Aminophylline Improves Ventilator-Induced Diaphragmatic Dysfunction by Targeting IGF-1-FOXO1-MURF1 Pathway.

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. lentiviral (LV) interference (LV--shRNA; controlled by lentiviral negative control LV-NC) was employed to inhibit expression and thereby block the IGF-1-FOXO1-MURF1 pathway. Protein and mRNA levels of , , and 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 (HO), 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 (), and Caspase-3. Levels of Atrogin-1, neuronally expressed developmentally downregulated 4 (), and muscle ubiquitin ligase of SCF complex in atrophy-1 () 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 while downregulated and ( < 0.05). AP administration reversed , and ( < 0.05), which was suppressed again by inhibition ( < 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 ( < 0.05). Additionally, oxidative stress, cell apoptosis, and protein degradation were increased and mitochondrial stability was decreased by MV treatment ( < 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.