ObjectiveTo 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. MethodsThe 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. ResultsThe 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). ConclusionATV-MSC-EXO enhances the viability and inhibits apoptosis of HREC under high glucose conditions by activating the AKT/eNOS signaling pathway.
Objective To investigate the expression of heme oxygenase-1 (HO-1) by high glucose treatment in human lung epithelial A549 cells. Methods The effect of high glucose on the expression of HO-1 in A549 cells was investigated with Western blot and reverse transcription PCR. HO-1 enzymic activity and reactive oxygen species (ROS) production were investigated with enzyme-linked immunosorbent test. Results Treatment with 25 mmol/L high glucose for 0, 24 h, 48 h, 72 h and in concentrations of 5 mmol/L, 10 mmol/L, 25 mmol/L, 40 mmol/L for 48 h induced increased expression on protein and mRNA level of HO-1 in a concentration- and time-dependent manner in A549 cells. High glucose treatment increased production of ROS and transforming growth factor-β1 (TGF-β1) in A549 cells, which thus mediated HO-1 expression. Following the increase in HO-1 expression, the enzymatic activity of HO-1 also increased in high glucose-treated cells. Pretreatment with N-acetyl-L-cysteine (NAC) and PI3K/Akt inhibitors attenuated the high glucose-induced increased HO-1 expression. Conclusions High glucose increases ROS and TGF-β1 production in A549 cells, which mediates HO-1 expression and increases HO-1 enzymic activity.