Department of Thoracic Surgery Medical Center, Nanfang Hospital, Southern Medical University, Guangzhou, China; Research Center for Tissue Repair and Regeneration Affiliated to the Medical Innovation Research Department, Chinese People's Liberation Army (PLA) General Hospital, Beijing, China.
Department of Thoracic Surgery Medical Center, Nanfang Hospital, Southern Medical University, Guangzhou, China.
Int Immunopharmacol. 2022 Sep;110:109017. doi: 10.1016/j.intimp.2022.109017. Epub 2022 Jul 2.
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.
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 HO. 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.
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.
These results show that GCLc overexpressing BMSCs with anti-apoptotic effects significantly improve acute lung injury.
急性肺损伤（ALI）是一种严重的异质性肺部疾病，死亡率高。骨髓间充质干细胞（BMSCs）对 ALI 有很好的治疗作用，但体内存活率不高。GCLc 具有谷氨酰胺半胱氨酸连接酶（GCL）的所有活性，可减少活性氧、抗氧化应激反应并提高细胞存活率。因此，在我们的研究中，通过慢病毒转导构建了过表达 GCLc 的 BMSCs，并通过气管内移植到 ALI 小鼠中评估其治疗效果，并探讨了 GCLc 在 BMSCs 中抗细胞凋亡的机制。
使用慢病毒载体构建过表达 GCLc 的 hBMSCs。通过 CCK-8 测定法检测 MSC 的细胞活力。通过试剂盒检测 GSH、MDA、SOD 和 ROS。Western blot 和 RT-qPCR 用于检测 BMSCs 中 GCLc、bax、bcl2、cleaved-caspase 3、caspase 3、cleaved-caspase 9、caspase 9 和 Foxo1 的表达。通过流式细胞术、JC-1 和 TUNEL 法分析 BMSCs 的凋亡。共聚焦显微镜观察 Foxo1 的核细胞外迁移。然后通过 Western blot 检测通路蛋白的表达水平。在 ALI 动物模型中，通过 H&E 染色、体外成像、肺组织湿/干重比以及从小鼠中提取支气管肺泡灌洗液来分析蛋白浓度、中性粒细胞、白细胞和巨噬细胞计数以及 ELISA 检测炎症因子，评估过表达 GCLc 的 BMSCs 的治疗效果。
我们证明，过表达 GCLc 可降低 MDA 和 ROS，并增加 GSH 和 SOD，同时降低 BMSCs 中促凋亡蛋白（bax、cleaved-caspase 3、caspase 3、cleaved-caspase 9、caspase 9）的表达，并升高抗凋亡蛋白（bcl-2）的表达。我们验证了它通过 PI3K/AKT/Foxo1 通路发挥作用。在 ALI 体内，与载体 BMSC 相比，过表达 GCLc 的 BMSCs 在肺部的保留时间更长，并改善肺水肿，降低肺泡蛋白浓度，并降低肺中的 TNF-α、IL-1β、IL-6 水平，增加 IL-10 水平。
这些结果表明，具有抗凋亡作用的过表达 GCLc 的 BMSCs 可显著改善急性肺损伤。
