Delphinidin-3-Glucoside Protects against Oxidized Low-Density Lipoprotein-Induced Mitochondrial Dysfunction in Vascular Endothelial Cells via the Sodium-Dependent Glucose Transporter SGLT1
Figure 4
Effects of Dp on mitochondrial dysfunction in oxLDL-induced HUVECs.
HUVECs were pretreated with various Dp concentrations (1, 10, 50 and 100 μM, respectively) for 2 h, and then exposed to 100 μg/mL of oxLDL for another 24 h. (A) Intracellular ROS levels were estimated using the probe DCFH-DA. Fluorescence was read at 485 nm for excitation and 520 nm for emission. (B) Intracellular O2·− levels were estimated using the probe DHE. Fluorescence was read at 355 nm for excitation and 420 nm for emission. (C) Determination of ΔΨm was carried out using CLSM and fluorescences were determined by spectrophotometry assay. Red fluorescence is emitted by JC-1 aggregates in healthy mitochondria with polarized inner mitochondrial membranes, while green fluorescence was emitted by cytosolic JC-1 monomers and indicates ΔΨm dissipation. Merged images indicated the co-localization of JC-1 aggregates and monomers. The ΔΨm in each group was calculated as the ratio of red to green fluorescence. (D) ΔΨm was detected by flow cytometry assay. The designated R2 and R3 regions represent cell populations that exhibit high (R2) or low (R4) red-to-green fluorescence ratio, consistent with high and low Δψm, respectively. Results shown are one representative of three separate experiments and the proportion of cells in R3 regions were quantified and expressed as percent cells with decreased ΔΨm. (E) The mPTP opening was assayed using the calcein–cobalt quenching method. Different HUVEC groups were used to measure the normalized relative fluorescence units (NRFU) of calcein. Fluorescences were determined using an InfiniteTM M200 Microplate Reader (Tecan Group Ltd., Männedorf, Switzerland). All results are presented as means ± SEM. and images were representative of at least three independent experiments. ##P<0.05, versus the control group; *P<0.05, **P<0.01 versus oxLDL-treated group.