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 HPMEC supernatant was withdrawn after modeling from each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.