摘要: |
目的 探讨纤溶酶原激活物抑制物-1(plasminogen activator inhibitor-1,PAI-1)对细菌脂多糖(lipopolysaccharide,LPS)诱导的肺泡Ⅱ型上皮细胞(alveolar epithelial type Ⅱ cell,AEC Ⅱ)损伤的调控作用及机制。方法 采用免疫印迹法和ELISA检测LPS处理后原代胎鼠AEC Ⅱ细胞内、外PAI-1的表达量。通过CCK8和细胞迁移实验(Transwell),用PAI-1重组蛋白和PAI-039(PAI-1的抑制剂)检测PAI-1对中性粒细胞活力、趋化的影响。收集Transwell下室的细胞培养液和AEC Ⅱ,采用ELISA检测肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素1β(interleukin-1β,IL-1β)、白细胞介素6(interleukin-6,IL-6)、髓过氧化物酶(myeloperoxidase,MPO)、基质金属蛋白酶-1(matrix metalloproteinase-1,MMP-1)和血小板/内皮黏附因子(platelet/endothelial cell adhesion molecule,PECAM)等促炎性因子水平。采用免疫印迹法检测AEC Ⅱ内STAT3 Tyr705和Ser727位点的磷酸化情况。同时通过台盼蓝染色观察AEC Ⅱ细胞损伤程度。用STAT3磷酸化位点抑制剂抑制其磷酸化后,观察LPS刺激后AEC Ⅱ的损伤程度。结果 LPS显著促进AEC Ⅱ死亡,促进PAI-1表达及释放(P<0.01);PAI-1重组蛋白能抑制中性粒细胞死亡,并促进其迁移和释放IL-6(P<0.001),使AEC Ⅱ中STAT3 Tyr705位点的磷酸化水平升高。分别用PAI-039和AZD1480抑制PAI-1的表达及JAK激活时,STAT3的Tyr705和Ser727位点磷酸化水平受到抑制,LPS诱导的AEC Ⅱ损伤缓解。结论 PAI-1能招募中性粒细胞促进其释放大量IL-6,提高JAK介导的STAT3 Tyr705位点的磷酸化水平,进而促进LPS诱导的AEC Ⅱ损伤。 |
关键词: 纤溶酶原激活物抑制物-1 中性粒细胞 肺泡Ⅱ型上皮细胞 脂多糖 IL6/JAK/STAT3通路 |
DOI:10.12025/j.issn.1008-6358.2022.20220507 |
分类号:R563 |
基金项目:国家自然科学基金(81800077),上海市卫生健康委员会科研课题计划项目(20204Y0082). |
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PAI-1 recruits neutrophil enrichments to promote LPS-induced lung cells injury through IL6/STAT3 signaling pathway |
DOU Mao-sen1, JIN Wen-ting1, SONG Yuan-lin2, PAN Jue1
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1.Department of Infectious Disease, Zhongshan Hospital, Fudan University, Shanghai 200032, China;2.Department of Pulmonary and Critical Care Medicine, Zhongshan Hospital, Fudan University, Shanghai 200032, China
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Abstract: |
Objective To explore the role and mechanism of plasminogen activator inhibitor-1 (PAI-1) in lipopolysaccharide (LPS)-induced injury of alveolar epithelial type Ⅱ cells (AEC Ⅱ). Methods The expression of intracellular and extracellular PAI-1 was measured by Western blotting and ELISA after LPS treatment of AEC Ⅱ. The effects of PAI-1 on neutrophil viability and chemotaxis were examined using PAI-1 recombinant protein and PAI-039 (an inhibitor of PAI-1) by CCK8 and Transwell assays. Pro-inflammatory factors including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), myeloperoxidase (MPO), matrix metalloproteinase-1 (MMP-1) and platelet/endothelial cell adhesion molecule (PECAM) were detected as well as phosphorylation of STAT3 Tyr705 and Ser727 at AEC Ⅱ. The extent of cell damage of AEC Ⅱ after LPS challenge with or without JAK inhibitor was observed using Trypan blue staining. Results LPS significantly promoted the death of AEC Ⅱ and announced the expression and release of PAI-1 (P<0.01). Moreover, PAI-1 recombinant protein inhibited the death of neutrophils, promoted their migration with IL-6 releasing (P<0.001), and increased the phosphorylation level of STAT3 Tyr305 in AEC Ⅱ. When PAI-039 and AZD1480 were used to inhibit the expression of PAI-1 and activation of JAK, respectively, the phosphorylation level of STAT3 was suppressed, and LPS-induced AEC Ⅱ injury alleviated. Conclusion PAI-1 recruits neutrophils to release large amounts of IL-6 and increases JAK-mediated phosphorylation level at the STAT3 Tyr705 site to promote LPS-induced AECⅡ injury. |
Key words: plasminogen activator inhibitor-1 neutrophils alveolar type Ⅱ epithelial cell lipopolysaccharide IL6/JAK/STAT3 pathway |