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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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Elevation of ΔΨm in 661W cells by SS31. (A) Treatment with 100 nM SS31 for 4 h increased the ΔΨm in the 100 µM t-BHP-treated 661W cells, demonstrated using flow cytometric analysis. (B) Quantitative analysis of the relative ratio of red/green fluorescence intensity of mitochondrial staining. Treatment with 100 nM SS31 significantly increased the red/green fluorescence intensity of the 661W cells (78.18±6.67% of the control; **P<0.05, vs. t-BHP; *P<0.05, vs. control). Data are presented as the mean ± standard error of the mean. ΔΨm, mitochondrial membrane potential.
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f5-mmr-12-04-5026: Elevation of ΔΨm in 661W cells by SS31. (A) Treatment with 100 nM SS31 for 4 h increased the ΔΨm in the 100 µM t-BHP-treated 661W cells, demonstrated using flow cytometric analysis. (B) Quantitative analysis of the relative ratio of red/green fluorescence intensity of mitochondrial staining. Treatment with 100 nM SS31 significantly increased the red/green fluorescence intensity of the 661W cells (78.18±6.67% of the control; **P<0.05, vs. t-BHP; *P<0.05, vs. control). Data are presented as the mean ± standard error of the mean. ΔΨm, mitochondrial membrane potential.

Mentions: To determine the involvement of the mitochondrial-mediated pathway in oxidative stress induced cell dysfunction, the present study measured ΔΨm using flow cytometry, using the cationic membrane potential indicator JC-1. Treatment with 100 nM SS31 for 4 h prevented the 100 µM t-BHP-induced loss of ΔΨm in the 661W cells (Fig. 5A). Compared with the untreated control cultures, exposure to 100 µM t-BHP for 4 h resulted in a rapid decrease in the red/green fluorescence intensity ratio to 51.49±7.59% of the control (P<0.01; Fig. 5B). Treatment with 100 nM SS31 significantly increased the red/green fluorescence intensity of the 661W cells to 78.18±6.67% of the control (P<0.05), which indicated that the ΔΨm was restored to baseline.


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)

Elevation of ΔΨm in 661W cells by SS31. (A) Treatment with 100 nM SS31 for 4 h increased the ΔΨm in the 100 µM t-BHP-treated 661W cells, demonstrated using flow cytometric analysis. (B) Quantitative analysis of the relative ratio of red/green fluorescence intensity of mitochondrial staining. Treatment with 100 nM SS31 significantly increased the red/green fluorescence intensity of the 661W cells (78.18±6.67% of the control; **P<0.05, vs. t-BHP; *P<0.05, vs. control). Data are presented as the mean ± standard error of the mean. ΔΨm, mitochondrial membrane potential.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5-mmr-12-04-5026: Elevation of ΔΨm in 661W cells by SS31. (A) Treatment with 100 nM SS31 for 4 h increased the ΔΨm in the 100 µM t-BHP-treated 661W cells, demonstrated using flow cytometric analysis. (B) Quantitative analysis of the relative ratio of red/green fluorescence intensity of mitochondrial staining. Treatment with 100 nM SS31 significantly increased the red/green fluorescence intensity of the 661W cells (78.18±6.67% of the control; **P<0.05, vs. t-BHP; *P<0.05, vs. control). Data are presented as the mean ± standard error of the mean. ΔΨm, mitochondrial membrane potential.
Mentions: To determine the involvement of the mitochondrial-mediated pathway in oxidative stress induced cell dysfunction, the present study measured ΔΨm using flow cytometry, using the cationic membrane potential indicator JC-1. Treatment with 100 nM SS31 for 4 h prevented the 100 µM t-BHP-induced loss of ΔΨm in the 661W cells (Fig. 5A). Compared with the untreated control cultures, exposure to 100 µM t-BHP for 4 h resulted in a rapid decrease in the red/green fluorescence intensity ratio to 51.49±7.59% of the control (P<0.01; Fig. 5B). Treatment with 100 nM SS31 significantly increased the red/green fluorescence intensity of the 661W cells to 78.18±6.67% of the control (P<0.05), which indicated that the ΔΨm was restored to baseline.

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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