线粒体自噬对肺泡巨噬细胞焦亡的调控作用及其机制

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

目的

初步探讨线粒体自噬对肺泡巨噬细胞（AM）焦亡的调控作用及其机制。

方法

选取大鼠肺泡巨噬细胞NR8383作为研究对象，使用1 μg/ml的脂多糖（LPS）刺激18 h构建脓毒症肺损伤体外模型，使用低浓度溴化乙锭（EtBr）处理大鼠AM构建胞质无线粒体DNA（mtDNA）的ρ0细胞，使用羟基氰氯苯腙（CCCP）增强线粒体自噬，通过转染上调胞质mtDNA水平。设立CON组、LPS组、LPS+ρ0组、LPS+CCCP组、LPS+CCCP+mtDNA组；采用qRT-PCR检测mtDNA拷贝数、胞质mtDNA水平；通过酶联免疫吸附测定（ELISA）检测细胞培养上清液中白介素（IL）-18、IL-1β等炎症因子的分泌水平；采用Western Blot检测NOD样受体热蛋白结构域相关蛋白3（NLRP3）、胱天蛋白酶1前体（pro-caspase-1）、胱天蛋白酶1剪切体（P20）、消皮素D（GSDMD）和消皮素D剪切体（cle-GSDMD）等焦亡相关蛋白的表达水平；采用免疫荧光观察细胞内caspase活性及细胞膜通透性；采用透射电镜观察细胞内线粒体自噬水平；采用Western Blot检测微管相关蛋白轻链B（LC3B）Ⅱ型、LC3BⅠ型等自噬相关蛋白表达水平。

结果

LPS刺激可诱导mtDNA释放至胞质，并促进炎症因子水平、焦亡相关蛋白表达水平、细胞内caspase活性及细胞膜通透性显著升高。通过CCCP增强线粒体自噬可抑制LPS诱导的IL-18（P＜0.01）、IL-1β（P＜0.01）等炎症因子及NLRP3（P＜0.001）、P20（P＜0.05）、cle-GSDMD（P＜0.01）等焦亡相关蛋白表达水平的上调，并抑制caspase活化及细胞膜透化。而转染外源性mtDNA可逆转CCCP的作用，诱导IL-18（P＜0.05）、IL-1β（P＜0.01）等炎症因子及NLRP3（P＜0.001）、P20（P＜0.05）、cle-GSDMD（P＜0.05）等焦亡相关蛋白表达水平升高，并活化caspase，增加细胞膜通透性。

结论

增强线粒体自噬可通过降低胞质mtDNA水平抑制LPS诱导的大鼠AM焦亡，减轻炎症反应。

关键词: 脓毒症肺损伤, 肺泡巨噬细胞, 线粒体自噬, 焦亡

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

表1 qRT-PCR引物序列

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

表1 qRT-PCR引物序列

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

表2 Western Blot相关抗体

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

表2 Western Blot相关抗体

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

图1 LPS通过促进mtDNA释放至胞质诱导AM炎症反应。图a：qRT-PCR检测大鼠AM内的mtDNA拷贝数；图b：qRT-PCR检测大鼠AM内胞质mtDNA水平；图c：ELISA检测细胞上清液中IL-18、IL-1β的分泌水平。**P＜0.01，***P＜0.001；n=3注：CON为空白对照；EtBr为溴化乙锭；LPS为脂多糖；mtDNA为线粒体DNA；AM为肺泡巨噬细胞；ρ0为胞质无线粒体DNA的肺泡巨噬细胞；qRT-PCR为实时荧光反转录聚合酶链反应；ELISA为酶联免疫吸附测定；IL-18为白介素18；IL-1β为白介素1β

图2 胞质mtDNA通过诱导AM焦亡加重炎症反应。图a、b：Western Blot检测NLRP3、pro-caspase-1、P20、GSDMD和cle-GSDMD的蛋白表达及水平；图c：大鼠AM的FAM-FLICA caspase荧光显微镜分析，放大倍数×200，比例尺=100 μm。*P＜0.05，**P＜0.01，***P＜0.001；n=3注：CON为空白对照；LPS为脂多糖；mtDNA为线粒体DNA；AM为肺泡巨噬细胞；ρ0为胞质无线粒体DNA的肺泡巨噬细胞；Western Blot为蛋白质免疫印迹实验；NLRP3为NOD样受体蛋白热蛋白结构域相关蛋白3；pro-caspase-1为胱天蛋白酶1前体；P20为胱天蛋白酶1剪切体；GSDMD为消皮素D；cle-GSDMD为消皮素D剪切体；GAPDH为甘油醛-3-磷酸脱氢酶；Hoechst为细胞核染料；FAM-FLICA为有活性caspase的荧光探针；PI为碘化丙啶

图3 透射电镜下大鼠AM的线粒体形态及CCCP对线粒体自噬的影响。图a：透射电镜下大鼠AM线粒体形态及MP、ML形成情况；黑色箭头代表细胞核，蓝色箭头代表正常线粒体，红色单箭头表示MP，是受损线粒体被早期自噬体包裹所形成，红色双箭头表示ML，是中期线粒体自噬体与溶酶体融合后，所形成的成熟线粒体自噬溶酶体；放大倍数×10 000。图b、c：Western Blot检测大鼠AM内LC3BⅡ、LC3BⅠ的蛋白表达及水平；*P＜0.05，**P＜0.01；n=3注：CON为空白对照组；LPS为脂多糖；CCCP为羟基氰氯苯腙；AM为肺泡巨噬细胞；MP为线粒体自噬体；ML为线粒体自噬溶酶体；Western Blot为蛋白质免疫印迹实验；LC3BⅡ为微管相关蛋白轻链BⅡ；LC3BⅠ为微管相关蛋白轻链BⅠ；GAPDH为甘油醛-3-磷酸脱氢酶

图4 线粒体自噬通过降低胞质mtDNA水平抑制LPS诱导的AM焦亡。图a：qRT-PCR检测大鼠AM内胞质mtDNA水平；图b：ELISA检测细胞上清液中IL-18、IL-1β的分泌水平；图c、d：Western Blot检测NLRP3、pro-caspase-1、P20、GSDMD和cle-GSDMD的蛋白表达水平；图e：大鼠AM的FAM-FLICA caspase荧光显微镜分析，放大倍数×200，比例尺=100 μm。*P＜0.05，**P＜0.01，***P＜0.001；n=3注：CON为空白对照；LPS为脂多糖；CCCP为羟基氰氯苯腙；mtDNA为线粒体DNA；AM为肺泡巨噬细胞；qRT-PCR为实时荧光反转录聚合酶链反应；ELISA为酶联免疫吸附测定；IL-18为白介素18；IL-1β为白介素1β；Western Blot为蛋白质免疫印迹实验；NLRP3为NOD样受体蛋白热蛋白结构域相关蛋白3；pro-caspase-1为胱天蛋白酶1前体；P20为胱天蛋白酶1剪切体；GSDMD为消皮素D；cle-GSDMD为消皮素D剪切体；GAPDH为甘油醛-3-磷酸脱氢酶；Hoechst为细胞核染料；FAM-FLICA为有活性caspase的荧光探针；PI为碘化丙啶

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Mitophagy inhibits alveolar macrophages pyroptosis by reducing cytosolic mtDNA level

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Mitophagy inhibits alveolar macrophages pyroptosis by reducing cytosolic mtDNA level