In vitro anti-bacterial activity and network pharmacology analysis of Sanguisorba officinalis L. against Helicobacter pylori infection

Abstract

Background: Helicobacter pylori (H. pylori) infection has become an international public health problem, and antibiotic-based triple or quadruple therapy is currently the mainstay of treatment. However, the effectiveness of these therapies decreases due to resistance to multiple commonly used antibiotics. Sanguisorba officinalis L. (S. officinalis), a traditional Chinese medicine clinically used for hemostasis and treatment of diarrhea, has various pharmacological activities. In this study, in vitro antimicrobial activity was used for the preliminary evaluation of S. officinalis against H. pylori. And a pharmacology analysis approach was also utilized to elucidate its underlying mechanisms against H. pylori infection.

Methods: Micro-broth dilution method, agar dilution method, checkerboard assay, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) were used for the assessment of anti-bacterial activity. Active ingredients screening, GO analysis, KEGG analysis, construction of PPI network, molecular docking, and RT-qPCR were used to elucidate the underlying pharmacological mechanisms of S. officinalis against H. pylori infection.

Results: The minimum inhibitory concentration (MIC) values of S. officinalis against multiple H. pylori strains including clinically isolated multi-drug resistant (MDR) strains were ranging from 160 to 320 µg/ml. These results showed that S. officinalis had additive interaction with four commonly used antibiotics and could exert antibacterial effect by changing the morphology of bacteria without developing drug resistance. Through network pharmacology analysis, 8 active ingredients in S. officinalis were screened out for subsequent studies. Among 222 putative targets of S. officinalis, 49 targets were identified as potential targets for treatment of H. pylori infection. And these 49 targets were significantly enriched in GO processes such as protein kinase B signaling, protein kinase activity, protein kinase binding, and KEGG pathways such as Pathways in cancer, MicroRNAs in cancer, and TNF signaling pathway. Protein-protein interaction analysis yielded 5 core targets (AKT1, VEGFA, EGFR, SRC, CCND1), which were validated by molecular docking and RT-qPCR.

Conclusions: Overall, this study confirmed the in vitro inhibitory activity of S. officinalis against H. pylori and explored the possible pharmacological mechanisms, laying the foundation for further research and clinical application.

Keywords: Active ingredients; Antibacterial; Helicobacter pylori; Network pharmacology; Sanguisorba officinalis L..

Fig. 1

Inhibiting kinetic curves and killing kinetic curves. a Inhibiting kinetic curve of S. officinalis against H. pylori 43,504 strain; b Inhibiting kinetic curve of S. officinalis against H. pylori 700,392 strain. Data represent medians $\pm$ standard deviation of the results from three independent experiments. c Killing kinetic curve of S. officinalis against H. pylori 43,504 strain; d Killing kinetic curve of S. officinalis against H. pylori 700,392 strain. The dotted line represents a 1,000-fold reduction in the number of bacteria compared to the initial inoculation

Fig. 2

Development of resistance to S. officinalis and metronidazole (MTZ) in 700,392. The fold change is the normalized ratio of the MIC obtained for a continuous subculture of sub-inhibitory concentration exposure to that MIC obtained for first-time exposure

Fig. 3

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images. a–c Morphological images of H. pylori cells on SEM after exposure to water, 2 MIC S. officinalis, and 4 MIC S. officinalis for 12 h. Each result is shown at three different scales. These results indicate that treatment of S. officinalis can result in cell shrinkage and cell wall damage in a dose-dependent manner. d-f Morphological images of H. pylori cells on TEM after exposure to water, 2 MIC S. officinalis, and 4 MIC S. officinalis for 12 h. Each result is shown at three different scales. Red arrows indicate separation between the cell wall and inner membrane, bleb formation, cell wall damage, and cell lysis. There only existed mild cellular damage at the concentration of 2 MIC S. officinalis, mainly separation between the cell wall and inner membrane and bleb formation. There appear significant cell wall damages and cell lysis at the concentration of 4 MIC S. officinalis

Fig. 4

Results figure of network pharmacology analysis. a Venn figure of S. officinalis and H. pylori infection; b Network figure of “Drug-Components-Targets; c Gene ontology analyses for potential targets, BP Biological process, CC Cellular components, MF Molecular function; d KEGG pathway enrichment analysis; e Network diagram of protein-protein interaction, the higher the degree value, the larger and darker the node

Fig. 5

Molecular docking results of three core targets with active ingredients of S. Officinalis. Active ingredients are represented by the ball-and-stick model, the secondary structure of the protein was represented by ribbon. The blue areas represent active pocket of the proteins. The serial numbers in the figure represent those in Table 6

Fig. 6

Cell viability (%) of AGS after 24 h treatment with S. officinalis aqueous extracts. IC₅₀, half-inhibitory concentration

Fig. 7

Gene expression of the core target genes VEGFA and CCDN1 under different conditions. Including the control group without any treatment, the group treated with S. officinalis alone (80 µg/ml), the group treated with H. pylori alone, and the group treated with both S. officinalis and H. pylori, were examined for 10 h, 24 and 48 h treatment times. *P < 0.05, **P < 0.01, ***P < 0.001, vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001, (S. officinalis + H. pylori) group vs. H. pylori group