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Sulfiredoxin-1 exerts anti-apoptotic and neuroprotective effects against oxidative stress-induced injury in rat cortical astrocytes following exposure to oxygen-glucose deprivation and hydrogen peroxide.

Zhou Y, Zhou Y, Yu S, Wu J, Chen Y, Zhao Y - Int. J. Mol. Med. (2015)

Bottom Line: We found that following exposure to OGD or H2O2, the knockdown of Srxn1 resulted in a decrease in cell viability, as shown by MTS assay, an increase in cell damage, as shown by lactate dehydrogenase assay and an increase in cellular apoptosis, as shown by Hoechst 33342 staining and flow cytometry.Furthermore, we found that following exposure to OGD or H2O2, the knockdown of Srxn1 resulted in a decrease in mitochondrial transmembrane potential (Δψm) as indicated by JC-1 staining, an increase in the cytoplasmic expression of cytochrome c (Cyt.C), caspase-3, caspase-9, poly(ADP-ribose) polymerase (PARP) and Bax protein at the protein level, but a decrease in the expression of the anti-apoptotic Bcl-2 protein; these effects were tightly associated with the mitochondrial apoptotic pathway.However, we found that there was no obvious change in the intracellular calcium ([Ca2+]i) levels and caspase-12 expression following the knockdown of Srxn1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Chongqing Medical University, Chongqing 400016, P.R. China.

ABSTRACT
Sulfiredoxin 1 (Srxn1), an endogenous antioxidant protein, plays an important neuroprotective role in cerebral ischemia. However, the exact mechanisms of action of Srxn1 in cerebral ischemia have not yet been fully elucidated. Therefore, in the present study, rat primary cortical astrocytes transfected with a lentiviral vector encoding short hairpin RNA (shRNA) were exposed to oxygen-glucose deprivation (OGD) for 4 h or to 100 µM hydrogen peroxide (H2O2) for 6 h, in order to construct an in vitro model of cerebral ischemia-induced damage. We found that following exposure to OGD or H2O2, the knockdown of Srxn1 resulted in a decrease in cell viability, as shown by MTS assay, an increase in cell damage, as shown by lactate dehydrogenase assay and an increase in cellular apoptosis, as shown by Hoechst 33342 staining and flow cytometry. Furthermore, we found that following exposure to OGD or H2O2, the knockdown of Srxn1 resulted in a decrease in mitochondrial transmembrane potential (Δψm) as indicated by JC-1 staining, an increase in the cytoplasmic expression of cytochrome c (Cyt.C), caspase-3, caspase-9, poly(ADP-ribose) polymerase (PARP) and Bax protein at the protein level, but a decrease in the expression of the anti-apoptotic Bcl-2 protein; these effects were tightly associated with the mitochondrial apoptotic pathway. However, we found that there was no obvious change in the intracellular calcium ([Ca2+]i) levels and caspase-12 expression following the knockdown of Srxn1. Taken together, the results from the present study demonstrate that Srxn1 protects primary rat cortical astrocytes from OGD- or H2O2-induced apoptosis and that involves the activation of the mitochondrial apoptotic pathway, which suggests that Srxn1 may be a potential target in the treatment of cerebral ischemia.

No MeSH data available.


Related in: MedlinePlus

Effect of exposure to oxygen-glucose deprivation (OGD) or hydrogen peroxide (H2O2) and the knockdown of sulfiredoxin 1 (Srxn1) on the expression levels of mitochondrial apoptotic pathway-related proteins. Representative western blots data quantification of (A and B) cytochrome c (Cyt.C), (A and C) caspase-9, (A and D) caspase-3, (A and E) poly(ADP-ribose) polymerase (PARP), (A and F) Bax and (A and G) Bcl-2 expression in astrocytes. The results revealed that the levels of these proteins increased in response to exposure to OGD or H2O2. Following exposure to OGD or H2O2, Srxn1 knockdown promoted these expression levels, apart from Bcl-2 expression; the level of Bcl-2 decreased. Data are the means ± SEM; *P<0.05 and **P<0.01 vs. controls (untreated cells); #P<0.05 and ##P<0.01 vs. control + OGD (or H2O2) group, n=4. NC, negative control.
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f5-ijmm-36-01-0043: Effect of exposure to oxygen-glucose deprivation (OGD) or hydrogen peroxide (H2O2) and the knockdown of sulfiredoxin 1 (Srxn1) on the expression levels of mitochondrial apoptotic pathway-related proteins. Representative western blots data quantification of (A and B) cytochrome c (Cyt.C), (A and C) caspase-9, (A and D) caspase-3, (A and E) poly(ADP-ribose) polymerase (PARP), (A and F) Bax and (A and G) Bcl-2 expression in astrocytes. The results revealed that the levels of these proteins increased in response to exposure to OGD or H2O2. Following exposure to OGD or H2O2, Srxn1 knockdown promoted these expression levels, apart from Bcl-2 expression; the level of Bcl-2 decreased. Data are the means ± SEM; *P<0.05 and **P<0.01 vs. controls (untreated cells); #P<0.05 and ##P<0.01 vs. control + OGD (or H2O2) group, n=4. NC, negative control.

