti-inflammatory effects through multiple targets and pathways. This preliminary study revealed the core target and the mechanism of action underlying its treatment of bronchial asthma. These findings provided valuable insights for further research on the pharmacological substances and quality control of GSF.