Department of Pulmonary and Critical Care Medicine, Hunan Provincial People's Hospital (The First Affiliated Hospital of Hunan Normal University), Changsha, Hunan 410016, China.
Department of Pulmonary and Critical Care Medicine, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China.
Oxid Med Cell Longev. 2023 Feb 2;2023:1493684. doi: 10.1155/2023/1493684. eCollection 2023.
Patients with severe asthma respond poorly to corticosteroids, and their care accounts for more than 60% of the total costs attributed to asthma. Neutrophils form neutrophil extracellular traps (NETs), which play a crucial role in severe asthma. Statins have shown anti-inflammatory effects by reducing NETosis. In this study, we investigate if simvastatin can attenuate severe asthma by reducing NETosis and the underlying mechanism.
Mice were concomitantly sensitized with ovalbumin (OVA), house dust mite (HDM), and lipopolysaccharide (LPS) during sensitization to establish a mouse model of severe asthma with neutrophil predominant inflammation (OVA+LPS mice) and treated with or without simvastatin. In inflammatory response, proportions of Th2, Th17, and Treg cells in lung tissue were detected by flow cytometry, and the levels of cytokines, dsDNA, and MPO-DNA in bronchoalveolar lavage fluid (BALF) were analyzed by ELISA. Citrullinated histone H3 (CitH3) and peptidyl arginine deiminase 4 (PAD4) in lung tissue were determined by Western blot and immunofluorescence imaging. PAD4 mRNA was determined by quantitative PCR (qPCR). HL-60 cells were differentiated into neutrophil-like cells by 1.25% DMSO. The neutrophil-like cells were treated with or without LPS, and simvastatin was then stimulated with PMA. CitH3 and PAD4 expressions were determined.
Sensitization with OVA, HDM, and LPS resulted in neutrophilic inflammation and the formation of NETs in the lungs. Simvastatin treatment reduced the inflammation score, cytokine levels, total cells, and neutrophil counts in the BALF and reduced proportions of Th2 and Th17 but increased Treg cells in lungs of OVA+LPS mice. Simvastatin-treated OVA+LPS mice show reduced NET formation in BALF and lung tissue compared to control mice. Adoptive transfer of neutrophils was sufficient to restore NETosis and neutrophilic inflammation in simvastatin-treated OVA+LPS mice. Simvastatin reduced PAD4 mRNA and protein expression in lung tissues and neutrophils isolated from lungs of OVA+LPS mice and consequent NET formation. In vitro, simvastatin reduced LPS-induced PAD4 upregulation and NETosis in HL-60-differentiated neutrophil-like cells. Furthermore, PAD4-overexpressed lentiviral transduction was sufficient to restore PAD4 protein expression and NETosis in simvastatin-treated HL-60-differentiated neutrophil-like cells.
Simvastatin reduces Th17-mediated neutrophilic inflammation and airway hyperreactivity by reducing PAD4 expression and inhibiting NETosis in a mouse model of severe asthma. Severe asthmatic patients with high levels of circulating NETs or sputum NETs may show improved responses to statin treatment.
严重哮喘患者对皮质类固醇反应不佳，其治疗费用占哮喘总费用的 60%以上。中性粒细胞形成中性粒细胞细胞外诱捕网（NETs），在严重哮喘中发挥关键作用。他汀类药物通过减少 NETosis 显示出抗炎作用。在这项研究中，我们研究了辛伐他汀是否可以通过减少 NETosis 来减轻严重哮喘，并探讨其潜在机制。
在致敏过程中，用卵清蛋白（OVA）、屋尘螨（HDM）和脂多糖（LPS）同时致敏，建立中性粒细胞为主的炎症的严重哮喘小鼠模型（OVA+LPS 小鼠），并用或不用辛伐他汀治疗。在炎症反应中，通过流式细胞术检测肺组织中 Th2、Th17 和 Treg 细胞的比例，通过 ELISA 分析支气管肺泡灌洗液（BALF）中细胞因子、dsDNA 和 MPO-DNA 的水平。通过 Western blot 和免疫荧光成像检测肺组织中瓜氨酸化组蛋白 H3（CitH3）和肽基精氨酸脱亚氨酶 4（PAD4）。通过定量 PCR（qPCR）测定 PAD4 mRNA。用 1.25% DMSO 将 HL-60 细胞分化为中性粒细胞样细胞。用 LPS 处理中性粒细胞样细胞，然后用 PMA 刺激辛伐他汀。检测 CitH3 和 PAD4 的表达。
OVA、HDM 和 LPS 致敏导致肺部中性粒细胞炎症和 NET 形成。辛伐他汀治疗降低了 OVA+LPS 小鼠肺部的炎症评分、细胞因子水平、总细胞数和中性粒细胞计数，并降低了 Th2 和 Th17 的比例，但增加了 Treg 细胞。与对照组小鼠相比，辛伐他汀治疗的 OVA+LPS 小鼠 BALF 和肺组织中的 NET 形成减少。在辛伐他汀治疗的 OVA+LPS 小鼠中，过继转移中性粒细胞足以恢复 NETosis 和中性粒细胞炎症。辛伐他汀降低了 OVA+LPS 小鼠肺组织和肺组织中 PAD4 mRNA 和蛋白表达，以及由此产生的 NET 形成。在体外，辛伐他汀降低了 LPS 诱导的 HL-60 分化的中性粒细胞样细胞中 PAD4 的上调和 NETosis。此外，过表达 PAD4 的慢病毒转导足以恢复辛伐他汀治疗的 HL-60 分化的中性粒细胞样细胞中的 PAD4 蛋白表达和 NETosis。
辛伐他汀通过降低 PAD4 表达和抑制严重哮喘小鼠模型中的 NETosis，减轻 Th17 介导的中性粒细胞炎症和气道高反应性。循环 NETs 或痰 NETs 水平高的严重哮喘患者可能对他汀类药物治疗有更好的反应。
