Supplementary Material for: Flavonoid Quercetin Enhances Nuclear factor erythroid 2-related factor 2 (Nrf2) Pathway Activation and Reduces Inflammatory Cytokines in Asthmatic Airway Epithelial cells

Background: Asthma is a chronic disease characterized by airway inflammation, oxidative stress, and bronchial hyperresponsiveness. Quercetin, a safe and well-tolerated flavonoid, reduces airway inflammation and has antioxidant effects, which are partly modulated by activating the nuclear factor erythroid 2–related factor 2 (Nrf2) pathway. This dual anti-inflammatory and antioxidant pharmacological effect makes this compound a potentially effective therapeutic agent. We used primary human bronchial epithelial cells (HBECs) from asthmatic and healthy controls to evaluate whether quercetin modulates Nrf2 expression and reduces inflammatory cytokines. Methods: Differentiated ciliated and mucus-producing HBECs were maintained for 21 days at the air–liquid interface (ALI), then were pretreated for 18 hours with 25 µM quercetin. Following a washout with phosphate-buffered saline, cells were exposed to IL-13 (10 ng/mL) for 3 hours. Cell culture supernatants were collected, and a cytokine panel was measured. Additionally, bulk RNA-seq differential expression testing was performed, where a significant between-group difference was defined by a false discovery rate (FDR) 0.5 Results: Human airway epithelial cells treated with quercetin showed a significant increase in Nrf2 protein levels compared to untreated cells (p=0.008). In addition, quercetin treatment was associated with a reduction in TNF-α expression in asthmatic cells. Although this decrease did not reach statistical significance, the observed trend may suggest a potential anti-inflammatory effect worth further investigation. Moreover, compared to control, quercetin significantly upregulated the gene expression of the γ-glutamate-cysteine ligase catalytic subunit (GCLC) subunit and NAD(P)H quinone oxidoreductase-1 (NQO1) (p=0.009 and p=0.04 respectively) in cultured HBECs. Conclusion: This study suggests that quercetin may be a promising therapeutic agent to improve health outcomes in asthma by activating the Nrf2 pathway to reduce oxidative stress and pro-inflammatory cytokines in airway epithelium, which warrants further mechanistic and clinical investigation.