2.3 Detection of changes of endothelial glycocalyx structure and functional activity
Flow cytometry To investigate HPMEC apoptosis and heparansulfate proteoglycan (HSPG) concentration, flow cytometry was performed in each group according to our previous methods. 15 Cells in each group were washed with PBS for 15 minutes, centrifuged, and resuspended with 500 μL AnnexinV-FITC (UElandy, China) binding buffer per tube. The cells were further stained with 5 μL AnnexinV-FITC, then mixed and incubated in the dark at 4 °C for 15 minutes. Lastly, 10 μL PI staining solution was added, and the flow cytometry (Becton, Dickinson and Company, American) was performed to detect after incubated in the dark for 5 min. Data compensation and analysis was performed using FlowJo version 7.6.1.
Measurements of reactive oxygen species (ROS) To examine the intracellular ROS levels, a ROS assay kit that sets DCFH-DA as the probe was used. HPMEC supernatant was withdrawn after modeling from each group, and DCFH-DA was diluted to 10 μmol/L by 1:1000 in a serum-free blank medium. Cells were stained with 2, 7-dichloride-hydrofluorescein diacetate (DCFH-DA, Nanjing Jiancheng, China) in a 37 ℃ cell incubator for 20 minutes. The cells were washed 3 times with a blank medium, observed and photographed by Olympus positive fluorescence system microscope (Olympus Corporation, Japan), and analyzed by Image J software.
Immunofluorescent Staining Cells from each group were washed with PBS for 3 minutes, permeabilized in precooled 0.25% Triton X-100 solution for 10 minutes on ice, and then were blocked in 1% bovine serum albumin (BSA, Sigma, American) for 1 h. Furthermore, rabbit anti-HSPG (Affinity Biosciences, American) was majorly used, which was diluted in PBS containing 1% BSA. Primary antibody incubation was performed at 4 °C for 2 h and washed with PBS; secondary antibody incubation was performed in the dark at room temperature for 1 h. Finally, the cells were incubated with DAPI in the dark for 5 minutes and then were observed in a fluorescence microscope.
Enzyme-linked immunosorbent assay test The level of major degraded products of endothelial glycocalyx, including Syndecans-1 (SDC-1) and heparin sulfate (HS), was measured by enzyme-linked immunosorbent assay (ELISA) based on an established procedure. Moreover, ELISA was also performed to detect the concentrations of Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6), Von Willebrand factor (vWF), and Endothelin-1 (ET-1).
Western blot The expression level of E-selectin, VCAM-1, and Occludin in HPMEC were investigated via western blot. Cells were collected and lysed in RIPA lysis buffer (LEAGENE, Beijing, China) which added with PMSF for 30 minutes on ice. The protein samples were determined using BCA Protein Assay Kit (Cat#: 23227, Pierce, USA), separated on 12% sulfate-polyacrylamide gel electrophoresis and were then transferred to PVDF membranes (BIO-RAD, American). The membranes were further blocked with 5% nonfat dry milk with 100 ml Tris-buffered saline with 0.05% Tween 20 (TBST) at room temperature and incubated with rabbit anti-E-selectin (SAB, American), rabbit anti-Occludin (SAB, American), rabbit anti-VCAM1 (SAB, American) primary antibodies at 4 °C overnight. TBST was used for rinsing for 10 minutes and shaking at room temperatures. Peroxidase-conjugated Affinipure Goat Anti-rabbit IgG/FITC and Goat Anti-Mouse IgG/PE were used for incubating in the dark at room temperatures for 1 h. The immunoblots were investigated by enhanced chemiluminescence (ECL), and the protein expression levels were analyzed by Image J software.