Platycodonis Radix Alleviates LPS-Induced Lung Inflammation through Modulation of TRPA1 Channels

Abstract

Platycodonis Radix (PR), a widely consumed herbal food, and its bioactive constituents, platycodins, have therapeutic potential for lung inflammation. Transient Receptor Potential Ankyrin 1 (TRPA1), which is essential for the control of inflammation, may be involved in the development of inflammation in the lungs. The aim of this study was to determine the TRPA1-targeted effects of PR against pulmonary inflammation and to investigate the affinity of PR constituents for TRPA1 and their potential mechanisms of action. Using a C57BL/6J mouse lipopolysaccharides (LPS) intratracheal instillation pneumonia model and advanced analytical techniques (UPLC-Q-TOF-MS/MS, molecular docking, immuno-fluorescence), five platycodins were isolated from PR, and the interaction between these platycodins and hTRPA1 was verified. Additionally, we analyzed the impact of platycodins on LPS-induced TRPA1 expression and calcium influx in BEAS-2B cells. The results indicated that PR treatment significantly reduced the severity of LPS-triggered inflammation in the mouse model. Interestingly, there was a mild increase in the expression of TRPA1 caused by PR in healthy mice. Among five isolated platycodins identified in the PR extract, Platycodin D₃ (PD₃) showed the highest affinity for hTRPA1. The interaction between platycodins and TRPA1 was verified through molecular docking methods, highlighting the significance of the S5-S6 pore-forming loop in TRPA1 and the unique structural attributes of platycodins. Furthermore, PD₃ significantly reduced LPS-induced TRPA1 expression and calcium ion influx in BEAS-2B cells, substantiating its own role as an effective TRPA1 modulator. In conclusion, PR and platycodins, especially PD₃, show promise as potential lung inflammation therapeutics. Further research should explore the precise mechanisms by which platycodins modulate TRPA1 and their broader therapeutic potential.

Keywords: TRPA1; lung inflammation; platycodins; platycodonis radix.

Figure 1

(A) Protective effects of PR on pneumonia in mice. (B) H&E staining of lung tissue and histologic scoring. (C) The concentrations of TNF-α and IL-1β were measured in BALF samples. (D) The mediating role of the TRPA1 receptor in pulmonary inflammation. (E) The TNF-α and IL-1β concentrations in the BALF were determined by ELISA. (F) TRPA1 protein expression was measured by Western blot analysis in lung tissue. (G) TRPA1 gene expression levels in lung tissue. All experiments were run in parallel (n = ≥3). Significant differences are indicated in the figures and legends as # p < 0.05, and #### p < 0.0001 vs. the Control; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. LPS-induced mice (LPS).

Figure 2

(A) PR inhibits TRPA1 expression to prevent LPS-induced pulmonary inflammation. (B) The expression levels of the TRPA1 gene in the lung tissue. TRPA1 (C) protein expression was measured by Western blot analysis in lung tissue. Since the number of mice in the experimental group amounted to 11, the high-dose group was analyzed in a second block of prefabricated gel. All experiments were run in parallel (n = ≥3). Significant differences are indicated in the figures and legends as ## p < 0.01, ### p < 0.001 vs. the Control; * p < 0.05, *** p < 0.001 and **** p < 0.0001 vs. LPS-induced mice (LPS). (D) Effect of PR on TRPA1 in mice. (E) The expression levels of the TRPA1 gene in the lung tissue. Experimental scheme (F) TRPA1 protein expression in lung tissue. Significant differences are indicated in the figures and legends as ## p < 0.01, ### p < 0.001 vs. the Control.

Figure 3

(A) The total ion chromatograms of PR by 2D UPLC/Xevo G2-XS-Q/TOF. (B) ESI-MS/MS spectrum of five platycodins.

Figure 4

The binding mode of the TRPA1 protein with the LPS, A967079, AITC, and platycodins.

Figure 5

(A) Immunofluorescence assay to detect the TRPA1 protein in BEAS-2B cells, scale bar, 20 μm. Green fluorescence corresponds to TRPA1, nuclei are stained in blue. (B) Histogram showing TRPA1 quantification in BEAS-2B cells. (C) Effect of PR on intracellular Ca²⁺ levels in BEAS-2B cells. Significant differences are indicated in the figures and legends as #### p < 0.0001 vs. the Control; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. LPS-induced mice (LPS).