Mentions: To evaluate whether the mitochondrial apoptotic pathway was activated, we determined Δψm using JC-1 staining (Fig. 4), as well as the expression levels of some mitochondrial apoptotic pathway-related proteins by western blot analysis (Fig. 5). At a high membrane potential, JC-1 enters the mitochondria and forms aggregates that have a fluorescence of bright red, whereas JC-1 exists as a green fluorescence at a low potential (21). As shown in Fig. 4A, the untreated cell mitochondria exhibited a high Δψm, as indicated by a bright, red fluorescence and a very low green fluorescence (Fig. 4A panels a and b). The knockdown of Srxn1 decreased Δψm, and thus the green fluorescence increased significantly compared with the red fluorescence (Fig. 4A panel c). In addition, following exposure to OGD or H2O2, the green fluorescence was stronger than the red fluorescence [control + OGD (or H2O2) group], compared with the untreated control group (Fig. 4A panels d, e, g and h). Following exposure to OGD or H2O2, Srxn1 knockdown resulted in a further decrease in Δψm, and thus the green fluorescence became much stronger and the red fluorescence became much weaker (Fig. 4A panels f and i). To quantify the level of Δψm, the JC-1 fluorescence ratio was calculated using the average optical density ratio of red/green. The JC-1 ratio decreased by ~38% in the sh-Srxn1 group, by 32% in the control + OGD group and by 31% in the control + H2O2 group, compared with the untreated control group (P<0.05; Fig. 4B). Moreover, following exposure to OGD or H2O2, Srxn1 knockdown markedly decreased the JC-1 ratio in the sh-Srxn1 + OGD (or H2O2) group compared with the control + OGD (or H2O2) group (P<0.05), representing a dissipation of Δψm. These data indicated that Srxn1 knockdown induced the dissipation of Δψm in the astrocytes following exposure to OGD or H2O2.


Sulfiredoxin-1 exerts anti-apoptotic and neuroprotective effects against oxidative stress-induced injury in rat cortical astrocytes following exposure to oxygen-glucose deprivation and hydrogen peroxide.

Zhou Y, Zhou Y, Yu S, Wu J, Chen Y, Zhao Y - Int. J. Mol. Med. (2015)

Effect of exposure to oxygen-glucose deprivation (OGD) or hydrogen peroxide (H2O2) and the knockdown of sulfiredoxin 1 (Srxn1) on the expression levels of mitochondrial apoptotic pathway-related proteins. Representative western blots data quantification of (A and B) cytochrome c (Cyt.C), (A and C) caspase-9, (A and D) caspase-3, (A and E) poly(ADP-ribose) polymerase (PARP), (A and F) Bax and (A and G) Bcl-2 expression in astrocytes. The results revealed that the levels of these proteins increased in response to exposure to OGD or H2O2. Following exposure to OGD or H2O2, Srxn1 knockdown promoted these expression levels, apart from Bcl-2 expression; the level of Bcl-2 decreased. Data are the means ± SEM; *P<0.05 and **P<0.01 vs. controls (untreated cells); #P<0.05 and ##P<0.01 vs. control + OGD (or H2O2) group, n=4. NC, negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4494579&req=5

