Mitophagy inhibits alveolar macrophages pyroptosis by reducing cytosolic mtDNA level

doi:10.3877/cma.j.issn.2096-1537.2023.01.012

Abstract

Abstract:

Objective

To preliminarily investigate the regulatory effect and mechanism of mitophagy on alveolar macrophages (AM) pyroptosis.

Methods

Rat AM NR8383 was selected to construct in vitro model of sepsis-associated acute lung injury (ALI) by stimulation with 1 μg/ml lipopolysaccharide (LPS) for 18 h. Rat AM was treated with low concentration of ethidium bromide (EtBr) to construct cytosolic mitochondrial DNA (mtDNA)-free ρ0 cells. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used to enhance mitophagy, and cytosolic mtDNA levels were up-regulated by transfection. CON group, LPS group, LPS+ρ0 group, LPS+CCCP group, LPS+CCCP+mtDNA group were set up. mtDNA copy number and cytosolic mtDNA level were detected by qRT-PCR. The secretion levels of inflammatory cytokines interleukin-1β (IL-18) and interleukin-18 (IL-1β) in cell culture supernatant were examined by ELISA. The expression levels of pyroptosis-related proteins NOD-like receptor thermal protein domain associated protein 3 (NLRP3), pro-cysteiny l aspartate-specific protease (pro-caspase-1), P20, gasdermin D (GSDMD) and cle-GSDMD were detected by Western Blot. The activity of caspase and the permeability of cell membrane were observed by immunofluorescence. The level of mitophagy was observed by transmission electron microscopy. The expression levels of mitophagy-related proteins such as light chain 3B (LC3B)Ⅱ and LC3BⅠ were determined by Western Blot.

Results

LPS stimulation could induce mtDNA release into the cytoplasm, and promote the levels of inflammatory factors, pyroptosis related protein expression, intracellular caspase activity and cell membrane permeability. The enhancement of mitophagy by CCCP could inhibit LPS-induced up-regulation of inflammatory factors such as IL-18 (P＜0.01) and IL-1β (P＜0.01) and pyroptosis related proteins such as NLRP3 (P＜0.001), P20 (P＜0.05) and Cle-GSDMD (P＜0.01), and inhibit caspase activation and cell membrane permeability. Transfection of exogenous mtDNA could reverse the effect of CCCP, induce the expression levels of inflammatory factors such as IL-18 (P＜0.05) and IL-1β (P＜0.01), and pyroptosis related proteins such as NLRP3 (P＜0.001), P20 (P＜0.05) and Cle-GSDMD (P＜0.05), activate caspase, and increase cell membrane permeability.

Conclusion

Enhancing mitophagy can inhibit LPS-induced AM pyroptosis and inflammation in rats by decreasing cytosolic mtDNA level.

Key words: Acute lung injury, Alveolar macrophages, Mitophagy, Pyroptosis

Ying Yang, Yamei Cui, Qiang Shao, Ning Zhao, Wenqiang Tao, Jiaquan Chen, Zeyao Xu, Kejian Qian, Fen Liu. Mitophagy inhibits alveolar macrophages pyroptosis by reducing cytosolic mtDNA level[J]. Chinese Journal of Critical Care & Intensive Care Medicine(Electronic Edition), 2023, 09(01): 69-77.

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URL: https://icu.cma-cmc.com.cn/EN/10.3877/cma.j.issn.2096-1537.2023.01.012

https://icu.cma-cmc.com.cn/EN/Y2023/V09/I01/69

Figures/Tables 6

References 22

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基因	引物序列（5’-3’）
线粒体基因（Mt-cyb）	正向：GCAGCTTAACATTCCGCCCAATCA
线粒体基因（Mt-cyb）	反向：TACTGGTTGGCCTCCGATTCATGT
核基因（Actb）	正向：ATCATGTTTGAGACCTTCAACACCC
核基因（Actb）	反向：CATCTCTTGCTCGAAGTCTAGG

基因	引物序列（5’-3’）
线粒体基因（Mt-cyb）	正向：GCAGCTTAACATTCCGCCCAATCA
线粒体基因（Mt-cyb）	反向：TACTGGTTGGCCTCCGATTCATGT
核基因（Actb）	正向：ATCATGTTTGAGACCTTCAACACCC
核基因（Actb）	反向：CATCTCTTGCTCGAAGTCTAGG

抗体	浓度	货号
NLRP3	1∶1000	ab263899
caspase-1	1∶1000	22915-1-AP
cle-GSDMD	1∶1000	39754
LC3	1∶2000	ab192890
GAPDH	1∶10 000	10494-1-AP
辣根过氧化物酶标记山羊抗兔IgG二抗	1∶20 000	SA00001-2

抗体	浓度	货号
NLRP3	1∶1000	ab263899
caspase-1	1∶1000	22915-1-AP
cle-GSDMD	1∶1000	39754
LC3	1∶2000	ab192890
GAPDH	1∶10 000	10494-1-AP
辣根过氧化物酶标记山羊抗兔IgG二抗	1∶20 000	SA00001-2

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