Nrf2 positively regulates autophagy antioxidant response in human bronchial epithelial cells exposed to diesel exhaust particles

Daniela Perroni Frias,Thais Mauad,Paulo Hilário Nascimento Saldiva,Daniela Cristina De Almeida Pereira,Mariangela Macchione,Paulo Roberto Da Conceição,Regiani Carvalho-Oliveira,Raquel Labiapari Nunes Gomes,Walcy Rosolia Teodoro,Pérola De Castro Vasconcellos,Mara De Souza Junqueira,Monique Matsuda,Kelly Yoshizaki

doi:10.1038/s41598-020-59930-3

Abstract

Diesel exhaust particles (DEP) are known to generate reactive oxygen species in the respiratory system, triggering cells to activate antioxidant defence mechanisms, such as Keap1-Nrf2 signalling and autophagy. The aim of this study was to investigate the relationship between the Keap1-Nrf2 signalling and autophagy pathways after DEP exposure. BEAS-2B cells were transfected with silencing RNA (siRNA) specific to Nrf2 and exposed to DEP. The relative levels of mRNA for Nrf2, NQO1, HO-1, LC3B, p62 and Atg5 were determined using RT-PCR, while the levels of LCB3, Nrf2, and p62 protein were determined using Western blotting. The autophagy inhibitor bafilomycin caused a significant decrease in the production of Nrf2, HO-1 and NQO1 compared to DEPs treatment, whereas the Nrf2 activator sulforaphane increased the LC3B (p = 0.020) levels. BEAS-2B cells exposed to DEP at a concentration of 50 μg/mL for 2 h showed a significant increase in the expression of LC3B (p = 0.001), p62 (p = 0.008), Nrf2 (p = 0.003), HO-1 (p = 0.001) and NQO1 (p = 0.015) genes compared to control. In siRNA-transfected cells, the LC3B (p < 0.001), p62 (p = 0.001) and Atg5 (p = 0.024) mRNA levels and the p62 and LC3II protein levels were decreased, indicating that Nrf2 modulated the expression of autophagy markers (R < 1). These results imply that, in bronchial cells exposed to DEP, the Nrf2 system positively regulates autophagy to maintain cellular homeostasis.

Highlights

Diesel exhaust particles (DEP) are known to generate reactive oxygen species in the respiratory system, triggering cells to activate antioxidant defence mechanisms, such as Kelch-like ECH-associated protein 1 (Keap1)-Nrf[2] signalling and autophagy
The results of the Trypan blue assay indicate that exposure to 50 μg/mL DEP significantly increased the percentage of dead cells compared to the number in the control (p = 0.007) and 10 μg/mL (p = 0.015) groups
The results indicate that DEP decrease cell viability and elicit the response of both biochemical pathways

Summary

Introduction

Diesel exhaust particles (DEP) are known to generate reactive oxygen species in the respiratory system, triggering cells to activate antioxidant defence mechanisms, such as Keap1-Nrf[2] signalling and autophagy. In siRNAtransfected cells, the LC3B (p < 0.001), p62 (p = 0.001) and Atg[5] (p = 0.024) mRNA levels and the p62 and LC3II protein levels were decreased, indicating that Nrf[2] modulated the expression of autophagy markers (R < 1). These results imply that, in bronchial cells exposed to DEP, the Nrf[2] system positively regulates autophagy to maintain cellular homeostasis. The transcription factor nuclear factor erythroid-derived 2-like 2 (NFE2L2, known as Nrf2) induces ARE genes as part of a protective response against oxidative challenge to organelles and macromolecules[21]

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