The oxidative stress of vessel endothelium is a major risk factor of cardiovascular disorders. Antioxidative stress drugs are widely used in cardiovascular therapy. Aspirin eugenol ester (AEE) is a new pharmaceutical compound synthesized by esterification reaction of aspirin with eugenols and possesses antioxidative activity. The present study was designed to investigate the mechanism how AEE protects human umbilical vein endothelial cells (HUVECs) from H2O2-induced oxidative stress. H2O2 was given to the HUVECs with or without AEE pretreatment. Changes in the oxidative stress-related factors, including those related to the mitochondria-lysosome axis, were determined with Western blotting, cellular immunofluorescence, and enzyme activity test. The results showed that, in the HUVECs, 300
The most global deaths and disability-adjusted life year events are associated with the cardiovascular diseases [
AEE transparent crystal with the purity of 99.5% by RE-HPLC was prepared in Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS. H2O2 solution (cat number: 323381), dimethyl sulfoxide (DMSO), reactive oxygen species assay kit, and trypsin-EDTA were supplied by Sigma (St. Louis, MO). Cell Counting Kit-8 (CCK-8) was from MedChemExpress (NJ, USA), DMEM/F12 (1 : 1), and fetal bovine serum was from Gibco (NY, USA). LysoTracker Red probe, Hoechst 33342 staining solution, cellular glutathione peroxidase assay kit, Cu/Zn-SOD and Mn-SOD assay kit, and mitochondrial membrane potential assay kit were purchased from Beyotime (Shanghai, China). MitoTracker Red CMXRos probe was from Thermo Scientific (MA, USA). Anti-Bid cleavage site, Anti-Bax, Anti-Bcl2, Anti-Bcl-XL, anti-Caspase3 (Cas3), anti-cytochrome C, and anti-Cathepsin D (CTSD) were purchased from Abcam (MA, USA) and the CTSD activity assay kit was from BioVision (CA, USA). An Annexin V/FITC apoptosis detection kit was from BD Biosciences (NY, USA). A Caspase-3 activity assay kit was from Cell Signaling Technology (MA, USA).
Human umbilical vein endothelial cells (ATCC® CRL-4053™) were purchased from ATCC (Rockville, MD). The cells were cultured in the cell culture flasks in DMEM/F12 (1 : 1) with 10% fetal bovine serum. Media were replaced every two days. Subcultures were performed with the trypsin-EDTA method. Experiments were subsequently conducted on 6-7 passages of cells.
HUVECs were randomly divided into 3 groups (
In the HUVECs, the apoptosis induced by H2O2 was detected quantitatively with Annexin V/FITC apoptosis detection kit and flow cytometry. Briefly, the cells were collected and washed three times with cold PBS. Then, they were incubated and stained with FITC-Annexin V and PI at room temperature for 15 min in the dark. The double stained cells were analyzed using flow cytometry, and the following controls were used to set up compensation and quadrants: unstained cells, cells stained with FITC-Annexin V, and cells stained with PI.
