PDF(Pubmed)
Abstract：
Neutrophil extracellular traps (NETs) can cause acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) by inducing macrophage pyroptosis. The purpose of this study was to find out whether pretreatment of alpha-linolenic acid (ALA) could inhibit NETs-induced macrophage pyroptosis in sepsis-induced ALI/ARDS, as well as to identify which inflammasome is involved in this process.
LPS was instilled into the trachea to establish sepsis-induced ALI/ARDS in a mouse model. Lung injury was assessed by microscopic examination of lung tissue after hematoxylin and eosin staining, pathology score, and bronchoalveolar lavage fluid (BALF) total protein concentration. The level of NETs in lung tissue was detected by MPO-DNA ELISA. Purified NETs, extracted from peritoneal neutrophils, induced macrophage pyroptosis in vitro. Expression of pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC) and IL-1β in the lung tissue and bone marrow-derived macrophages (BMDMs) were determined by western blotting or ELISA. Specks of Pyrin/ASC were examined by confocal immunofluorescence microscopy. Mefv (Pyrin)-/- mice were used to study the role of Pyrin in the process of sepsis-induced ALI/ARDS.
ALA alleviated LPS-induced lung injury. ALA reduced the level of NETs, pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC), and IL-1β in the lung tissue of sepsis mice. In vitro, NETs increased the expression of pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC) and IL-1β significantly in BMDMs. Pyrin protein was found to be higher and form the inflammasome with ASC in NETs challenged-BMDMs. Knockout of Mefv (Pyrin) gene fully restored the increased expression of pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC) and IL-1β in vitro and in vivo. Lung injury was alleviated significantly in Mefv (Pyrin)-/- mice as well. ALA suppresses all the NETs-induced changes as mentioned above.
Our study is the first to demonstrate Pyrin inflammasome driving NETs-induced macrophage pyroptosis, and ALA may reduce ALI/ARDS by inhibiting the activation of the Pyrin inflammasome-driven macrophage pyroptosis.
LPS was instilled into the trachea to establish sepsis-induced ALI/ARDS in a mouse model. Lung injury was assessed by microscopic examination of lung tissue after hematoxylin and eosin staining, pathology score, and bronchoalveolar lavage fluid (BALF) total protein concentration. The level of NETs in lung tissue was detected by MPO-DNA ELISA. Purified NETs, extracted from peritoneal neutrophils, induced macrophage pyroptosis in vitro. Expression of pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC) and IL-1β in the lung tissue and bone marrow-derived macrophages (BMDMs) were determined by western blotting or ELISA. Specks of Pyrin/ASC were examined by confocal immunofluorescence microscopy. Mefv (Pyrin)-/- mice were used to study the role of Pyrin in the process of sepsis-induced ALI/ARDS.
ALA alleviated LPS-induced lung injury. ALA reduced the level of NETs, pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC), and IL-1β in the lung tissue of sepsis mice. In vitro, NETs increased the expression of pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC) and IL-1β significantly in BMDMs. Pyrin protein was found to be higher and form the inflammasome with ASC in NETs challenged-BMDMs. Knockout of Mefv (Pyrin) gene fully restored the increased expression of pyroptosis-related proteins (Cl-caspase-1, Cl-GSDMD, ASC) and IL-1β in vitro and in vivo. Lung injury was alleviated significantly in Mefv (Pyrin)-/- mice as well. ALA suppresses all the NETs-induced changes as mentioned above.
Our study is the first to demonstrate Pyrin inflammasome driving NETs-induced macrophage pyroptosis, and ALA may reduce ALI/ARDS by inhibiting the activation of the Pyrin inflammasome-driven macrophage pyroptosis.
