关键词: 红景天素, 幽门螺杆菌, 脲酶, 分子对接, 炎症, 氧化应激

Abstract: Objective  To screen natural small-molecule compounds with anti-Helicobacter pylori urease (HPU) activity based on traditional Chinese medicine active ingredients and to systematically investigate their inhibitory mechanisms against HPU, as well as their regulatory effects on cellular inflammation and oxidative stress following Helicobacter pylori (Hp) infection. Methods  A multi-dimensional screening strategy was adopted. Firstly, virtual screening was performed on the traditional Chinese medicine monomer compound database based on the crystal structure of HPU, and the candidate molecules were selected in combination with bibliometric analysis. Subsequently, the modified Berthelot method was applied to verify urease inhibitory activity in vitro. Inhibition kinetics were analyzed with Lineweaver-Burk plots. The inhibitory sites were explored through sulfhydryl blocking agents and Ni²⁺ competitive inhibitors, followed by molecular docking simulations with AutoDock Vina (version 1.2.3). A Hp-infected human gastric mucosal epithelial cells (GES-1) model was established. The compound's cytotoxicity was assessed with the CCK-8 assay and lactate dehydrogenase release assay. The mRNA expression levels of interleulain (IL)-6, IL-8, and IL-1β were quantified with quantitative real-time PCR (qRT-PCR). Intracellular reactive oxygen species (ROS) levels were measured with a DCFH-DA fluorescent probe. Results  According to the screening results, the natural small-molecule compound rhodiosin (RHO) significantly inhibited HPU activity with a half-maximal inhibitory concentration (IC50) of (82.38±5.45) μmol/L. Enzyme kinetics analysis revealed that RHO acted as an anti-competitive inhibitor, showing an inhibition constant of (146.40±2.19) μmol/L; RHO-sulfhydryl/Ni²⁺-HPU interaction experiments confirmed that its target was located in the sulfhydryl group in the active center of HPU. Molecular docking simulations suggested that RHO is bound exactly to the Flap domain of the urease active pocket, with a binding energy of -8.678 kcal/mol. No significant cytotoxicity towards GES-1 cells was observed with RHO at 80 μmol/L in cellular experiments. Furthermore, RHO significantly down-regulates the mRNA overexpression of IL-6, IL-8, and IL-1β induced by Hp and reduces the production of ROS by 95%. Conclusion  The monomer RHO of traditional Chinese medicine inhibits HPU through anti-competitive binding to the sulfhydryl site. It can effectively alleviate the inflammatory response and oxidative stress injury of GES-1 caused by Hp infection, providing a theoretical foundation for developing novel anti-Hp treatment strategies.

Key words: rhodiosin, Helicobacter pylori, urease, molecular docking, inflammation, oxidative stress