Quercetin attenuates inflammation in LPS-induced lung epithelial cells via the Nrf2 signaling pathway

Abstract

Background: Pneumonia is the leading cause of death among children under five, and kill almost two million children each year. Quercetin, a flavonoid polyphenolic compound, exerts many beneficial biological activities, including anti-inflammatory functions. Our study aimed to investigate the possibility of quercetin as a therapeutic agent for pneumonia and its role in the inflammatory response induced by lipopolysaccharide (LPS).

Methods: LPS induced human alveolar epithelial cell A549 as a lung inflammation model in vitro. The effects of quercetin on the production of cytokines and the expression of related-proteins were detected by Enzyme-Linked ImmunoSorbent Assay and Western Blot, respectively. Cell Counting Kit-8 assay was used to detect cell viability. flow cytometry was used to measure cell apoptosis. NO levels were also analyzed through NO kit.

Results: Our results found that quercetin attenuated the release of IL-1β, IL-6, PGE2, and nitrite in LPS-induced A549 cells. In addition, quercetin inhibits cell apoptosis and relieves ROS generation in LPS-induced A549 cells. Quercetin also inhibits LPS-induced NF-κB activation. They have upregulated the expression of nuclear factor erythroid 2 (Nrf2) and HO-1.

Conclusion: In conclusion, these results suggested that quercetin attenuates LPS-induced inflammation in A549 by activating the Nrf2 signaling pathway.

Keywords: LPS; Nrf2; inflammatory cytokine; pneumonia; quercetin.

Figure 1

Quercetin attenuates cell damage of LPS‐induced A549 cells. (A) The chemical structure of quercetin. (B) the cell viability was detected by CCK8 assay after being treated with the different conditions of quercetin. (C) the cell viability was detected by CCK8 assay after being treated with the different conditions of quercetin in LPS‐induced cells. Data are shown as mean ± SEM (n = 3 independent experiments). Statistics: one‐way analysis of variances with Tukey's multiple comparison correction. *p < .05 and ***p < .001. compared to the control group.

Figure 2

Quercetin inhibits inflammation of LPS‐induced A549 cells. a secreted level of IL‐1β (A), IL‐6 (B), PGE2 (C), and nitrite (D) ELISA assay detected in cultured cells. Data are shown as mean ± SEM (n = 3 independent experiments). Statistics: one‐way analysis of variances with Tukey's multiple comparison correction. *p < .05 and ***p < .001. compared to the control group.

Figure 3

Quercetin Inhibits Cell Apoptosis and cellular ROS level in LPS‐Induced A549 Cells (A) the apoptosis rate of cultured A549 cells detected by Flow cytometry. (B) the apoptosis rate of LPS‐Induced A549 Cells was detected by Flow cytometry. (C) The apoptotic rate was quantified after LPS induction and gradient Quercetin treatment (D) Relative levels of intracellular ROS in A549 cells. *p < .05 and ***p < .001. The data presented are the means ± SEM of three independent experiments.

Figure 4

Quercetin inhibits NF‐κB activation and induces Nrf2 activation in LPS‐induced A549 cells. (A) images for p‐p65, Nrf2, and HO‐1 proteins expression in cultured cells after quercetin treatment. (B−D) Quantification of the relative expression level of p‐p65, Nrf2, and HO‐1. *p < .05 and ***p < .001. The data presented are the means ± SEM of three independent experiments.

Figure 5

The anti‐inflammatory effects of quercetin were Nrf2 dependent. (A) Representative images for the expression of Nrf2 proteins in transfected cells. (B) Quantification of the relative expression level of Nrf2. ELISA assay detects a secreted level of IL‐1β (C), IL‐6 (D), PGE2(E), and nitrite (F) in cultured A549 cells. Data are shown as mean ± SEM (n = 3 independent experiments). Statistics: one‐way analysis of variances with Tukey's multiple comparison correction. *p < .05 and ***p < .001. compared to the control group.