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Protective effects of SS31 on t‑BHP induced oxidative damage in 661W cells.

Ma W, Zhu X, Ding X, Li T, Hu Y, Hu X, Yuan L, Lei L, Hu A, Luo Y, Tang S - Mol Med Rep (2015)

Bottom Line: The viability of the cells improved following treatment with SS31 between 100 nM and 1 µM, compared with untreated control group.Compared with the t‑BHP treatment group (20.0±3.8%), the number of annexin V‑positive cells decreased dose‑dependently to 13.6±2.6, 9.8±0.5 and 7.4±2.0% in the SS‑31 treated group at concentrations of 10 nM, 100 nM and 1 µM, respectively.Treatment with SS‑31 significantly prevented the t‑BHP‑induced expression of nitrotyrosine and 8‑OHdG, decreased the quantity of mitochondrial ROS, increased mitochondrial potential, and prevented the release of cytochrome c from mitochondria into the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat‑Sen University, Guangzhou, Guangdong 510060, P.R. China.

ABSTRACT
The present study aimed to investigate the ability of SS31, a novel mitochondria‑targeted peptide to protect against t‑BHP‑induced mitochondrial dysfunction and apoptosis in 661W cell lines. The 661W cells were treated with various concentrations of SS‑31 and an MTT assay was used to determine cell viability. The expression of nitrotyrosine and 8‑hydroxydeoxyguanosine (8‑OHdG) was detected using immunofluorescent staining. Apoptosis were assessed using Hoechst staining and an annexin V/propidium iodide flow cytometer. Reactive oxygen species (ROS) were detected using MitoSOXTM with confocal microscopy. Changes in mitochondrial membrane potential were analyzed using flow cytometry. In addition, the release of cytochrome c was analyzed using confocal microscopy. The viability of the cells improved following treatment with SS31 between 100 nM and 1 µM, compared with untreated control group. Compared with the t‑BHP treatment group (20.0±3.8%), the number of annexin V‑positive cells decreased dose‑dependently to 13.6±2.6, 9.8±0.5 and 7.4±2.0% in the SS‑31 treated group at concentrations of 10 nM, 100 nM and 1 µM, respectively. Treatment with SS‑31 significantly prevented the t‑BHP‑induced expression of nitrotyrosine and 8‑OHdG, decreased the quantity of mitochondrial ROS, increased mitochondrial potential, and prevented the release of cytochrome c from mitochondria into the cytoplasm. Therefore, the SS31 mitochondria‑targeted peptide protected the 661W cells from the sustained oxidative stress induced by t‑BHP.

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Inhibitory effect of SS31 on t-BHP-induced mitochondrial release of ROS. (A and B) t-BHP increased mitochondrial superoxide generation in the 661W cells in a time- and dose-dependent manner. (C) Representative confocal images of 661W cells, revealing an increase in mitochondrial MitoSOX™ fluorescence (red) following treatment with 100 µM t-BHP for 1 h. Treatment with 100 nM SS31 significantly ameliorated the t-BHP- induced increase in mitochondrial Immunofluorescence was analyzed using LSM510 META confocal microscopy (magnification, ×1,000). ROS, reactive oxygen species.
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f4-mmr-12-04-5026: Inhibitory effect of SS31 on t-BHP-induced mitochondrial release of ROS. (A and B) t-BHP increased mitochondrial superoxide generation in the 661W cells in a time- and dose-dependent manner. (C) Representative confocal images of 661W cells, revealing an increase in mitochondrial MitoSOX™ fluorescence (red) following treatment with 100 µM t-BHP for 1 h. Treatment with 100 nM SS31 significantly ameliorated the t-BHP- induced increase in mitochondrial Immunofluorescence was analyzed using LSM510 META confocal microscopy (magnification, ×1,000). ROS, reactive oxygen species.

Mentions: To further examine the SS31 protective mechanisms, the present study investigated its effect on mitochondrial ROS. MitoSOX™, a specific dye for mitochondrial ROS, was used to detect mitochondrial ROS. Confocal microscopy was used to localize the oxidative stress-induced increase of ROS production in the mitochondria of the 661W cells using MitoSOX™. Quantitative measurements of the mean fluorescence intensities from the samples demonstrated that t-BHP increased mitochondrial superoxide generation in the 661W cells in a dose- and time-dependent manner (Fig. 4A and B). The MitoSOX™ fluorescence intensity per mm2 cell area increased over time, beginning at 1 h (11.0±2.0; P<0.001, vs. control). The mitochondrial ROS was markedly increased following incubation with 100 µM t-BHP for 1 h, whereas treatment with 100 nM SS31 significantly ameliorated the oxidative stress-induced increase in mitochondrial ROS (Fig. 4C).


