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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

Knockdown of sulfiredoxin 1 (Srxn1) increases apoptosis following exposure to oxygen-glucose deprivation (OGD) or hydrogen peroxide (H2O2). (A and B) Apoptosis was assessed by Hoechst 33342 staining and visualized by fluorescence. Exposure to OGD or H2O2 increased cell apoptosis. Following exposure to OGD or H2O2, the knockdown of Srxn1 increased apoptosis increased more significantly. Scale bar, 20 μm. Results are expressed as the means ± SEM; **P<0.01 vs. controls (untreated cells); ##P<0.01 vs. control + OGD (or H2O2) group, n=4. (C and D) Flow cytometric analysis of apoptosis and necrosis. (C) Typical representative dot plots of the distribution of normal (Annexin V−/PI−), early apoptotic (Annexin V+/PI−), late apoptotic (Annexin V+/PI+) and necrotic (Annexin V−/PI+) cells. (D) Late-apoptotic and early-apoptotic astrocytes were calculated from the total astrocytes. Following exposure to OGD or H2O2, the knockdown of Srxn1 markedly increased apoptosis. Data are the means ± SEM; **P<0.01 vs. controls (untreated cells); ##P<0.01 vs. control + OGD (or H2O2) group, n=4. NC, negative control (unreated cells).
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f3-ijmm-36-01-0043: Knockdown of sulfiredoxin 1 (Srxn1) increases apoptosis following exposure to oxygen-glucose deprivation (OGD) or hydrogen peroxide (H2O2). (A and B) Apoptosis was assessed by Hoechst 33342 staining and visualized by fluorescence. Exposure to OGD or H2O2 increased cell apoptosis. Following exposure to OGD or H2O2, the knockdown of Srxn1 increased apoptosis increased more significantly. Scale bar, 20 μm. Results are expressed as the means ± SEM; **P<0.01 vs. controls (untreated cells); ##P<0.01 vs. control + OGD (or H2O2) group, n=4. (C and D) Flow cytometric analysis of apoptosis and necrosis. (C) Typical representative dot plots of the distribution of normal (Annexin V−/PI−), early apoptotic (Annexin V+/PI−), late apoptotic (Annexin V+/PI+) and necrotic (Annexin V−/PI+) cells. (D) Late-apoptotic and early-apoptotic astrocytes were calculated from the total astrocytes. Following exposure to OGD or H2O2, the knockdown of Srxn1 markedly increased apoptosis. Data are the means ± SEM; **P<0.01 vs. controls (untreated cells); ##P<0.01 vs. control + OGD (or H2O2) group, n=4. NC, negative control (unreated cells).

Mentions: Apoptosis was detected by combining microscopic analysis with Hoechst 33342 staining (Fig. 3A and B) and FCM (Fig. 3C and D). Compared with the control group, exposure to OGD (or H2O2) induced apoptosis (control + OGD group and control + H2O2 group) (P<0.01), and following the knockdown of Srxn1, the cell apoptotic rate increased more significantly (sh-Srxn1 + OGD group and sh-Srxn1 + H2O2 group) compared with the control + OGD (or H2O2) group (P<0.01; Fig. 3A and B). Annexin V-FITC/PI double staining revealed a significant increase in apoptotis by ~29% in the control + OGD group and by 31% in the control + H2O2 group, compared with the control group (P<0.01; Fig. 3C and D). In addition, following exposure to OGD or H2O2, the knockdown of Srxn1 resulted in an even more significant increase in apoptotis (P<0.01), compared with the control + OGD (or H2O2) group. Taken together, these results indicate that Srxn1 plays a vital role in protecting astrocytes from apoptosis induced by 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)

Knockdown of sulfiredoxin 1 (Srxn1) increases apoptosis following exposure to oxygen-glucose deprivation (OGD) or hydrogen peroxide (H2O2). (A and B) Apoptosis was assessed by Hoechst 33342 staining and visualized by fluorescence. Exposure to OGD or H2O2 increased cell apoptosis. Following exposure to OGD or H2O2, the knockdown of Srxn1 increased apoptosis increased more significantly. Scale bar, 20 μm. Results are expressed as the means ± SEM; **P<0.01 vs. controls (untreated cells); ##P<0.01 vs. control + OGD (or H2O2) group, n=4. (C and D) Flow cytometric analysis of apoptosis and necrosis. (C) Typical representative dot plots of the distribution of normal (Annexin V−/PI−), early apoptotic (Annexin V+/PI−), late apoptotic (Annexin V+/PI+) and necrotic (Annexin V−/PI+) cells. (D) Late-apoptotic and early-apoptotic astrocytes were calculated from the total astrocytes. Following exposure to OGD or H2O2, the knockdown of Srxn1 markedly increased apoptosis. Data are the means ± SEM; **P<0.01 vs. controls (untreated cells); ##P<0.01 vs. control + OGD (or H2O2) group, n=4. NC, negative control (unreated cells).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4494579&req=5

f3-ijmm-36-01-0043: Knockdown of sulfiredoxin 1 (Srxn1) increases apoptosis following exposure to oxygen-glucose deprivation (OGD) or hydrogen peroxide (H2O2). (A and B) Apoptosis was assessed by Hoechst 33342 staining and visualized by fluorescence. Exposure to OGD or H2O2 increased cell apoptosis. Following exposure to OGD or H2O2, the knockdown of Srxn1 increased apoptosis increased more significantly. Scale bar, 20 μm. Results are expressed as the means ± SEM; **P<0.01 vs. controls (untreated cells); ##P<0.01 vs. control + OGD (or H2O2) group, n=4. (C and D) Flow cytometric analysis of apoptosis and necrosis. (C) Typical representative dot plots of the distribution of normal (Annexin V−/PI−), early apoptotic (Annexin V+/PI−), late apoptotic (Annexin V+/PI+) and necrotic (Annexin V−/PI+) cells. (D) Late-apoptotic and early-apoptotic astrocytes were calculated from the total astrocytes. Following exposure to OGD or H2O2, the knockdown of Srxn1 markedly increased apoptosis. Data are the means ± SEM; **P<0.01 vs. controls (untreated cells); ##P<0.01 vs. control + OGD (or H2O2) group, n=4. NC, negative control (unreated cells).
Mentions: Apoptosis was detected by combining microscopic analysis with Hoechst 33342 staining (Fig. 3A and B) and FCM (Fig. 3C and D). Compared with the control group, exposure to OGD (or H2O2) induced apoptosis (control + OGD group and control + H2O2 group) (P<0.01), and following the knockdown of Srxn1, the cell apoptotic rate increased more significantly (sh-Srxn1 + OGD group and sh-Srxn1 + H2O2 group) compared with the control + OGD (or H2O2) group (P<0.01; Fig. 3A and B). Annexin V-FITC/PI double staining revealed a significant increase in apoptotis by ~29% in the control + OGD group and by 31% in the control + H2O2 group, compared with the control group (P<0.01; Fig. 3C and D). In addition, following exposure to OGD or H2O2, the knockdown of Srxn1 resulted in an even more significant increase in apoptotis (P<0.01), compared with the control + OGD (or H2O2) group. Taken together, these results indicate that Srxn1 plays a vital role in protecting astrocytes from apoptosis induced by 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