The role and mechanism of autophagy in lipopolysaccharide-induced inflammatory response of A549 cells.
10.7499/j.issn.1008-8830.2202135
- Author:
Jia SHI
1
;
Hui-Xian TAO
;
Yan GUO
1
;
Yun-Su ZOU
1
;
Mu-Zi WANG
1
;
Zhi-Tao LU
;
Yi-Fang DING
;
Wei-Dong XU
;
Xiao-Guang ZHOU
1
Author Information
1. Neonatal Medical Center, Children's Hospital of Nanjing Medical University, Nanjing 210008, China.
- Publication Type:Journal Article
- Keywords:
Autophagy;
Human alveolar epithelial A549 cell;
Inflammatory response;
Lipopolysaccharide
- MeSH:
Humans;
A549 Cells;
Autophagy;
Beclin-1/metabolism*;
Caspase 1/metabolism*;
Inflammation;
Lipopolysaccharides/pharmacology*;
NLR Family, Pyrin Domain-Containing 3 Protein/metabolism*;
Toll-Like Receptor 4/metabolism*
- From:
Chinese Journal of Contemporary Pediatrics
2022;24(10):1161-1170
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVES:To study the role and mechanism of autophagy in lipopolysaccharide (LPS)-induced inflammatory response of human alveolar epithelial A549 cells.
METHODS:A549 cells were stimulated with LPS to establish a cell model of inflammatory response, and were then grouped (n=3 each) by concentration (0, 1, 5, and 10 μg/mL) and time (0, 4, 8, 12, and 24 hours). The A549 cells were treated with autophagy inhibitor 3-methyladenine (3-MA) to be divided into four groups (n=3 each): control, LPS, 3-MA, and 3-MA+LPS. The A549 cells were treated with autophagy agonist rapamycin (RAPA) to be divided into four groups (n=3 each): control, LPS, RAPA, and RAPA+LPS. The A549 cells were transfected with the Toll-like receptor 4 (TLR4) overexpression plasmid to be divided into four groups (n=3 each): TLR4 overexpression control, TLR4 overexpression, TLR4 overexpression control+LPS, and TLR4 overexpression+LPS. The A549 cells were transfected with TLR4 siRNA to be divided into four groups (n=3 each): TLR4 silencing control,TLR4 silencing, TLR4 silencing control+LPS, and TLR4 silencing+LPS. CCK-8 assay was used to measure cell viability. Western blot was used to measure the protein expression levels of inflammatory indicators (NLRP3, Caspase-1, and ASC), autophagic indicators (LC3B, Beclin-1, and P62), and TLR4.
RESULTS:After stimulation with 1 μg/mL LPS for 12 hours, the levels of inflammatory indicators (NLRP3, Caspase-1, and ASC), autophagic indicators (LC3B, Beclin-1, and P62), and TLR4 increased and reached the peak (P<0.05). Compared with the LPS group, the 3-MA+LPS group had reduced expression of autophagy-related proteins and increased expression of inflammation-related proteins and TLR4, while the RAPA+LPS group had increased expression of autophagy-related proteins and reduced inflammation-related proteins and TLR4 (P<0.05). The TLR4 overexpression+LPS group had reduced autophagy-related proteins and increased inflammation-related proteins compared with the TLR4 overexpression control+LPS group, and the TLR4 silencing+LPS group had increased autophagy-related proteins and reduced inflammation-related proteins compared with the TLR4 silencing control+LPS group (P<0.05).
CONCLUSIONS:In the LPS-induced inflammatory response of human alveolar epithelial A549 cells, autophagic flux has a certain protective effect on A549 cells. TLR4-mediated autophagic flux negatively regulates the LPS-induced inflammatory response of A549 cells.