f5-ijmm-36-01-0043: Effect of exposure to oxygen-glucose deprivation (OGD) or hydrogen peroxide (H2O2) and the knockdown of sulfiredoxin 1 (Srxn1) on the expression levels of mitochondrial apoptotic pathway-related proteins. Representative western blots data quantification of (A and B) cytochrome c (Cyt.C), (A and C) caspase-9, (A and D) caspase-3, (A and E) poly(ADP-ribose) polymerase (PARP), (A and F) Bax and (A and G) Bcl-2 expression in astrocytes. The results revealed that the levels of these proteins increased in response to exposure to OGD or H2O2. Following exposure to OGD or H2O2, Srxn1 knockdown promoted these expression levels, apart from Bcl-2 expression; the level of Bcl-2 decreased. Data are the means ± SEM; *P<0.05 and **P<0.01 vs. controls (untreated cells); #P<0.05 and ##P<0.01 vs. control + OGD (or H2O2) group, n=4. NC, negative control.
Mentions: To evaluate whether the mitochondrial apoptotic pathway was activated, we determined Δψm using JC-1 staining (Fig. 4), as well as the expression levels of some mitochondrial apoptotic pathway-related proteins by western blot analysis (Fig. 5). At a high membrane potential, JC-1 enters the mitochondria and forms aggregates that have a fluorescence of bright red, whereas JC-1 exists as a green fluorescence at a low potential (21). As shown in Fig. 4A, the untreated cell mitochondria exhibited a high Δψm, as indicated by a bright, red fluorescence and a very low green fluorescence (Fig. 4A panels a and b). The knockdown of Srxn1 decreased Δψm, and thus the green fluorescence increased significantly compared with the red fluorescence (Fig. 4A panel c). In addition, following exposure to OGD or H2O2, the green fluorescence was stronger than the red fluorescence [control + OGD (or H2O2) group], compared with the untreated control group (Fig. 4A panels d, e, g and h). Following exposure to OGD or H2O2, Srxn1 knockdown resulted in a further decrease in Δψm, and thus the green fluorescence became much stronger and the red fluorescence became much weaker (Fig. 4A panels f and i). To quantify the level of Δψm, the JC-1 fluorescence ratio was calculated using the average optical density ratio of red/green. The JC-1 ratio decreased by ~38% in the sh-Srxn1 group, by 32% in the control + OGD group and by 31% in the control + H2O2 group, compared with the untreated control group (P<0.05; Fig. 4B). Moreover, following exposure to OGD or H2O2, Srxn1 knockdown markedly decreased the JC-1 ratio in the sh-Srxn1 + OGD (or H2O2) group compared with the control + OGD (or H2O2) group (P<0.05), representing a dissipation of Δψm. These data indicated that Srxn1 knockdown induced the dissipation of Δψm in the astrocytes following exposure to OGD or H2O2.

Bottom Line: We found that following exposure to OGD or H2O2, the knockdown of Srxn1 resulted in a decrease in cell viability, as shown by MTS assay, an increase in cell damage, as shown by lactate dehydrogenase assay and an increase in cellular apoptosis, as shown by Hoechst 33342 staining and flow cytometry.Furthermore, we found that following exposure to OGD or H2O2, the knockdown of Srxn1 resulted in a decrease in mitochondrial transmembrane potential (Δψm) as indicated by JC-1 staining, an increase in the cytoplasmic expression of cytochrome c (Cyt.C), caspase-3, caspase-9, poly(ADP-ribose) polymerase (PARP) and Bax protein at the protein level, but a decrease in the expression of the anti-apoptotic Bcl-2 protein; these effects were tightly associated with the mitochondrial apoptotic pathway.However, we found that there was no obvious change in the intracellular calcium ([Ca2+]i) levels and caspase-12 expression following the knockdown of Srxn1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Chongqing Medical University, Chongqing 400016, P.R. China.

ABSTRACT
Sulfiredoxin 1 (Srxn1), an endogenous antioxidant protein, plays an important neuroprotective role in cerebral ischemia. However, the exact mechanisms of action of Srxn1 in cerebral ischemia have not yet been fully elucidated. Therefore, in the present study, rat primary cortical astrocytes transfected with a lentiviral vector encoding short hairpin RNA (shRNA) were exposed to oxygen-glucose deprivation (OGD) for 4 h or to 100 µM hydrogen peroxide (H2O2) for 6 h, in order to construct an in vitro model of cerebral ischemia-induced damage. We found that following exposure to OGD or H2O2, the knockdown of Srxn1 resulted in a decrease in cell viability, as shown by MTS assay, an increase in cell damage, as shown by lactate dehydrogenase assay and an increase in cellular apoptosis, as shown by Hoechst 33342 staining and flow cytometry. Furthermore, we found that following exposure to OGD or H2O2, the knockdown of Srxn1 resulted in a decrease in mitochondrial transmembrane potential (Δψm) as indicated by JC-1 staining, an increase in the cytoplasmic expression of cytochrome c (Cyt.C), caspase-3, caspase-9, poly(ADP-ribose) polymerase (PARP) and Bax protein at the protein level, but a decrease in the expression of the anti-apoptotic Bcl-2 protein; these effects were tightly associated with the mitochondrial apoptotic pathway. However, we found that there was no obvious change in the intracellular calcium ([Ca2+]i) levels and caspase-12 expression following the knockdown of Srxn1. Taken together, the results from the present study demonstrate that Srxn1 protects primary rat cortical astrocytes from OGD- or H2O2-induced apoptosis and that involves the activation of the mitochondrial apoptotic pathway, which suggests that Srxn1 may be a potential target in the treatment of cerebral ischemia.

No MeSH data available.


Related in: MedlinePlus