Intracellular ROS was measured with reactive oxygen species assay kits following the manufacturer’s instructions. Briefly, the HUVECs were cultured on the 24-well glass bottom cell culture plate and treated with AEE and H2O2. Then, the cells were incubated with the ROS detection work solutions (10
The mitochondrial ROS was detected by a MitoSOX probe following the manufacturer’s instructions. Briefly, the HUVECs were cultured on the 24-well glass bottom cell culture plate and treated with AEE and H2O2. Then, the cells were incubated with the MitoSOX probe work solutions (4
The concentrations of MDA, GSH/GSSG ratio, SOD, and glutathione peroxidase activity in the HUVECs were determined using the commercial kits according to the manufacturer’s protocols (see Section
A mitochondrial membrane potential assay kit was used to determine the mitochondrial membrane potential. Briefly, the HUVECs were cultured on the 24-well glass bottom cell culture plate and treated with AEE and H2O2. Then, the cells were incubated with JC-1 work solutions (5
Lysosomal membrane stability was determined with a LysoTracker Red probe. Briefly, the HUVECs were cultured on the 24-well glass bottom cell culture plate and treated with AEE and H2O2. Then, the cells were incubated with LysoTracker Red work solutions (10 nM) at 37°C for 40 min in the dark. The relative fluorescence intensity of the cells was measured using a laser scanning confocal microscope (ZEISS LSM-800, Jena, Germany). With the positive control, the HUVECs were treated with chloroquine (50
CTSD activity in the HUVECs was determined with an enzymatic assay method using a commercial kit according to the manufacturer’s protocol (see Section
The expression of Bid, Bcl2, Bcl-XL, Cas3, and CTSD was evaluated by Western blot analysis. In brief, the total protein of the HUVECs was isolated with RIPA lysis buffer. The concentration was quantified using the BCA method. SDS-PAGE (10%) electrophoresis and transfer of the separated proteins onto polyvinylidene fluoride membrane (Merck Millipore) were performed using standard procedures. The blots were incubated with primary antibodies against Bid, Bcl2, Bcl-XL, Cas3, CTSD, and internal control
The expression of cytochrome C and Bax was evaluated with immunofluorescence. Briefly, the cells were cultured on glass coverslips in the medium and the mitochondria were labeled with the CMXRos probe; then, immunofluorescence analyzes 4% paraformaldehyde–fixed. 0.1% Triton X-100 permeabilized the cells labeling the primary antibody against cytochrome C and Bax, and then, they were incubated with the Alexa Fluor®-conjugated secondary antibody. The relative fluorescence intensity of the cells was measured using a laser scanning confocal microscope (ZEISS LSM-800, Jena, Germany). The colocalization data of cytochrome c and mitochondria have been acquired by ZEN blue software (ZEISS, Jena, German) following with the colocalization analysis.
Cas3 activity in the HUVECs was determined with the enzymatic assay method using the Cas3 assay kit according to the manufacturer’s protocol.
The lentivirus labeled ubiquitin IRES-puromycin containing short interference (si) RNA oligonucleotides against Bcl2 (siBcl2) or control siRNA was used to transfect HUVECs for knockdown experiments, and the HUVECs were transduced with a lentivirus expressing Bcl2 gene labeled ubiquitin IRES-puromycin for overexpression experiments. The positive cells were selected using puromycin.
All experiments and data analyses were carried out according to the blinding principles. Statistical analysis was performed using the SAS 9.2 (SAS Institute Inc., NC, USA). Where applicable, the values from treatment groups were normalized to the corresponding control values. All data were presented as
To verify the effects of AEE on apoptosis in HUVECs, cells were incubated with AEE at 0.5, 1.0, 2.0, and 4.0
AEE reduced the apoptosis of HUVECs induced by H2O2. (a) The double stained with Annexin V and PI results among different treatment groups. (b) The apoptosis rate among different treatment groups. Values were expressed as
The levels of MDA were not different in the HUVECs incubated with different concentrations of AEE (0, 0.5, 1.0, 2.0, and 4.0
AEE enhanced the antioxidant capacity of HUVECs. (a) AEE decreased lipid peroxidation in the HUVECs. (b) AEE enhanced the SOD activity. (c) AEE raised the GSH-Px activity. (d) AEE increased the ratio of GSH to GSSG. Values were expressed as
After the HUVECs were incubated with different concentrations of AEE (0.5, 1.0, 2.0, and 4.0
The expression and activity of CTSD were examined with Western blotting and enzyme activity test kit. In the control group, the expression of mature CTSD and the activity of CTSD were very low. After treatment with 300
