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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

Evaluation of the efficiency of short hairpin RNA (shRNA) against sulfiredoxin 1 (Srxn1) following exposure to oxygen-glucose deprivation (OGD) or hydrogen peroxide (H2O2). (A and B) Srxn1 protein expression was measured by western blot analysis. (C) Srxn1 mRNA expression was determined by RT-qPCR. The results showed that Srxn1 protein and mRNA expression were decreased by shRNA Srxn1 knockdown and that sensitivity to OGD or H2O2 was increased. The exposure to astrocytes to OGD or H2O2 resulted in a significant increase in Srxn1 protein and mRNA expression, and Srxn1 knockdown prevented this increase. 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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f1-ijmm-36-01-0043: Evaluation of the efficiency of short hairpin RNA (shRNA) against sulfiredoxin 1 (Srxn1) following exposure to oxygen-glucose deprivation (OGD) or hydrogen peroxide (H2O2). (A and B) Srxn1 protein expression was measured by western blot analysis. (C) Srxn1 mRNA expression was determined by RT-qPCR. The results showed that Srxn1 protein and mRNA expression were decreased by shRNA Srxn1 knockdown and that sensitivity to OGD or H2O2 was increased. The exposure to astrocytes to OGD or H2O2 resulted in a significant increase in Srxn1 protein and mRNA expression, and Srxn1 knockdown prevented this increase. 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: The inhibition of Srxn1 expression in the presence of OGD or H2O2 was confirmed by western blot analysis (Fig. 1A and B) and RT-qPCR (Fig. 1C). Compared with the control group, Srxn1 knockdown resulted in a ~61% decrease in protein and a ~47% decrease in Srxn1 mRNA expression (Fig. 1). The exposure of the cells to OGD (or H2O2) resulted in a significant increase in Srxn1 protein and mRNA expression, and Srxn1 knockdown prevented this increase (sh-Srxn1 + OGD group and sh-Srxn1 + H2O2 group), compared with the control + OGD (or H2O2) group (P<0.05 and P<0.01). These results suggest that Srxn1 plays an important role in protecting astrocytes from damage 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)

Evaluation of the efficiency of short hairpin RNA (shRNA) against sulfiredoxin 1 (Srxn1) following exposure to oxygen-glucose deprivation (OGD) or hydrogen peroxide (H2O2). (A and B) Srxn1 protein expression was measured by western blot analysis. (C) Srxn1 mRNA expression was determined by RT-qPCR. The results showed that Srxn1 protein and mRNA expression were decreased by shRNA Srxn1 knockdown and that sensitivity to OGD or H2O2 was increased. The exposure to astrocytes to OGD or H2O2 resulted in a significant increase in Srxn1 protein and mRNA expression, and Srxn1 knockdown prevented this increase. 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

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

f1-ijmm-36-01-0043: Evaluation of the efficiency of short hairpin RNA (shRNA) against sulfiredoxin 1 (Srxn1) following exposure to oxygen-glucose deprivation (OGD) or hydrogen peroxide (H2O2). (A and B) Srxn1 protein expression was measured by western blot analysis. (C) Srxn1 mRNA expression was determined by RT-qPCR. The results showed that Srxn1 protein and mRNA expression were decreased by shRNA Srxn1 knockdown and that sensitivity to OGD or H2O2 was increased. The exposure to astrocytes to OGD or H2O2 resulted in a significant increase in Srxn1 protein and mRNA expression, and Srxn1 knockdown prevented this increase. 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: The inhibition of Srxn1 expression in the presence of OGD or H2O2 was confirmed by western blot analysis (Fig. 1A and B) and RT-qPCR (Fig. 1C). Compared with the control group, Srxn1 knockdown resulted in a ~61% decrease in protein and a ~47% decrease in Srxn1 mRNA expression (Fig. 1). The exposure of the cells to OGD (or H2O2) resulted in a significant increase in Srxn1 protein and mRNA expression, and Srxn1 knockdown prevented this increase (sh-Srxn1 + OGD group and sh-Srxn1 + H2O2 group), compared with the control + OGD (or H2O2) group (P<0.05 and P<0.01). These results suggest that Srxn1 plays an important role in protecting astrocytes from damage 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