Gclc overexpression inhibits apoptosis of bone marrow mesenchymal stem cells through the PI3K/AKT/Foxo1 pathway to alleviate inflammation in acute lung injury

Abstract

Background: Acute lung injury (ALI) represents a serious heterogenous pulmonary disorder with high mortality. Bone marrow mesenchymal stem cells (BMSCs) have a good therapeutic effect on ALI, but their survival rate in vivo is not high. GCLc has all the activities of Glutamate cysteine ligase (GCL) and can reduce reactive oxygen species, antioxidant stress response and improve cell survival. Therefore, in our study, overexpressing GCLc BMSCs were constructed by lentiviral transduction and intratracheally transplanted into ALI mice to evaluate their therapeutic effects, and we explored the mechanism of anti-apoptosis of GCLc in BMSCs.

Methods: Overexpressing GCLc hBMSCs were constructed using lentiviral vectors. The cell viability of MSCs was detected by CCK-8 assay. GSH, MDA, SOD and ROS were detected by the manufacturer's kit. Western blot and RT-qPCR were used to detect the expression of GCLc, bax, bcl2, cleaved-caspase 3, caspase 3, cleaved-caspase 9, caspase 9 and Foxo1 in BMSCs stimulated by H₂O₂. Apoptosis of BMSCs was analyzed by flow cytometry, JC-1 and TUNEL method. Confocal microscopy was to observe the nuclear extracellular migration of Foxo1. We then examined the expression levels of the pathway proteins by Western blot. In ALI animal model, we evaluated the therapeutic effect of the overexpressing GCLc BMSCs by H&E staining, in vitro imaging, wet/dry weight ratio of lung tissue, and extraction of bronchoalveolar lavage fluid from mice to analyze protein concentrations, neutrophil, leukocyte and macrophage counts and ELISA for inflammatory factors.

Results: We demonstrated that overexpression of GCLc reduced MDA and ROS and increased GSH and SOD, while GCLc reduced the expression of pro-apoptotic proteins (bax, cleaved-caspase 3, caspase 3, cleaved-caspase 9, caspase 9) and elevated the expression of anti-apoptotic proteins (bcl-2) in BMSCs. We verified that it acts through the PI3K/AKT/Foxo1 pathway. In ALI vivo, overexpression of GCLc BMSCs had a longer retention time in the lung compared to vector BMSC and improved pulmonary edema, decreased alveolar protein concentration and reduced TNF-α, IL-1β, IL-6 levels and increased IL-10 levels in the lung.

Conclusions: These results show that GCLc overexpressing BMSCs with anti-apoptotic effects significantly improve acute lung injury.

Keywords: ALI; Cell apoptosis; GCLc; Mesenchymal stem cells.