Fig 1.
BGP-15 is enriched in mitochondria and reduces membrane potential (ΔΨ) in isolated mitochondria.
(A) Membrane potential enhanced the mitochondrial uptake of BGP-15 (50 μM) in isolated rat liver mitochondria. Uncoupling was found to occur with 50 μM 2,4-dinitrophenol. Data are presented as the mean ± SEM of three independent experiments. ***P < 0.001 compared to coupled mitochondria, ###P < 0.001 compared to the glucose-6-phosphate signal. (B) Mitochondrial membrane potential was monitored by measuring the fluorescence intensity of R123, a cationic fluorescent dye. Isolated rat liver mitochondria, represented by the first arrow, took up the dye in a voltage-dependent manner, resulting in fluorescent quenching. At the second arrow either 1 mM, 2.5 mM or 5 mM BGP-15 was added. A representative plot of three independent concurrent experiments is presented.
Fig 2.
BGP-15 protects against reactive oxygen species-induced depolarization of mitochondria in WRL-68 cells, as determined by JC-1 and TMRM.
(A) Effect of BGP-15 on H2O2-induced mitochondrial membrane depolarization in WRL-68 cells. Cells were exposed to 50 μM H2O2 in the absence or presence of 50 μM BGP-15 for 3 hours, then stained with 100 ng/mL of JC-1, a membrane potential-sensitive fluorescent dye. The dye was loaded, and after a 15 minute incubation fluorescent microscopic images were taken using both the red and green channels. The inserts show the homogenous red fluorescence in H2O2-treated cells, and the dotted labelling represents the H2O2 + BGP-15 treated cells, showing that BGP-15 protected the mitochondrial integrity in the presence of H2O2. Inserts are expanded from the area indicated by dashed rectangles. Representative merged images of three independent experiments are presented. (B) Quantitative analysis of mitochondrial depolarization induced by H2O2 (50 μM) and its reduction by BGP-15 (50 μM) in WRL-68 cells. Results are presented as the mean ± SEM. ***P < 0.001 compared to control cells, #P < 0.05 compared to H2O2-treated cells. (C) Effect of BGP-15 on H2O2-induced mitochondrial membrane depolarization in WRL-68 cells. Cells were treated with 50 μM H2O2 in the absence or presence of 50 μM BGP-15 for 3 hours, then stained with 50 ng/mL of TMRM, a cationic, cell-permeant, red fluorescent dye. After a 15 minutes incubation fluorescent signal was measured by the GloMax Multi Detection System, then remeasured after the application of 1 μM FCCP ΔΨ was calculated as the difference of fluoresescence signal before and after FCCP-treatment. Data are presented as the mean ± SEM of three independent experiments. **P < 0.01, ***P < 0.001 compared to control cells; ##P < 0.01 compared to H2O2-treated cells.
Fig 3.
BGP-15 attenuates hydrogen peroxide-induced mitochondrial reactive oxygen species production in WRL-68 cells.
(A) Effect of hydrogen peroxide and BGP-15 pretreatment (for 30 minutes) on mitochondrial ROS production, as determined by the oxidation of the mitochondrial enriched dye from DHR123 to R123 in WRL-68 cells that had been labelled with mitochondrial directed red fluorescent protein. High magnification fluorescent microscopic images show the different localization of the produced R123. Inserts are expanded from the area indicated by dashed rectangles. (B) Quantification of R123 production. Data are presented as the mean ± SEM of three independent experiments. **P < 0.01 and ***P < 0.001 compared to control cells; ##P < 0.01 compared to H2O2-treated cells.
Fig 4.
BGP-15 attenuates oxidative stress-induced DHR123 oxidation and superoxide formation in complexes I-III in WRL-68 and H9c2 cells.
