The Anti-Inflammatory Effect of Callicarpa nudiflora Extract on H. Pylori-Infected GES-1 Cells through the Inhibition of ROS/NLRP3/Caspase-1/IL-1 β Signaling Axis

Abstract

Helicobacter pylori (H. pylori) is the main pathogenic factor of gastric cancer, chronic gastritis, and other gastric diseases. It has been found that Callicarpa nudiflora (CN) as an air-dried leaf extract has a broad-spectrum antibacterial effect. This study aims to examine the effect of CN on H. pylori-infected GES-1 cells in vitro and elucidate its underlying mechanism by extracting active ingredients from air-dried leaves. GES-1 cells were cocultured with HPSS1 at MOI = 100 : 1 and treated with different concentrations of CN (100 and 200 μg/ml). Results showed that CN can significantly reduce cellular LDH leakage and attenuate H. pylori-induced cell apoptosis and ROS production in GSE-1 cells, so as to protect gastric epithelial cells from damage by H. pylori. CN can also inhibit the secretion of inflammatory factors, such as TNF-α, IL-1β, IL-6, and IL-8. After CN treatment, the expression levels of active caspase-1, PYCARD, and NLRP3 were remarkably decreased in the treatment groups compared with the model group. To sum up, CN is highly protective against H. pylori-induced cell damage and apoptosis; CN can depress NLRP3 inflammasome activation and ROS production via the ROS/NLRP3/caspase-1/IL-1β signaling axis to suppress H. pylori-triggered inflammatory response and pyroptosis.

Figure 1

CN showed anti-HPSS1 activity in vitro. (a) The MIC value of CN on HPSS1. (b) Toxicity determination of CN to GES-1. (c) Determination of the proportion of GES-1 infected with HPSS1. (d) Comparison of the effects of CN before and after HPSS1 infection. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.001 compared with the control group.

Figure 2

CN reduced the damage of H. pylori to GES-1. (a) Morphological characterization of GES-1. Scale bar, 100 µm. (b) The effects of CN on cell viability of H. pylori-infected GES-1. (c) The effects of CN on the percentage of LDH leakage of H. pylori-infected GES-1. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, compared with the specified group.

Figure 3

CN reduced the apoptosis of H. pylori-infected GES-1. (a, b) Apoptosis detected by flow cytometry. (c, d) Western blotting results of PARP and caspase-3 proteins. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, compared with the specified group.

Figure 4

CN decreased the ROS production of H. pylori-induced GES-1. (a) ROS production detected by flow cytometry. (b) Quantitative analysis of DCFH-DA accumulation by FACS.

Figure 5

CN alleviated inflammatory response of H. pylori-infected GES-1. (a-d) Cellular cytokine productions detected by ELISA. (e-f) Western blotting results of NLRP3, PYCARD, and caspase-1 proteins. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, compared with the specified group.