Human umbilical cord mesenchymal stem cell exosomes pretreated with atorvastatin alleviate high glucose-induced injury of human retinal vascular endothelial cells through the protein kinase B/endothelial nitric oxide synthase pathway
10.3760/cma.j.cn511434-20241210-00479
- VernacularTitle:阿托伐他汀预处理的人脐带间充质干细胞外泌体经蛋白激酶B/内皮型一氧化氮合成酶通路缓解高糖诱导的人视网膜血管内皮细胞损伤
- Author:
Bin WU
1
;
Yangchen LIU
1
;
Wei ZHANG
1
;
Guanghui HE
1
Author Information
1. 天津市眼科医院 天津市眼科学与视觉科学重点实验室, 天津 300020
- Publication Type:Journal Article
- Keywords:
Atorvastatin;
Mesenchymal stem cell;
Exosomes;
High glucose;
Cell experiment
- From:
Chinese Journal of Ocular Fundus Diseases
2025;41(9):707-716
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate whether exosomes derived from atorvastatin (ATV)-pretreated human umbilical cord mesenchymal stem cells (ATV-MSC-EXO) alleviate high glucose-induced injury in human retinal vascular endothelial cells (HREC) via the protein kinase B (AKT)/endothelial nitric oxide synthase (eNOS) signaling pathway.Methods:The optimal pretreatment concentration of ATV was determined using the cell counting Kit-8 (CCK-8) assay. Exosomes derived from mesenchymal stem cells (MSC-EXO) and ATV-pretreated MSC (ATV-MSC-EXO) were isolated and extracted, and their morphology and surface markers were characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting (WB). The uptake capacity of exosomes by human retinal vascular endothelial cells (HREC) was evaluated using a fluorescence labeling assay. In vitro cultured HREC were divided into the following groups: normal control group (NC group), high glucose group (HG group), high glucose+MSC-EXO group (MSC-EXO group), high glucose+ATV-MSC-EXO group (ATV-MSC-EXO group), high glucose+ATV-MSC-EXO+AKT inhibitor group (ATV-MSC-EXO-MK-2206-2HCL group), and high glucose+ATV-MSC-EXO+eNOS inhibitor group (ATV-MSC-EXO-L-NAME group). Cell proliferation and apoptosis were detected using CCK-8 and flow cytometry, respectively. The protein expression levels of B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated protein (Bax), and Caspase-3 were measured by WB. In addition, the regulatory effects of ATV-MSC-EXO on the AKT/eNOS signaling pathway and its downstream functional molecules were analyzed by detecting the phosphorylation levels of AKT (P-AKT/AKT) and eNOS (P-eNOS/eNOS) via WB, the mRNA expression levels of AKT and eNOS by quantitative real-time polymerase chain reaction, and the concentrations of nitric oxide (NO) and endothelin-1 (ET-1) using commercial NO and ET-1 assay kits.Results:The optimal pretreatment concentration of ATV was 1 μmol/L. ATV-MSC-EXO exhibited similar morphology and particle size to MSC-EXO and were efficiently taken up by HREC. Under high glucose conditions, ATV-MSC-EXO significantly enhanced the viability of HREC ( F=83.24, P<0.000 1) and inhibited apoptosis ( F=77.39, P<0.000 1). WB analysis further confirmed that ATV-MSC-EXO upregulated the expression of the anti-apoptotic protein Bcl-2 ( F=53.17), while downregulating the pro-apoptotic proteins Bax ( F=36.49) and Caspase-3 ( F=60.75) ( P<0.001). In addition, ATV-MSC-EXO markedly increased the protein levels of P-AKT/AKT ( F=107.60) and P-eNOS/eNOS ( F=38.59), as well as the relative mRNA expression of AKT, eNOS ( F=203.60, 315.00; P<0.000 1). Furthermore, ATV-MSC-EXO promoted NO production ( F=407.40) and suppressed the relative expression of ET-1 ( F=49.76) ( P<0.000 1). Conclusion:ATV-MSC-EXO enhances the viability and inhibits apoptosis of HREC under high glucose conditions by activating the AKT/eNOS signaling pathway.
