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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 the knockdown of sulfiredoxin 1 (Srxn1) on endoplasmic reticulum stress pathway-mediated apoptosis. (A and B) Intracellular calcium ([Ca2+]i) levels were measured by laser scanning confocal microscopy. Images showed that green fluorescence had a fluo-3 fluorescence intensity. The stronger the fluorescence for [Ca2+]i, the higher the expression. Compared with the controls (untreated cells), the level of [Ca2+]i markedly increased in the control + oxygen-glucose deprivation (OGD) [or hydrogen peroxide (H2O2)] group, and the level of [Ca2+]i did not change significantly after the knockdown of Srxn1. Data are the means ± SEM; *P<0.05 vs. controls (untreated cells), n=4; (C and D) Western blot analysis for caspase-12. Exposure to OGD or H2O2 increased the level of caspase-12, but it did not affect caspase-12 expression after the knockdown of Srxn1. Data are the means ± SEM; *P<0.05 vs. controls (untreated cells), n=4.
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f6-ijmm-36-01-0043: Effect of the knockdown of sulfiredoxin 1 (Srxn1) on endoplasmic reticulum stress pathway-mediated apoptosis. (A and B) Intracellular calcium ([Ca2+]i) levels were measured by laser scanning confocal microscopy. Images showed that green fluorescence had a fluo-3 fluorescence intensity. The stronger the fluorescence for [Ca2+]i, the higher the expression. Compared with the controls (untreated cells), the level of [Ca2+]i markedly increased in the control + oxygen-glucose deprivation (OGD) [or hydrogen peroxide (H2O2)] group, and the level of [Ca2+]i did not change significantly after the knockdown of Srxn1. Data are the means ± SEM; *P<0.05 vs. controls (untreated cells), n=4; (C and D) Western blot analysis for caspase-12. Exposure to OGD or H2O2 increased the level of caspase-12, but it did not affect caspase-12 expression after the knockdown of Srxn1. Data are the means ± SEM; *P<0.05 vs. controls (untreated cells), n=4.

Mentions: In order to determine whether endoplasmic reticulum stress pathway-mediated apoptosis had occurred, we detected [Ca2+]i and caspase-12 levels based on Fluo-3 AM staining (Fig. 6A and B) and western blot analysis (Fig. 6C and D). Of note, we found that the levels of caspase-12 and [Ca2+]i increased following exposure to OGD or H2O2 compared with the control group; however, after the knockdown of Srxn1, these levels did not differ from those of the control + OGD (or H2O2) group. Hence, we deduced that the neuroprotective effects of Srxn1 are not related to endoplasmic reticulum stress pathway-mediated apoptosis.


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 the knockdown of sulfiredoxin 1 (Srxn1) on endoplasmic reticulum stress pathway-mediated apoptosis. (A and B) Intracellular calcium ([Ca2+]i) levels were measured by laser scanning confocal microscopy. Images showed that green fluorescence had a fluo-3 fluorescence intensity. The stronger the fluorescence for [Ca2+]i, the higher the expression. Compared with the controls (untreated cells), the level of [Ca2+]i markedly increased in the control + oxygen-glucose deprivation (OGD) [or hydrogen peroxide (H2O2)] group, and the level of [Ca2+]i did not change significantly after the knockdown of Srxn1. Data are the means ± SEM; *P<0.05 vs. controls (untreated cells), n=4; (C and D) Western blot analysis for caspase-12. Exposure to OGD or H2O2 increased the level of caspase-12, but it did not affect caspase-12 expression after the knockdown of Srxn1. Data are the means ± SEM; *P<0.05 vs. controls (untreated cells), n=4.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6-ijmm-36-01-0043: Effect of the knockdown of sulfiredoxin 1 (Srxn1) on endoplasmic reticulum stress pathway-mediated apoptosis. (A and B) Intracellular calcium ([Ca2+]i) levels were measured by laser scanning confocal microscopy. Images showed that green fluorescence had a fluo-3 fluorescence intensity. The stronger the fluorescence for [Ca2+]i, the higher the expression. Compared with the controls (untreated cells), the level of [Ca2+]i markedly increased in the control + oxygen-glucose deprivation (OGD) [or hydrogen peroxide (H2O2)] group, and the level of [Ca2+]i did not change significantly after the knockdown of Srxn1. Data are the means ± SEM; *P<0.05 vs. controls (untreated cells), n=4; (C and D) Western blot analysis for caspase-12. Exposure to OGD or H2O2 increased the level of caspase-12, but it did not affect caspase-12 expression after the knockdown of Srxn1. Data are the means ± SEM; *P<0.05 vs. controls (untreated cells), n=4.
Mentions: In order to determine whether endoplasmic reticulum stress pathway-mediated apoptosis had occurred, we detected [Ca2+]i and caspase-12 levels based on Fluo-3 AM staining (Fig. 6A and B) and western blot analysis (Fig. 6C and D). Of note, we found that the levels of caspase-12 and [Ca2+]i increased following exposure to OGD or H2O2 compared with the control group; however, after the knockdown of Srxn1, these levels did not differ from those of the control + OGD (or H2O2) group. Hence, we deduced that the neuroprotective effects of Srxn1 are not related to endoplasmic reticulum stress pathway-mediated apoptosis.

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