背景:使用挽救生命的机械通气(MV)可能会导致呼吸机引起的膈肌功能障碍(VIDD),增加死亡率和发病率。氨茶碱(AP)具有增强动物骨骼肌纤维收缩力和改善人体呼吸肌活动的潜力,胰岛素样生长因子-1(IGF-1)-叉头框蛋白O1(FOXO1)-肌肉环指-1(MURF1)通路在骨骼肌功能障碍中起着至关重要的作用。本研究旨在探讨AP对VIDD的影响,并阐明IGF-1-FOXO1-MURF1通路作为潜在机制的作用。
方法:通过MV处理建立VIDD大鼠模型。采用IGF-1慢病毒(LV)干扰(LV-IGF-1-shRNA;由慢病毒阴性对照LV-NC控制)来抑制IGF-1表达,从而阻断IGF-1-FOXO1-MURF1途径。使用蛋白质印迹和实时逆转录酶聚合酶链反应(RT-qPCR)评估IGF-1,FOXO1和MURF1的蛋白质和mRNA水平,分别。通过测量复合肌肉动作电位(CMAP)和苏木精和伊红(H&E)染色来检查膈肌收缩性和形态计量学。氧化应激通过过氧化氢(H2O2)的水平来评估,超氧化物歧化酶(SOD),抗氧化剂谷胱甘肽(GSH),和羰基化的蛋白质.线粒体稳定性通过测量线粒体膜电位(MMP)来评估,通过动态蛋白相关蛋白1(DRP1)的蛋白质水平检查线粒体裂变和线粒体自噬,mitofusin2蛋白(MFN2),磷酸酶和张力蛋白同源物(PTEN)诱导的激酶1(PINK1),和Parkin(westernblot)。使用末端脱氧核苷酸转移酶介导的尿苷5'-三磷酸(UTP)缺口末端标记(TUNEL)测定和Bax水平评估细胞凋亡,B细胞淋巴瘤2(BCL-2),和Caspase-3。Atrogin-1的水平,神经元表达的发育下调4(NEDD4),和萎缩-1(MUSA1)mRNA中SCF复合物的肌肉泛素连接酶,以及泛素化蛋白,用于确定蛋白质降解。此外,使用SUnSET(翻译的表面感测)方法来确定蛋白质合成的速率。
结果:MV治疗上调IGF-1,而下调FOXO1和MURF1(p<0.05)。AP给药逆转IGF-1,FOXO1和MURF1(p<0.05),再次被IGF-1抑制抑制(p<0.05),证明IGF-1-FOXO1-MURF1途径的阻断。MV治疗导致CMAP和膈肌纤维横截面积减少,CMAP的时程增加(p<0.05)。此外,氧化应激,细胞凋亡,MV处理增加了蛋白质降解,降低了线粒体稳定性(p<0.05)。相反,AP给药逆转了MV引起的所有这些变化,但这种逆转被IGF-1-FOXO1-MURF1通路的阻断所破坏。
结论:在这项研究中,MV治疗诱发大鼠VIDD症状,这些都被AP调节IGF-1-FOXO1-MURF1通路有效逆转,证明AP改善VIDD的潜力。
BACKGROUND: The usage of life-saving mechanical ventilation (MV) could cause ventilator-induced diaphragmatic dysfunction (VIDD), increasing both mortality and morbidity.
Aminophylline (AP) has the potential to enhance the contractility of animal skeletal muscle fibers and improve the activity of human respiratory muscles, and the insulin-like growth factor-1 (IGF-1)- forkhead box protein O1 (FOXO1)-muscle RING finger-1 (MURF1) pathway plays a crucial role in skeletal muscle dysfunction. This study aimed to investigate the impact of AP on VIDD and to elucidate the role of the IGF-1-FOXO1-MURF1 pathway as an underlying mechanism.
METHODS: Rat models of VIDD were established through MV treatment. IGF-1 lentiviral (LV) interference (LV-IGF-1-shRNA; controlled by lentiviral negative control LV-NC) was employed to inhibit IGF-1 expression and thereby block the IGF-1-FOXO1-MURF1 pathway. Protein and mRNA levels of IGF-1, FOXO1, and MURF1 were assessed using western blot and real-time reverse transcriptase-polymerase chain reaction (RT-qPCR), respectively. Diaphragm contractility and morphometry were examined through measurement of compound muscle action potentials (CMAPs) and hematoxylin and eosin (H&E) staining. Oxidative stress was evaluated by levels of hydrogen peroxide (H2O2), superoxide dismutase (SOD), antioxidant glutathione (GSH), and carbonylated protein. Mitochondrial stability was assessed by measuring the mitochondrial membrane potential (MMP), and mitochondrial fission and mitophagy were examined through protein levels of dynamin-related protein 1 (DRP1), mitofusin 2 protein (MFN2), phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1), and Parkin (western blot). Apoptosis was evaluated using the terminal deoxynucleotidyl transferase-mediated uridine 5\'-triphosphate (UTP) nick-end labeling (TUNEL) assay and levels of Bax, B-cell lymphoma 2 (BCL-2), and Caspase-3. Levels of Atrogin-1, neuronally expressed developmentally downregulated 4 (NEDD4), and muscle ubiquitin ligase of SCF complex in atrophy-1 (MUSA1) mRNA, as well as ubiquitinated protein, were utilized to determine protein degradation. Furthermore, the SUnSET (surface sensing of translation) method was employed to determine rates of protein synthesis.
RESULTS: MV treatment upregulated IGF-1 while downregulated FOXO1 and MURF1 (p < 0.05). AP administration reversed IGF-1, FOXO1 and MURF1 (p < 0.05), which was suppressed again by IGF-1 inhibition (p < 0.05), demonstrating the blockage of the IGF-1-FOXO1-MURF1 pathway. MV treatment caused decreased CMAP and cross-sectional areas of diaphragm muscle fibers, and increased time course of CMAP (p < 0.05). Additionally, oxidative stress, cell apoptosis, and protein degradation were increased and mitochondrial stability was decreased by MV treatment (p < 0.05). Conversely, AP administration reversed all these changes induced by MV, but this reversal was disrupted by the blockage of the IGF-1-FOXO1-MURF1 pathway.
CONCLUSIONS: In this study, MV treatment induced symptoms of VIDD in rats, which were all effectively reversed by AP regulating the IGF-1-FOXO1-MURF1 pathway, demonstrating the potential of AP in ameliorating VIDD.