Inhibition of TRPA1, Endoplasmic Reticulum Stress, Human Airway Epithelial Cell Damage, and Ectopic MUC5AC Expression by Vasaka (Adhatoda vasica; Malabar Nut) Tea

Abstract

This study tested whether a medicinal plant, Vasaka, typically consumed as a tea to treat respiratory malaise, could protect airway epithelial cells (AECs) from wood smoke particle-induced damage and prevent pathological mucus production. Wood/biomass smoke is a pneumotoxic air pollutant. Mucus normally protects the airways, but excessive production can obstruct airflow and cause respiratory distress. Vasaka tea pre- and co-treatment dose-dependently inhibited mucin 5AC (MUC5AC) mRNA induction by AECs treated with wood smoke particles. This correlated with transient receptor potential ankyrin-1 (TRPA1) inhibition, an attenuation of endoplasmic reticulum (ER) stress, and AEC damage/death. Induction of mRNA for anterior gradient 2, an ER chaperone/disulfide isomerase required for MUC5AC production, and TRP vanilloid-3, a gene that suppresses ER stress and wood smoke particle-induced cell death, was also attenuated. Variable inhibition of TRPA1, ER stress, and MUC5AC mRNA induction was observed using selected chemicals identified in Vasaka tea including vasicine, vasicinone, apigenin, vitexin, isovitexin, isoorientin, 9-oxoODE, and 9,10-EpOME. Apigenin and 9,10-EpOME were the most cytoprotective and mucosuppressive. Cytochrome P450 1A1 (CYP1A1) mRNA was also induced by Vasaka tea and wood smoke particles. Inhibition of CYP1A1 enhanced ER stress and MUC5AC mRNA expression, suggesting a possible role in producing protective oxylipins in stressed cells. The results provide mechanistic insights and support for the purported benefits of Vasaka tea in treating lung inflammatory conditions, raising the possibility of further development as a preventative and/or restorative therapy.

Keywords: MUC5AC; TRPA1; Vasaka; airway epithelium; biomass smoke; lung injury; mucus hypersecretion; wood smoke.

Figure 1

Concentration-dependent inhibition of MUC5AC mRNA expression by Vasaka tea. HBEC3-KT cells were pre- (2 h) and cotreated with Vasaka tea diluted in treatment medium with (solid circles) and without (open circles) WSPM (2.5 mg/mL) for 24 h (n = 3). MUC5AC mRNA expression was quantified using qPCR. Data were modeled using the log (antagonist) vs. response equation in GraphPad 9.5 software to predict the IC₅₀ value.

Figure 2

Inhibition of TRPA1-mediated calcium flux by Vasaka tea in HBEC3-KT cells treated with the TRPA1 agonist AITC (25 μM; white bars) or WSPM (2.5 mg/mL or 195 μg/cm²; gray bars). Replicates within each group are shown as open circles. HC-030031 is a TRPA1 antagonist. Vasaka tea was diluted in medium, and cells were pretreated with HC-030031 and Vasaka tea for 30 min prior to agonist (AITC or WSPM) application. Data were analyzed by one-way ANOVA with post hoc testing for significance among preselected groups using the Bonferroni test (n = 4–8). *** p < 0.001, **** p < 0.0001.

Figure 3

(A) Heatmap comparing the expression of the 1000 most changed transcripts in HBEC3-KT cells treated with WSPM compared to the vehicle control, as a function of treatment group. (B) Quantitative comparison of normalized enrichment score values multiplied by −1 (−1*NES) from pathway/GSEA of HBEC3-KT cells treated with vehicle (cell culture medium + 0.2% DMSO), WSPM (10 μg/cm²), Vasaka tea (1.25 mg/mL), or WSPM and Vasaka tea. Differential gene expression was determined using hciR and DESeq2 (1.30.1). Pathway analysis was performed using fgsea (1.18.0) using the Hallmarks Molecular Signatures Database. A negative value indicates enrichment in the comparison control group.

Figure 4

Heatmaps comparing the expression of significantly altered genes associated with the (A) hallmark ROS/oxidative stress response pathway, (B) the unfolded protein response pathway, (C) the hallmark G2M checkpoint pathway, and (D) the hallmark E2F targets pathway in HBEC3-KT cells treated with vehicle (cell culture medium + 0.2% DMSO), WSPM (10 μg/cm²), Vasaka tea (1.25 mg/mL), or WSPM and Vasaka tea.

Figure 5

Comparison of AGR2 and TRPV3 mRNA expression in HBEC3-KT cells treated with vehicle (cell culture medium + 0.2% DMSO), WSPM (10 μg/cm²), Vasaka tea (1.25 mg/mL), or WSPM and Vasaka tea using qPCR. Replicates within each group are shown as open circles. **** p < 0.0001 using 2-way ANOVA and Tukey post-test to compare the effects of treatment on mRNA expression for each gene (n = 4).

