Ginsenoside Ro improves Salmonella Typhimurium-induced colitis through inhibition of the virulence factors SopB and SopE2 via the RAC1/CDC42/ARP2/3 pathway

Abstract

Salmonella enterica serovar Typhimurium (S. Typhimurium) poses a serious threat to human and animal health, and there is an urgent need to develop new therapeutic agents. In our in vivo study, ginsenoside Ro (Ro) reduced the mortality rate of S. Typhimurium-infected mice by effectively improving three key disease activity index (DAI) indicators. In particular, ginsenoside Ro inhibited S. Typhimurium-induced colitis by reversing colon length shortening; alleviating pathological damage to the colon; decreasing the levels of IL-1β, TNF-α, IFN-γ, and IL-6; and decreasing the activities of MPO and EPO, while increasing the levels of IL-10 as well as the colon epithelial barrier and tight junction-related genes (Mucin 1, Mucin 2, Occludin, Claudin-3, and ZO-1). Furthermore, ginsenoside Ro reduced CFUs in the liver, spleen, colon, and feces. In a mechanistic in vitro study, ginsenoside Ro reduced CFUs in HeLa and Raw264.7 cells, which was associated with ginsenoside Ro inhibition of the recruited S. Typhimurium-containing vacuole (SCV) biomarkers LC3, Rab7, GAL8, and NDP52. Molecular docking results revealed that the binding energies of ginsenoside Ro and SopB and ginsenoside Ro and SopE2 were as high as -11.3 and -9.7 kcal/mol, respectively, as verified by CETSA and DARTS assays. Moreover, ginsenoside Ro at 100 and 200 μM significantly decreased the enzyme activities and expression of SopB and SopE2. Finally, ginsenoside Ro inhibited the membrane ruffling caused by SopB-regulated Arf6/Cyth2/Arf1-, RAC1-, and CDC42-driven Arp2/3-dependent actin polymerization and the SopE2-regulated CDC42/Arp2/3 signaling pathway. In summary, our findings suggest that ginsenoside Ro is a potential lead compound for therapeutic use against S. Typhimurium infection, and these findings lay a foundation for its further development.

Keywords: S. Typhimurium; colitis; ginsenoside Ro; invasion; virulence factors.

FIGURE 1

Ginsenoside Ro protected mice from S. Typhimurium‐induced colitis. (A) Flow chart of ginsenoside Ro treatment of mice. (B) Survival rate (n = 10). (C) Disease activity index (DAI) score. (D) Colon length analysis (n = 5 in each group) and representative H&E‐stained pathological images of the colon. Images were obtained at a magnification of 200×; scale bar: 20 μm. (E) The levels of the inflammatory factors IL‐1β, TNF‐α, IFN‐γ, IL‐6, and IL‐10. (F) MPO and EPO activities. (G) qPCR results. “*,” “^#,” “^##,” “**,” “***,” and “^###” indicate significant differences according to two‐way ANOVA with Tukey's multiple comparisons test (p < .05, p < .01 and p < .001, respectively).

FIGURE 2

Ginsenoside Ro reduced the replication of S. Typhimurium and regulated the inflammatory factors. (A) Bacterial colonization numbers in the colon, liver, spleen, and feces of the mice. Each data point represents one animal, and the results from three independent experiments (n = 5–10 in each group) were pooled. (B) IC₅₀ values of ginsenoside Ro in HeLa and Raw264.7 cells. (C) Bacterial colonization numbers detected in HeLa and Raw264.7 cells. (D, E) Representative images of colocalization of Rab7 and LC3; and NDP52 and GAL8, respectively (white arrows). Rab7 and NDP52 are labeled with Alexa Fluor 594 (red), and LC3 and GAL8 are labeled with Alexa Fluor 647 (yellow). The scale bars represent 20 μm. (F, G) The levels of inflammatory factors in HeLa and Raw264.7 cells. “^###,” “***,” “**,” and “*” indicate significant differences according to ordinary one‐way ANOVA (p < .001, p < .01 and p < .05, respectively).

FIGURE 3

Molecular docking shows ginsenoside Ro interacting with dual targets, which is verified in vitro. (A, B) 2D and 3D diagrams of the interaction between ginsenoside Ro and SopB and between ginsenoside Ro and SopE2, respectively. The green, pink, and orange circles represent hydrogen bonds and hydrophobic interactions, respectively. (C) CETSA results of ginsenoside Ro binding to the SopB and SopE2 proteins. (D) DARTS results of ginsenoside Ro binding to the SopB and SopE2 proteins. The Pronase® E:protein ratios used were as follows: 1:300, 1:500, and 1:1000. “*” and “**” indicate a significant difference (p < .05 and p < .01, respectively) according to ANOVA.

FIGURE 4

Ginsenoside Ro inhibited the enzyme activity and expression of STM SopB and SopE2 in vitro. (A) Malachite green experiment. (B) IL‐8 production detection. (C) SopB and SopE2 expression was detected in the STM‐infected groups and ginsenoside Ro‐treated STM‐infected groups. To ensure equal loading of the lanes, the result was reprobed with an anti‐α‐DnaK antibody. (D, E) Representative CLSM images of SopB and SopE2, respectively. The secondary antibody was labeled with Alexa Fluor 594 (red). DNA was stained with Hoechst 33342 (blue), and S. Typhimurium was labeled with MycoLight™ Green JJ99 stain (green). Images represent projections of confocal optical Z‐sections obtained via Nikon CLSM. The bar at the bottom right represents 20 μm. The results represent the mean ± SD of three independent experiments. “^###”, “***”, and “**” indicate significant differences according to ordinary one‐way ANOVA (p < .001, p < .001, and p < .01 respectively).

FIGURE 5

Ginsenoside Ro inhibited membrane ruffling through the RAC1/CDC42/ARP2/3 pathway mediated by SopB and SopE2. (A) RAC1 and CDC42 knockdown assays. HeLa cells were transfected with RAC1 siRNA, CDC42 siRNA or negative control siRNA for 6 h. Western blotting and qPCR was used to analyze silencing effect of siRNA on the proteins and mRNAs of RAC1 and CDC42, respectively. The membrane ruffle rates were determined from 100 randomly chosen HeLa cells. Scale bars = 20 μm. (B) Intracellular CFU counting experiment. (C) Membrane ruffling rate. The white triangles point to membrane ruffling. Cytoskeletal Actin was labeled with TRITC‐phalloidin (red), DNA was stained with Hoechst 33342 (blue), and Salmonella was labeled with MycoLight™ Green JJ99 stain (green). The results represent three independent experiments (mean ± SD). The scale bars represent 20 μm. (D) The expression of Rac1‐GTP/Cdc42‐GTP/Arf6/Cyth2/Arf1/Arp2/3. The relative levels of activated RAC1 and Cdc42 were determined by the amount bound to GST‐PAK‐PBD. GAPDH was used as a loading control. (“^###” p < .001, “***” p < .001, “**” p < .01 determined by one‐way ANOVA).