Abstract：Objective 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.
SHI Jia,TAO Hui-Xian,GUO Yan et al. The role and mechanism of autophagy in lipopolysaccharide-induced inflammatory response of A549 cells[J]. CJCP, 2022, 24(10): 1161-1170.
Fukatsu M, Ohkawara H, Wang X, et al. The suppressive effects of Mer inhibition on inflammatory responses in the pathogenesis of LPS-induced ALI/ARDS[J]. Sci Signal, 2022, 15(724): eabd2533. PMID: 35258998. DOI: 10.1126/scisignal.abd2533.
Shi X, An X, Yang L, et al. Reticulocalbin 3 deficiency in alveolar epithelium attenuated LPS-induced ALI via NF-κB signaling[J]. Am J Physiol Lung Cell Mol Physiol, 2021, 320(4): L627-L639. PMID: 33625944. DOI: 10.1152/ajplung.00526.2020.
Hu Q, Hao C, Tang S. From sepsis to acute respiratory distress syndrome (ARDS): emerging preventive strategies based on molecular and genetic researches[J]. Biosci Rep, 2020, 40(5): BSR20200830. PMID: 32319516. PMCID: PMC7199454. DOI: 10.1042/BSR20200830.
Zhang Z, Chen Z, Liu R, et al. Bcl-2 proteins regulate mitophagy in lipopolysaccharide-induced acute lung injury via PINK1/parkin signaling pathway[J]. Oxid Med Cell Longev, 2020, 2020: 6579696. PMID: 32148654. PMCID: PMC7054785. DOI: 10.1155/2020/6579696.
Li NN, Cao T, Yu F, et al. MiR-520c-3p alleviates LPS-induced A549 cell and mice lung injury via targeting NLRC5[J]. Pharmazie, 2020, 75(6): 275-278. PMID: 32539925. DOI: 10.1692/ph.2020.0355.
Lamark T, Svenning S, Johansen T. Regulation of selective autophagy: the p62/SQSTM1 paradigm[J]. Essays Biochem, 2017, 61(6): 609-624. PMID: 29233872. DOI: 10.1042/EBC20170035.
Bi X, Li J, Fan X, et al. GSTP1 inhibits LPS-induced inflammatory response through regulating autophagy in THP-1 cells[J]. Inflammation, 2020, 43(3): 1157-1169. PMID: 32128658. DOI: 10.1007/s10753-020-01202-3.
Kim S, Lee JY, Shin SG, et al. ESRRA (estrogen related receptor alpha) is a critical regulator of intestinal homeostasis through activation of autophagic flux via gut microbiota[J]. Autophagy, 2021, 17(10): 2856-2875. PMID: 33172329. PMCID: PMC8525935. DOI: 10.1080/15548627.2020.1847460.
Farré-Alins V, Narros-Fernández P, Palomino-Antolín A, et al. Melatonin reduces NLRP3 inflammasome activation by increasing α7 nAChR-mediated autophagic flux[J]. Antioxidants (Basel), 2020, 9(12):1299. PMID: 33353046. PMCID: PMC7767051. DOI: 10.3390/antiox9121299.