Figure 6

Comparison of HBEC3-KT morphological features and monolayer integrity as a function of WSPM and Vasaka tea treatments and time. Confluent monolayers of HBEC3-KT cells were treated with WSPM (20 μg/cm²) or WSPM + Vasaka tea (2.5 mg/mL) and imaged using an Incucyte live cell imaging system. (A) Representative images 12 h and 36 h post treatment with a cell mask overlaid in yellow. Red boxes indicate expanded regions of the image. Quantification of (B,C) cell area, (D,E) cell perimeter, and (F,G) internal density of cells at 12 and 36 h. **** p < 0.0001 using ANOVA and Tukey post-test to compare the average feature values as a function of treatments. Movies showing changes associated with the treatments are shown in the Supplementary Materials.

Figure 7

(A) Estimated concentrations of vasicine, vasicinone, isoorientin, isovitexin, and apigenin in a 1 mg/mL solution of Vasaka tea. Analytes were quantified by LC/MS/MS (n = 4 extracts). The dashed line and arrows indicate which y-axis the data are plotted on and replicates within each group are shown as open circles. A representative LC/MS/MS chromatogram is shown in Figure S4. (B) Inhibition of AITC-induced (TRPA1-dependent) calcium flux in HBEC3-KT cells treated with increasing concentrations of apigenin, vasicine, and vasicinone and stimulated with AITC (25 μM). Data were modeled using the log (antagonist) vs. response equation in GraphPad 9.5 software to estimate the IC₅₀ value (n = 3–4).

Figure 8

Comparison of (A) DDIT3, (B) HMOX1, and (C) MUC5AC mRNA expression in HBEC3-KT cells treated with vehicle (cell culture medium + 0.2% DMSO), WSPM (20 μg/cm²), and various concentrations of Vasaka tea components using qPCR (vasicine = 300 μM; vasicinone = 50 μM; vitexin = 2.5 μM; isovitexin = 2.5 μM; isoorientin = 2.5 μM; apigenin = 2.5 μM, based on Figure 7A. replicates within each group are shown as open circles. * p < 0.05, ** p < 0.01 and **** p < 0.0001 using two-way ANOVA and Dunnett post-test to compare the effects of treatment on mRNA expression for each gene compared to the control (n = 3). (D) Representative images 24 h post treatment collected using a Molecular Devices Image Express PICO with cell masks overlaid in yellow. Red boxes indicate expanded regions of the image. (E) Cell area as a function of treatments. ** p < 0.01 and **** p < 0.0001 using ANOVA and Dunnett post-test to compare to the effects of WSPM treatment.

Figure 9

(A) Estimated concentrations of 13(S)- and 9(S)-HODE, 13-oxo and 9-oxoODE, 12,13-and 9,10-DiHOME, and 12,13- and 9,10-EpOME in a 1 mg/mL solution of Vasaka tea. Analytes were quantified by LC/MS/MS (n = 4 extracts). The dashed line and arrows indicate which y-axis the data are plotted on and replicates within each group are shown as open circles. A representative LC/MS/MS chromatogram is shown in Figure S6F. (B) Inhibition of AITC-induced (TRPA1-dependent) calcium flux in HBEC3-KT cells treated with various concentrations of oxylipins and stimulated with AITC (25 μM). Data were modeled with the log (antagonist) vs. response equation in GraphPad 9.5 software to estimate the IC₅₀ value (n = 3–6). (C) DDIT3 and (D) MUC5AC mRNA expression in HBEC3-KT cells treated with WSPM (20 μg/cm²) with and without pre- (30 min) and cotreatment with 9-oxoODE or 9,10-EpOME (1 μM). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 using two-way ANOVA and a Tukey post hoc test comparing all groups (panel D) and ANOVA with a Dunnett post-test comparing the effects of treatment relative to WSPM treatment (panel C). n = 4–7. (E) Representative images 24 h post treatment collected using a Molecular Devices Image Express PICO with cell masks overlaid in yellow. Red boxes indicate expanded regions of the image. (F) Cell area as a function of treatments. **** p < 0.0001 using ANOVA and Dunnett post-test to compare to the effects of WSPM treatment. Data showing TRPA1 agonist and antagonist effects of 9-oxoODE, 9-HpODE, 9,10-EpOME, and 9-HODE at 50 μM are shown in Figure S7.

Figure 10

(A) Comparison of CYP1A1, 1A2, and 1B1 mRNA expression by HBEC3-KT cells treated with vehicle (cell culture medium + 0.2% DMSO), WSPM (10 μg/cm²), Vasaka tea (1.25 mg/mL), or WSPM and Vasaka tea. Replicates within each group are shown as open circles. Data were analyzed using two-way ANOVA with a Tukey post-test comparing the effects of treatments (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Expression of mRNA for (B) CYP1A1, (C) DDIT3, (D) TRPV3, and (E) MUC5AC as a function of WSPM treatment with and without pre- (2 h) and cotreatment with the CYP1A1 inhibitor 7-ethoxyresorufin (7-ER) (n = 3). Data were analyzed using two-way ANOVA and a Bonferroni post-test to compare the effects of each treatment. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.