(A) Effect of BGP-15 on oxidative stress-induced DHR123 oxidation in WRL-68 cells. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 compared to control cells; #P < 0.05 and ##P < 0.01 compared to H2O2-treated cells. (B) Effect of BGP-15 on oxidative stress-induced DHR123 oxidation in H9c2 cardiomyocytes. Results are presented as the mean ± SEM of three independent experiments. *P < 0.05 compared to control cells, #P < 0.05 compared to H2O2-treated cells. (C) BGP-15 in chemical reactions does not inhibit DHR123 oxidation induced by H2O2 (500 μM). Data are presented as the mean ± SEM of three independent experiments. ***P < 0.001 compared to control group. (D) BGP-15 in chemical reactions does not inhibit DHR123 oxidation induced by H2O2 (50 μM) and Fe(II)-EDTA (66 μM) (Fenton reaction system). Results are presented as the mean ± SEM of three independent experiments. ***P < 0.001 compared to the control group. (E) Effect of BGP-15 on oxidative stress-induced superoxide production in WRL-68 cells in the absence or presence of 20 μM MitoTEMPO as determined by MitoSOX (0.3 μM). Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01 compared to control cells, #P < 0.05 compared to H2O2-treated cells. (F) Effect of BGP-15 on the oxidative stress-induced superoxide production in H9c2 cardiomyocytes in the absence or presence of 20 μM MitoTEMPO as determined by MitoSOX (0.3 μM). Results are presented as the mean ± SEM of three independent experiments. *P < 0.05 compared to control cells, #P < 0.05 compared to H2O2-treated cells. (G) Effect of BGP-15 on mitochondrial DHR123 oxidation using glutamate-malate as substrate and with complex III inhibited by antimycin A. Data are presented as the mean ± SEM of three independent experiments. **P < 0.01 compared to the control group. (H) Effect of BGP-15 on mitochondrial DHR123 oxidation using succinate as substrate and with complex IV inhibited by CN-. Results are presented as the mean ± SEM of three independent experiments. *P < 0.05 compared to the control group.
Fig 5.
BGP-15 attenuates reactive oxygen species-induced apoptotic and necrotic cell death in WRL-68 cells.
(A) BGP-15 protects against H2O2-induced cell death (sulforhodamine B assay). Data are presented as the mean ± SEM of eight independent experiments. ***P < 0.001 compared to control cells; #P < 0.05 and ##P < 0.01 compared to H2O2-treated cells. (B) Determination of the effect of BGP-15 on H2O2-induced apoptosis (fluorescein-labelled annexin V) and necrosis (propidium iodide) pathways. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05 and ***P < 0.001 compared to control cells; #P < 0.05 and ##P < 0.01 compared to H2O2-treated cells.
Fig 6.
BGP-15 attenuates lipopolysaccharide-induced mitochondrial depolarization and production of reactive oxygen species.
(A) Effect of BGP-15 on LPS-induced mitochondrial membrane depolarization in U-251 MG cells. Cells were exposed to 1 μg/mL LPS in the absence or presence of 50 μM BGP-15 for 1 hour, then stained with 100 ng/mL of JC-1. Fluorescent microscopic images were taken using both the red and green channels. Representative merged images of three independent experiments are presented. (B) Quantitative analysis of LPS-induced (1 μg/mL) mitochondrial depolarization and its reduction by BGP-15 (50 μM) in U-251 MG cells. Results are presented as the mean ± SEM. **P < 0.01 and ***P < 0.001 compared to control cells; #P < 0.05 compared to LPS-treated cells. (C) Effect of BGP-15 on LPS-induced mitochondrial membrane depolarization in U-251 MG cells. Cells were treated with 1 μg/mL LPS in the absence or presence of 50 μM BGP-15 for 1 hour, then stained with 50 nM of TMRM. After a 15 minutes incubation fluorescent signal was measured by the GloMax Multi Detection System, then remeasured after the application of 1 μM FCCP. ΔΨ was calculated as the difference of fluoresescence signal before and after FCCP-treatment. Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, ***P < 0.001 compared to control cells; ##P < 0.01 compared to LPS-treated cells. (D) Effect of BGP-15 on the LPS-induced ROS production in U-251 MG cells (containing the TLR4 receptor). Cells were treated with 1 mg/mL LPS in the presence or absence of 50 μM BGP-15 for 30 minutes. LPS-induced ROS production was determined by the oxidation of DHR123 (1 μM) to R123, measured with fluorescent microscopy. Cell nuclei were labelled using Hoechst 33342. Representative merged images of three independent experiments are presented. (E) Quantitative analysis of LPS-induced (1 μg/mL) ROS production and the protective effect of BGP-15 (50 μM). Data are presented as the mean ± SEM of three independent experiments. ***P < 0.001 compared to control cells; ###P < 0.001 compared to LPS-treated cells. (F) Effect of BGP-15 on oxidative stress-induced superoxide production in U-251 MG cells in the absence or presence of 20 μM MitoTEMPO as determined by MitoSOX (0.3 μM). Data are presented as the mean ± SEM of three independent experiments. *P < 0.05, ***P < 0.001 compared to control cells, ##P < 0.01 compared to LPS-treated cells.
Fig 7.
Possible mitochondrial molecular mechanism of BGP-15 cytoprotective action.
BGP-15 reduces mitochondrial ROS production at complex I and at complex III, and so reduces ROS induced mitochondrial damage, as well as cell death.