Protective effects of SS31 on t‑BHP induced oxidative damage in 661W cells.

Ma W, Zhu X, Ding X, Li T, Hu Y, Hu X, Yuan L, Lei L, Hu A, Luo Y, Tang S - Mol Med Rep (2015)

Inhibitory effect of SS31 on t-BHP-induced mitochondrial release of ROS. (A and B) t-BHP increased mitochondrial superoxide generation in the 661W cells in a time- and dose-dependent manner. (C) Representative confocal images of 661W cells, revealing an increase in mitochondrial MitoSOX™ fluorescence (red) following treatment with 100 µM t-BHP for 1 h. Treatment with 100 nM SS31 significantly ameliorated the t-BHP- induced increase in mitochondrial Immunofluorescence was analyzed using LSM510 META confocal microscopy (magnification, ×1,000). ROS, reactive oxygen species.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581771&req=5

f4-mmr-12-04-5026: Inhibitory effect of SS31 on t-BHP-induced mitochondrial release of ROS. (A and B) t-BHP increased mitochondrial superoxide generation in the 661W cells in a time- and dose-dependent manner. (C) Representative confocal images of 661W cells, revealing an increase in mitochondrial MitoSOX™ fluorescence (red) following treatment with 100 µM t-BHP for 1 h. Treatment with 100 nM SS31 significantly ameliorated the t-BHP- induced increase in mitochondrial Immunofluorescence was analyzed using LSM510 META confocal microscopy (magnification, ×1,000). ROS, reactive oxygen species.
Mentions: To further examine the SS31 protective mechanisms, the present study investigated its effect on mitochondrial ROS. MitoSOX™, a specific dye for mitochondrial ROS, was used to detect mitochondrial ROS. Confocal microscopy was used to localize the oxidative stress-induced increase of ROS production in the mitochondria of the 661W cells using MitoSOX™. Quantitative measurements of the mean fluorescence intensities from the samples demonstrated that t-BHP increased mitochondrial superoxide generation in the 661W cells in a dose- and time-dependent manner (Fig. 4A and B). The MitoSOX™ fluorescence intensity per mm2 cell area increased over time, beginning at 1 h (11.0±2.0; P<0.001, vs. control). The mitochondrial ROS was markedly increased following incubation with 100 µM t-BHP for 1 h, whereas treatment with 100 nM SS31 significantly ameliorated the oxidative stress-induced increase in mitochondrial ROS (Fig. 4C).

Bottom Line: The viability of the cells improved following treatment with SS31 between 100 nM and 1 µM, compared with untreated control group.Compared with the t‑BHP treatment group (20.0±3.8%), the number of annexin V‑positive cells decreased dose‑dependently to 13.6±2.6, 9.8±0.5 and 7.4±2.0% in the SS‑31 treated group at concentrations of 10 nM, 100 nM and 1 µM, respectively.Treatment with SS‑31 significantly prevented the t‑BHP‑induced expression of nitrotyrosine and 8‑OHdG, decreased the quantity of mitochondrial ROS, increased mitochondrial potential, and prevented the release of cytochrome c from mitochondria into the cytoplasm.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat‑Sen University, Guangzhou, Guangdong 510060, P.R. China.

ABSTRACT
The present study aimed to investigate the ability of SS31, a novel mitochondria‑targeted peptide to protect against t‑BHP‑induced mitochondrial dysfunction and apoptosis in 661W cell lines. The 661W cells were treated with various concentrations of SS‑31 and an MTT assay was used to determine cell viability. The expression of nitrotyrosine and 8‑hydroxydeoxyguanosine (8‑OHdG) was detected using immunofluorescent staining. Apoptosis were assessed using Hoechst staining and an annexin V/propidium iodide flow cytometer. Reactive oxygen species (ROS) were detected using MitoSOXTM with confocal microscopy. Changes in mitochondrial membrane potential were analyzed using flow cytometry. In addition, the release of cytochrome c was analyzed using confocal microscopy. The viability of the cells improved following treatment with SS31 between 100 nM and 1 µM, compared with untreated control group. Compared with the t‑BHP treatment group (20.0±3.8%), the number of annexin V‑positive cells decreased dose‑dependently to 13.6±2.6, 9.8±0.5 and 7.4±2.0% in the SS‑31 treated group at concentrations of 10 nM, 100 nM and 1 µM, respectively. Treatment with SS‑31 significantly prevented the t‑BHP‑induced expression of nitrotyrosine and 8‑OHdG, decreased the quantity of mitochondrial ROS, increased mitochondrial potential, and prevented the release of cytochrome c from mitochondria into the cytoplasm. Therefore, the SS31 mitochondria‑targeted peptide protected the 661W cells from the sustained oxidative stress induced by t‑BHP.

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