Flavokavains A- and B-Free Kava Enhances Resilience against the Adverse Health Effects of Tobacco Smoke in Mice

Abstract

Tobacco smoke remains a serious global issue, resulting in serious health complications, contributing to the onset of numerous preventive diseases and imposing significant health burdens. Despite regulatory policies and cessation measures aimed at curbing its usage, novel interventions are urgently needed for effective damage reduction. Our preclinical and pilot clinical studies showed that AB-free kava has the potential to reduce tobacco-smoking-induced lung cancer risk, mitigate tobacco dependence, and reduce tobacco use. To understand the scope of its benefits in damage reduction and potential limitations, this study evaluated the effects of AB-free kava on a panel of health indicators in mice exposed to 2-4 weeks of daily tobacco smoke exposure. Our assessments included global transcriptional profiling of the lung and liver tissues, analysis of lung inflammation, evaluation of lung function, exploration of tobacco nicotine withdrawal, and characterization of the causal protein kinase A (PKA) signaling pathway. As expected, tobacco smoke exposure perturbed a wide range of biological processes and compromised multiple functions in mice. Remarkably, AB-free kava demonstrated the ability to globally mitigate tobacco smoke-induced deficits at the molecular and functional levels with promising safety profiles, offering AB-free kava unique promise to mitigate tobacco smoke-related health damages. Further preclinical evaluations are warranted to fully harness the potential of AB-free kava in combating tobacco smoke-related harms in the preparation of its clinical translation.

Figure 1

Structures of the six major kavalactones and the two chalcone-based flavokavain compounds in kava.

Figure 2

RNA-seq results of the lung and liver tissues from mice with different treatment regimens. Air: Filtered Air; TS: tobacco smoke; AB-free Kava: AB-free kava. (A, B) PCA of the RNA-Seq data, showing the first two principal components (PCs) for individual mice in lung and liver tissues, respectively. The variance explained by each PC is indicated on the x-axis and y-axis labels. (C, D) Correlation of the activation z scores of the top 40 signaling pathways perturbed by TS in comparison to Air (y-axis) and by TS + AB-free kava in comparison to TS (x-axis). β represents the regression coefficient of the scatter plot. These pathways are categorized into three groups: those significant only when TS is compared to Air, those significant only when TS + AB-free kava is compared to TS, and those significant in both comparisons. (E, F) Correlation of log2Fold Change among the top 300 genes perturbed by TS in comparison to Air, and by TS + AB-free kava in comparison to TS.

Figure 3

Effect of TS and/or AB-free kava on proinflammatory immune cells in the BAL and lung tissues. (A–C) Flow cytometry analysis of neutrophils in the BAL and lung tissues. (A) Gating schematic for neutrophils showing the Ly6C⁺Ly6G⁺ population. The effect of TS exposure and/or AB-free kava via dietary supplementation on neutrophils in mouse BAL, after (B) 2 weeks or (C) 4 weeks of TS and/or AB-free kava (n = 4 mice/group). (D) BAL smear-based pathological determination of neutrophil numbers in BAL after 2 weeks of TS and/or AB-free kava via gavage (n = 5 mice/group).

Figure 4

Effect of TS and/or AB-free kava on proinflammatory cytokines and lung tissues. (A) TNF-α or (B) IL-6 in mouse BAL or serum samples of different treatment groups after 2 weeks of TS and/or AB-free kava via dietary supplementation (n = 4 mice per group). (C, D) Effects of tobacco smoke and/or AB-free kava via dietary supplementation on (C) total protein and (D) albumin levels in mouse BAL after 2 weeks of TS and/or AB-free kava (n = 4 mice per group). (E) Representative lung tissue pathological images from mice from different groups with no obvious lesions related to different treatment regimens (top, TS treated lung; bottom, TS + AB-free Kava).

Figure 5

AB-free kava diminishes the somatic withdrawal signs in TS-exposed mice. Asterisks indicate more signs compared to the air group during the same week. Plus signs indicate fewer signs compared to the TS group during the same week. Ampersand signs indicate more signs compared to the TS group during week 2. **, p < 0.01; ***, +++, &&& p < 0.001.

Figure 6

Effects of TS exposure with/without AB-free kava dietary supplementation on lung function characterization. (A) Basal airway resistance. (B) Normalized resistance in response to methacholine in airways. (C) Basal airway elastance. (D) Normalized elastance in airways in response to methacholine. * = compared Air. #; compared to TS. (E) Basal airway Newtonian resistance. (F) Normalized Newtonian resistance in airways in response to methacholine. (G) Basal tissue damping. (H) Normalized tissue damping in airways in response to methacholine. (I) Basal tissue elastance. (J) Normalized tissue elastance in airways in response to methacholine. Air (n = 3), Tobacco (n = 4); Tobacco + AB-free kava (n = 4). Abbreviations: M.E.T., main effect of treatment.

Figure 7

Western blotting analyses of the lung tissues upon TS exposure with and without AB-free kava supplementation, centering on the β-AR mediated PKA/CREB/LKB1/COX-2 pathway.