Intensive Care Unit, Children's Hospital of Soochow University, Suzhou, Jiangsu Province, People's Republic of China.
Department of Pediatric Surgery, Children's Hospital of Soochow University, Suzhou, Jiangsu Province, People's Republic of China.
Infect Immun. 2023 Jan 24;91(1):e0046522. doi: 10.1128/iai.00465-22. Epub 2022 Nov 30.
Acute lung injury (ALI) caused by sepsis is a common respiratory critical illness with high morbidity and mortality. Protein kinase C-alpha (PRKCA) plays a protective role in sepsis-induced ALI. However, the detailed molecular mechanism of PRKCA in ALI caused by sepsis is unclear. Animal and cell models of sepsis were established by cecal ligation and puncture (CLP)-surgery and lipopolysaccharide (LPS)/interferon-gamma (IFN-γ) treatment, respectively. Lentivirus transfection was used to overexpress PRKCA. H&E staining and lung injury in CLP-surgery mice were evaluated. Gene expression was evaluated using qPCR and Western blotting. The expression of TNF-α, IL-1β, and IL-6 was examined using qPCR and ELISA. The expression of LC3 and TOM20 was evaluated using immunofluorescence assays. Cell apoptosis was assessed using a flow cytometry assay. The bond between miR-15a-5p and PDK4 was confirmed by dual-luciferase reporter gene and RNA immunoprecipitation assays. and in vitro, PRKCA overexpression reduced lung injury to prompt mitophagy and inhibit the inflammatory response, ROS production, and cell apoptosis. miR-15a-5p was highly expressed in macrophages treated with LPS/IFN-γ and was negatively mediated by PRKCA. The overexpression of miR-15a-5p reduced the effects of PRKCA upregulation in macrophages. miR-15a-5p could restrain mitophagy in LPS/IFN-γ-treated macrophages by directly targeting PDK4. Furthermore, PDK4 knockdown reversed the inhibition of cell apoptosis and inflammatory factor release caused by miR-15a-5p silencing. The PRKCA/miR-15a-5p/PDK4 axis alleviated ALI caused by sepsis by promoting mitophagy and repressing anti-inflammatory response.
脓毒症引起的急性肺损伤 (ALI) 是一种常见的呼吸系统危重症，具有高发病率和死亡率。蛋白激酶 C-α (PRKCA) 在脓毒症诱导的 ALI 中发挥保护作用。然而，PRKCA 在脓毒症引起的 ALI 中的详细分子机制尚不清楚。通过盲肠结扎穿孔术 (CLP) 手术和脂多糖 (LPS)/干扰素-γ (IFN-γ) 处理分别建立脓毒症动物和细胞模型，并使用慢病毒转染过表达 PRKCA。通过 qPCR 和 Western blot 评估 CLP 手术小鼠的 H&E 染色和肺损伤。使用 qPCR 和 ELISA 检测基因表达。通过 qPCR 和 ELISA 检测 TNF-α、IL-1β 和 IL-6 的表达。通过免疫荧光测定法评估 LC3 和 TOM20 的表达。通过流式细胞术评估细胞凋亡。通过双荧光素酶报告基因和 RNA 免疫沉淀测定证实 miR-15a-5p 与 PDK4 之间的结合。体内外实验均表明，PRKCA 过表达可减轻肺损伤，促进噬线粒体，并抑制炎症反应、ROS 产生和细胞凋亡。miR-15a-5p 在 LPS/IFN-γ 处理的巨噬细胞中高表达，并受 PRKCA 负调控。miR-15a-5p 的过表达可减弱巨噬细胞中 PRKCA 上调的作用。miR-15a-5p 可通过直接靶向 PDK4 抑制 LPS/IFN-γ 处理的巨噬细胞中的噬线粒体。此外，PDK4 敲低可逆转 miR-15a-5p 沉默引起的细胞凋亡和炎症因子释放的抑制作用。PRKCA/miR-15a-5p/PDK4 轴通过促进噬线粒体和抑制抗炎反应来缓解脓毒症引起的 ALI。
