Possible mitochondrial molecular mechanism of BGP-15 cytoprotective action.

<p>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.</p

BGP-15 is enriched in mitochondria and reduces membrane potential (ΔΨ) in isolated mitochondria.

<p>(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.</p

BGP-15 protects against reactive oxygen species-induced depolarization of mitochondria in WRL-68 cells, as determined by JC-1 and TMRM.

<p>(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.</p

BGP-15 attenuates hydrogen peroxide-induced mitochondrial reactive oxygen species production in WRL-68 cells.

<p>(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.</p

BGP-15 attenuates lipopolysaccharide-induced mitochondrial depolarization and production of reactive oxygen species.

<p>(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.</p

BGP-15 attenuates oxidative stress-induced DHR123 oxidation and superoxide formation in complexes I-III in WRL-68 and H9c2 cells.

<p>(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.</p

Effect of PARP inhibitor on the PI-3-kinase – Akt pathway and Akt level in transplanted and control kidney samples.

<p>(<b>A</b>) Effects of PARP inhibitor on the Akt1 protein, and phosphorylation of Akt1 and GSK-3 β in control and transplanted kidneys determined using immunoblotting with protein and phospho-specific primary antibodies. Actin was used as loading control. Representative blots of at least three parallel experiments are presented. <b>B</b>. The bar diagrams represent pixel volumes of phosphorylated Akt (serine 473) in kidney samples. The bands were normalized to the appropriate actin band. *p<0.01 transplanted PARP inhibitor treated samples compared to other samples. Difference between control samples (independently of PARP treatment) and transplanted untreated kidney samples were not significant. The bar diagrams represent pixel volumes of GSK-3β (serine 9) phosphorylation bands. The bands were normalized to the appropriate actin band. *p<0.001 transplanted PARP inhibitor treated samples compared to control PARP inhibitor treated, or untreated samples. **p<0.05 untreated transplanted kidney samples compared to control samples (independently of PARP treatment. *** p<0.01 transplanted PARP inhibitor treated samples compared untreated transplanted samples. The bar diagrams represent pixel volumes of Akt1 protein bands. The bands were normalized to the appropriate actin band. *p<0.001 transplanted kidney samples (independently of PARP inhibitor treatment) compared to control kidney samples (independently of PARP inhibitor treatment). (<b>C</b>) Effects of PARP inhibitor on nuclear NF-kappaB and p-NF-kappaB leveles in control and transplanted kidneys determined by immunoblotting with protein and phospho-specific primary antibodies. Actin was used as loading control. Representative blots of at least three parallel experiments are presented. (<b>D</b>) The bar diagrams represent pixel volumes of nuclear NF-kappaB protein bands. The bands were normalized to the appropriate actin band. *p<0.01 transplanted PARP inhibitor treated, or untreated, samples compared to control PARP inhibitor treated, or untreated control samples. **p<0.05 untreated transplanted kidney samples compared to PARP inhibitor treated transplanted kidney samples. The bar diagrams represent pixel volumes of nuclear p-NF-kappaB bands. The bands were normalized to the appropriate actin band. *p<0.001 transplanted PARP inhibitor treated, or untreated, samples compared to control PARP inhibitor treated, or untreated control samples. **p<0.05 untreated transplanted kidney samples compared to PARP inhibitor treated transplanted kidney samples. The vertical axes represent pixel volume means±SEM of the scanned bands of the immunoblots in arbitrary units.</p

Effect of PARP inhibitor on activation and phosphorylation of ERK1/2, JNK1/2 and p38 MAP kinase pathways in transplanted and control kidney samples.

<p>(A) Effects of PARP inhibitor on the ERK1/2, JNK1/2 and p38 MAP kinase phosphorylation and activation in control and transplanted kidneys determined by immunoblotting with phospho-specific primary antibodies. Actin was used as loading control (B) The bar diagrams represent pixel volumes of ERK1/2 phosphorylation bands. The bands were normalized to the appropriate actin band. (p-ERK1) *p<0.001 control PARP inhibitor treated samples compared to control samples. **p<0.01 control PARP inhibitor treated samples compared to transplanted kidney samples independently from PARP inhibitor treatment. ***p<0.05 control untreated sample compared to transplanted kidney samples independently from PARP inhibitor treatment. (p-ERK2) *p<0.001 control PARP inhibitor treated samples compared to control samples. **p<0.01 control PARP inhibitor treated samples compared to transplanted kidney samples independently from PARP inhibitor treatment. ***p<0.05 control untreated sample compared to transplanted kidney samples independently from PARP inhibitor treatment. The bar diagrams represent pixel volumes of p-p38 MAK kinase phosphorylation bands. The bands were normalized to the appropriate actin band. *p<0.001 control samples compared to transplanted kidney samples (independently of PARP inhibitor treatment). **p<0.05 untreated transplanted kidney samples compared to control PARP inhibitor treated transplanted kidney samples. The bar diagrams represent pixel volumes of phosphorylated JNK1/2 bands. The bands were normalized to the appropriate actin band. (p-JNK1) *p<0.05 transplanted untreated kidney samples compared to all others samples. (p-JNK2) **p<0.001 control kidney samples (independently of PARP inhibitor treatment) comparing to transplanted samples. ***p<0.05 transplanted untreated kidney samples compared to transplanted PARP inhibitor treated kidney samples. The vertical axes represent pixel volume means±SEM of the scanned bands of the immunoblots in arbitrary units.</p

Effect of PARP inhibitor on the Bax and Bcl-2 immunohistology in transplanted and control kidneys.

<p>(A) All images show Bax immunohistochemistry, brown color indicates Bax positivity, scale bar: 20 µm. (B). Quantitative analysis of Bax immunohistochemistry samples *: p<0.0001 untreated transplanted kidneys compared to others. ; **: p<0.0001 PARP inhibitor treated transplanted kidneys compared to others. ANOVA, Bonferroni post hoc test. Mean±SD. (C) All images show Bcl-2 immunohistochemistry, brown color indicates Bcl-2 positivity, scale bar: 20 µm. All details described under Materials and Methods. (D) Quantitative analysis of the Bcl-2 immunohistochemistry. *: p<0,0001 transplanted and PARP-treated kidneys compared to others. ANOVA, Bonferroni post hoc test. Mean±SD. All details described under Materials and Methods.</p

Effect of PARP inhibitor on the structure of tubulo-interstitial system of transplanted kidneys.

<p>Kidneys were fixed in 10% formalin, embedded into paraffin and 5 µm thin sections were cut with microtome. Sections were stained with hematoxylin–eosin (HE). Two representative images of different magnifications are presented for untreated unoperated (Cont.), 4OHQ treated unoperated (Cont.+4OHQ), untreated transplanted (Transp.), and 4OHQ treated transplanted kidneys.Scale bar: 100 and 20 µm for upper and lower row, respectively.</p