Department of Intensive Care Unit, The First Affiliated Hospital of Nanchang University, No. 17 Yongwaizheng Street, Dong Lake District, Nanchang, 330000, Jiangxi Province, China.
Medical Innovation Center, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province, China.
Inflamm Res. 2023 Feb;72(2):329-346. doi: 10.1007/s00011-022-01675-y. Epub 2022 Dec 20.
The present study was designed to explore the potential regulatory mechanism between mitophagy and pyroptosis during sepsis-associated acute lung injury (ALI).
In vitro or in vivo models of sepsis-associated ALI were established by administering lipopolysaccharide (LPS) or performing caecal ligation and puncture (CLP) surgery. Pyroptosis levels were detected by electron microscopy, immunofluorescence, flow cytometry, western blotting and immunohistochemistry. Dual-luciferase reporter gene assay was applied to verify the targeting relationship between miR-138-5p and NLRP3. Methylation-specific PCR and chromatin immunoprecipitation assays were used to determine methylation of the miR-138-5p promoter. Mitophagy levels were examined by transmission electron microscopy and western blotting.
NLRP3 inflammasome silencing alleviated alveolar macrophage (AM) pyroptosis and septic lung injury. In addition, we confirmed the direct targeting relationship between miR-138-5p and NLRP3. Overexpressed miR-138-5p alleviated AM pyroptosis and the pulmonary inflammatory response. Moreover, the decreased expression of miR-138-5p was confirmed to depend on promoter methylation, while inhibition of miR-138-5p promoter methylation attenuated AM pyroptosis and pulmonary inflammation. Here, we discovered that an increased cytoplasmic mtDNA content in sepsis-induced ALI models induced the methylation of the miR-138-5p promoter, thereby decreasing miR-138-5p expression, which may activate the NLRP3 inflammasome and trigger AM pyroptosis. Mitophagy, a form of selective autophagy that clears damaged mitochondria, reduced cytoplasmic mtDNA levels. Furthermore, enhanced mitophagy might suppress miR-138-5p promoter methylation and relieve the pulmonary inflammatory response, changes that were reversed by treatment with isolated mtDNA.
In summary, our study indicated that mitophagy induced the demethylation of the miR-138-5p promoter, which may subsequently inhibit NLRP3 inflammasome, AM pyroptosis and inflammation in sepsis-induced lung injury. These findings may provide a promising therapeutic target for sepsis-associated ALI.
本研究旨在探讨脓毒症相关急性肺损伤（ALI）中自噬与细胞焦亡之间的潜在调控机制。
通过给予脂多糖（LPS）或进行盲肠结扎穿孔（CLP）手术，建立脓毒症相关 ALI 的体外或体内模型。通过电子显微镜、免疫荧光、流式细胞术、Western blot 和免疫组织化学检测细胞焦亡水平。应用双荧光素酶报告基因实验验证 miR-138-5p 与 NLRP3 之间的靶向关系。采用甲基化特异性 PCR 和染色质免疫沉淀实验确定 miR-138-5p 启动子的甲基化。通过透射电镜和 Western blot 检测自噬水平。
NLRP3 炎性小体沉默减轻肺泡巨噬细胞（AM）焦亡和脓毒症肺损伤。此外，我们证实了 miR-138-5p 与 NLRP3 之间的直接靶向关系。过表达 miR-138-5p 减轻 AM 细胞焦亡和肺部炎症反应。此外，下调 miR-138-5p 的表达被证实依赖于启动子甲基化，而抑制 miR-138-5p 启动子甲基化可减轻 AM 细胞焦亡和肺炎症。在这里，我们发现脓毒症诱导的 ALI 模型中细胞质 mtDNA 含量增加诱导 miR-138-5p 启动子的甲基化，从而降低 miR-138-5p 的表达，可能激活 NLRP3 炎性小体并触发 AM 细胞焦亡。自噬是一种选择性清除受损线粒体的自噬形式，可降低细胞质 mtDNA 水平。此外，增强自噬可能抑制 miR-138-5p 启动子甲基化并缓解肺部炎症反应，而用分离的 mtDNA 处理则可逆转这些变化。
综上所述，我们的研究表明，自噬诱导 miR-138-5p 启动子去甲基化，进而可能抑制 NLRP3 炎性小体、AM 细胞焦亡和脓毒症肺损伤中的炎症。这些发现可能为脓毒症相关 ALI 提供有希望的治疗靶点。