摘要：
■中性粒细胞胞外诱捕网（NETs）可通过诱导巨噬细胞焦凋亡而引起急性肺损伤（ALI）/急性呼吸窘迫综合征（ARDS）。本研究的目的是发现α-亚麻酸（ALA）预处理是否可以抑制NETs诱导的脓毒症ALI/ARDS中巨噬细胞的焦亡。以及确定哪个炎症小体参与了这个过程。
■将LPS滴入气管以在小鼠模型中建立脓毒症诱导的ALI/ARDS。通过苏木精和曙红染色后对肺组织进行显微镜检查来评估肺损伤，病理学评分,支气管肺泡灌洗液（BALF）总蛋白浓度。MPO-DNAELISA法检测肺组织中NETs的含量。纯化NET,从腹膜中性粒细胞中提取，体外诱导巨噬细胞焦亡。焦亡相关蛋白的表达（Cl-caspase-1，Cl-GSDMD，通过蛋白质印迹或ELISA测定肺组织和骨髓源性巨噬细胞（BMDMs）中的ASC）和IL-1β。通过共聚焦免疫荧光显微镜检查了Pyrin/ASC的斑点。用Mefv(Pyrin)-/-小鼠研究Pyrin在脓毒症诱导的ALI/ARDS过程中的作用。
■ALA减轻LPS诱导的肺损伤。ALA降低了NET的水平，细胞凋亡相关蛋白（Cl-caspase-1，Cl-GSDMD，ASC)，和IL-1β在脓毒症小鼠肺组织中的表达。体外，NETs增加了焦亡相关蛋白的表达（Cl-caspase-1，Cl-GSDMD，ASC）和IL-1β显着在BMDMs中。在NET攻击的BMDMs中，Pyrin蛋白被发现更高，并与ASC形成炎症小体。Mefv（Pyrin）基因的敲除完全恢复了细胞凋亡相关蛋白的表达增加（Cl-caspase-1，Cl-GSDMD，ASC）和IL-1β在体外和体内。Mefv（Pyrin）-/-小鼠的肺损伤也明显减轻。ALA抑制如上所述的所有NET诱导的变化。
■我们的研究首次证明了Pyrin炎性体驱动NETs诱导的巨噬细胞焦亡，ALA可能通过抑制Pyrin炎性体驱动的巨噬细胞焦亡的激活来减少ALI/ARDS。
■将LPS滴入气管以在小鼠模型中建立脓毒症诱导的ALI/ARDS。通过苏木精和曙红染色后对肺组织进行显微镜检查来评估肺损伤，病理学评分,支气管肺泡灌洗液（BALF）总蛋白浓度。MPO-DNAELISA法检测肺组织中NETs的含量。纯化NET,从腹膜中性粒细胞中提取，体外诱导巨噬细胞焦亡。焦亡相关蛋白的表达（Cl-caspase-1，Cl-GSDMD，通过蛋白质印迹或ELISA测定肺组织和骨髓源性巨噬细胞（BMDMs）中的ASC）和IL-1β。通过共聚焦免疫荧光显微镜检查了Pyrin/ASC的斑点。用Mefv(Pyrin)-/-小鼠研究Pyrin在脓毒症诱导的ALI/ARDS过程中的作用。
■ALA减轻LPS诱导的肺损伤。ALA降低了NET的水平，细胞凋亡相关蛋白（Cl-caspase-1，Cl-GSDMD，ASC)，和IL-1β在脓毒症小鼠肺组织中的表达。体外，NETs增加了焦亡相关蛋白的表达（Cl-caspase-1，Cl-GSDMD，ASC）和IL-1β显着在BMDMs中。在NET攻击的BMDMs中，Pyrin蛋白被发现更高，并与ASC形成炎症小体。Mefv（Pyrin）基因的敲除完全恢复了细胞凋亡相关蛋白的表达增加（Cl-caspase-1，Cl-GSDMD，ASC）和IL-1β在体外和体内。Mefv（Pyrin）-/-小鼠的肺损伤也明显减轻。ALA抑制如上所述的所有NET诱导的变化。
■我们的研究首次证明了Pyrin炎性体驱动NETs诱导的巨噬细胞焦亡，ALA可能通过抑制Pyrin炎性体驱动的巨噬细胞焦亡的激活来减少ALI/ARDS。
