Ren Guangdong, Liu Ran, Mai Huiqiang, Yin Gang, Ding Fulai, Wang Chunmei, Chen Shuxin, Lan Xianqi
Emergency Department, Zhongshan City People's Hospital, Zhongshan, Guangdong 528403, P.R. China.
Exp Ther Med. 2024 Aug 14;28(4):400. doi: 10.3892/etm.2024.12689. eCollection 2024 Oct.
Endothelial dysfunction is a crucial pathogenetic mechanism for sepsis. GRB2-associated binder 1 (GAB1) alleviates sepsis-induced multi-organ damage; however, to the best of our knowledge, its function in endothelial dysfunction in sepsis remains unclear. HUVECs were induced by lipopolysaccharide (LPS) to simulate endothelial cell injury under sepsis. Cell transfection was conducted to achieve GAB1 overexpression or suppressor of cytokine signaling 3 (SOCS3) knockdown. The expression levels of GAB1 and SOCS3 were detected by reverse transcription-quantitative PCR and western blotting. Cell viability, apoptosis and migration were assessed using Cell Counting Kit-8, TUNEL and wound healing assays, respectively. The production of cytokines and nitric oxide (NO) was detected using commercial kits. The interaction between GAB1 and SOCS3 was confirmed using a co-immunoprecipitation assay. GAB1 was downregulated in LPS-induced HUVECs. However, GAB1 overexpression significantly mitigated LPS-induced cell viability decrease and apoptosis in HUVECs, accompanied by upregulation of Bcl2 expression, and downregulation of Bax and cleaved caspase-3 expression. GAB1 also inhibited the production of pro-inflammatory cytokines and increased NO level, increased the levels of endothelial NO synthase (eNOS) and phosphorylated (p)-eNOS, and promoted migration in LPS-induced HUVECs. However, SOCS3 knockdown partially weakened the effects of GAB1 overexpression on cell viability, apoptosis, inflammation, p-eNOS, eNOS expression and NO levels in LPS-induced HUVECs. In addition, GAB1 and SOCS3 regulated Janus kinase 2 (JAK2)/STAT3 signaling in LPS-induced HUVECs. In conclusion, GAB1 exerted a protective effect against LPS-induced endothelial cell apoptosis, inflammation and dysfunction by modulating the SOCS3/JAK2/STAT3 signaling pathway.
内皮功能障碍是脓毒症的关键发病机制。生长因子受体结合蛋白2相关结合蛋白1（GAB1）可减轻脓毒症诱导的多器官损伤；然而，据我们所知，其在脓毒症内皮功能障碍中的作用仍不清楚。用脂多糖（LPS）诱导人脐静脉内皮细胞（HUVECs）以模拟脓毒症时的内皮细胞损伤。进行细胞转染以实现GAB1过表达或细胞因子信号转导抑制因子3（SOCS3）敲低。通过逆转录定量PCR和蛋白质印迹法检测GAB1和SOCS3的表达水平。分别使用细胞计数试剂盒-8、TUNEL和伤口愈合试验评估细胞活力、凋亡和迁移。使用商业试剂盒检测细胞因子和一氧化氮（NO）的产生。使用免疫共沉淀试验证实GAB1和SOCS3之间的相互作用。在LPS诱导的HUVECs中GAB1表达下调。然而，GAB1过表达显著减轻了LPS诱导的HUVECs细胞活力下降和凋亡，同时Bcl2表达上调，Bax和裂解的caspase-3表达下调。GAB1还抑制促炎细胞因子的产生并增加NO水平，增加内皮型一氧化氮合酶（eNOS）和磷酸化（p）-eNOS的水平，并促进LPS诱导的HUVECs迁移。然而，SOCS3敲低部分削弱了GAB1过表达对LPS诱导的HUVECs细胞活力、凋亡、炎症、p-eNOS、eNOS表达和NO水平的影响。此外，GAB1和SOCS3在LPS诱导的HUVECs中调节Janus激酶2（JAK2）/信号转导和转录激活因子3（STAT3）信号通路。总之，GAB1通过调节SOCS3/JAK2/STAT3信号通路对LPS诱导的内皮细胞凋亡、炎症和功能障碍发挥保护作用。