AEE ameliorated lysosomal disorder induced by H2O2. (a, b) AEE reduced the expression of mature CTSD induced by H2O2. (c) AEE reduced the increase in CTSD activity induced by H2O2. The high-density fluorescence of LysoTracker Red was indicated by green arrows. (d, e) AEE enhanced the stability of the lysosomal membrane disrupted by H2O2 ((d)
A JC-1 probe was used to examine the mitochondrial membrane potential. As presented in Figures
AEE alleviated the mitochondrial dysfunction induced by H2O2. (a, b) AEE reduced the collapse of the mitochondrial membrane potential induced by H2O2 ((a)
The expression of Bcl2, Bcl-xl, Bax, and Bid was examined with Western blotting and immunofluorescence. In the H2O2-treated HUVECs, the expression of proapoptotic proteins (Bax and activated Bid) was significantly upregulated while the antiapoptotic proteins (Bcl2 and Bcl-xl) were significantly downregulated. Those changes were reversed by pretreating HUVECs with 1
AEE treatment reduced the effect of H2O2 on antiapoptosis or proapoptosis proteins. (a, b) AEE reduced H2O2-induced expression of mature Cas3 and Bid and reversed the H2O2-inhibited expression of Bcl2 and Bcl-XL. (c, d) AEE reduced H2O2-induced expression of Bax ((c)
The roles of Bcl2 in the protective effect of AEE on H2O2-induced mitochondrial and lysosomal dysfunction were investigated. The lentivirus labeled with ubiquitin IRES-puromycin containing siRNA oligonucleotides was used to silence Bcl2 expression. There were over 85% of positive HUVECs transfected with the lentivirus at 5 MOI (multiplicity of infection). The Bcl2 expression was significantly downregulated in the positive HUVECs compared with normal HUVECs. After the HUVECs with downregulated Bcl2 were given 300
Genetic inhibition of Bcl2 reduced the effect of AEE on H2O2-induced mitochondrial and lysosomal dysfunction. (a, b) The changes in the mitochondrial membrane potential between different treatment groups ((a)
To confirm the role of Bcl2 in the mitochondrial lysosomal dysfunction induced by H2O2, HUVECs were transduced with lentivirus to overexpress Bcl2. Bcl2 overexpression significantly ameliorated mitochondrial and lysosomal disorders manifested as the increase in ROS generation and activity of Cas3 and CTSD induced by H2O2 (Figures
Vascular endothelial cells provide a haemocompatible vessel lining via regulating procoagulant and anticoagulant balance of endothelium [
Various events involved in oxidative stress and apoptosis are closely related with mitochondrial dysfunction, including generation of ROS, cellular unbalance of the redox status, changes in electron transport and mitochondrial transmembrane potential, release of apoptosis activators (such as cytochrome c), changes in the pro- and antiapoptotic Bcl-2 family proteins, and activation of downstream caspase family proteins [
To investigate the role of Bcl2 in the protective effect of AEE on H2O2-induced oxidative stress and lysosomal-mitochondrial axis of apoptosis in the HUVECs, the Bcl2 was knocked down or overexpressed by genetic intervention of Bcl2 proteins and this showed alteration in a variety of mitochondrial events, including mitochondrial transmembrane potential and release of proapoptotic and antiapoptotic factor from mitochondria. Bcl2 proteins inhibited caspase-dependent apoptosis; however, there were few reports showing influence of Bcl2 in lysosomal function in the apoptosis. In the present study, we demonstrated that Bcl2 overexpression significantly reduced H2O2-induced dysfunctions in mitochondria and lysosome systems, including increase in mitochondrial transmembrane potential and stabilizing lysosomal membrane and decrease in CTSD and Cas3 activity. The knockdown of Bcl2 did not cause the apoptosis in HUVECs but enhanced the mitochondrial and lysosomal dysfunctions induced by H2O2. It is interesting to note that those changes were not reversed by pretreating HUVECs with AEE. These findings suggested that Bcl2 play a vital role in the protective effect of AEE on H2O2-induced oxidative stress and apoptosis in the lysosomal-mitochondrial axis in HUVECs. Previous studies have confirmed that AEE has antithrombotic and antiatherosclerotic effects [
Our study demonstrated that AEE treatment significantly reduced H2O2-induced oxidative stress in HUVECs via mitochondria-lysosome axis and Bcl2 was an important regulation target of AEE to protect cells from oxidative stress.
The data used to support the findings of this study are included within the article.
The authors declare no conflict of interest.
This study was supported by the Special Fund for National Natural Science Foundation (Grant/Award numbers: 31572573 and 31872518). The authors would like to thank the personnel in the Key Laboratory of Veterinary Pharmaceutical Development of Ministry of Agriculture, Key Laboratory of New Animal Drug Project of Gansu Province for their contribution to this project.