Study on the effect and mechanism of verapamil on the growth of human airway granulosa fibroblasts

Abstract

Objective: This study aimed to investigate the inhibitory effect of the calcium channel blocker verapamil on human airway sarcomeric fibroblasts through network pharmacology molecular docking combined with experimental validation, so as to provide a theoretical and experimental basis for clinical use of calcium channel blockers against benign airway stenosis. Methods: The relevant targets of verapamil, airway injury, sarcoidosis and fibroblast were collected and intersected by a public database, respectively. The protein-protein interaction network was constructed by combining STRING; the DAVID database was used for gene function GO and KEGG pathway enrichment analysis. Molecular docking validation of verapamil and key targets was performed using AutoDock. Meanwhile, human lung bronchial tissue and human airway granulation tissue were treated with different antibiotic regimens to prevent contamination, in which human normal airway fibroblasts were treated with 92.81 μg/ml verapamil, while airway granulosa fibroblasts were treated with 69.57 μg/ml verapamil. The flow cytometry test was performed to detect the apoptotic effect of verapamil on human airway granulosa fibroblasts. The relative expression of mRNA of relevant factors between human normal airway fibroblasts and human airway granulosa fibroblasts was detected using polymerase chain reaction (PCR). Meanwhile, the difference in mRNA expression between human airway fibroblasts in normal and disease conditions was measured by PCR, and the change in mRNA expression of related factors after treatment with verapamil was determined to verify the findings of network pharmacology analysis. Results: This study predicted that verapamil affects 162 targets in human airway granulosa fibroblasts, among which, the most concerned and core target proteins are TGF-β1, VEGFA and IL-6; mainly involved in biological processes such as apoptosis, angiogenesis, energy metabolism and inflammation, and AGE-RAGE, JAK-STAT, PI3K-AKT, HIF as well as IL-17 signaling pathways. Molecular docking results showed that verapamil can bind to key targets. The apoptosis rate of human airway granulomatous fibroblasts treated with verapamil was higher than that of the control group, and the difference was statistically significant (P < 0.05). PCR showed that the relative expression of TGF-β1, VEGFA, IL-6 and other mRNAs in human airway granulomatous fibroblasts was significantly higher than that in human normal airway fibroblasts (P < 0.05), whereas the relative expression of TGF-β1 mRNA was down-regulated (P < 0.05) and the mRNA expression of VEGFA, IL-6 did not change significantly (P > 0.05) in human airway granulosa fibroblasts after treatment with verapamil. Conclusions: Increasing the antibiotic concentration and combining multiple antibiotics to soak the tissue can effectively kill the tissue pathogens. Verapamil can inhibit the proliferation of airway granulomatous fibroblasts through multiple targets and pathways, and it may be a method to prevent and treat benign airway stenosis.