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Global proteomic analysis of brain tissues in transient ischemia brain damage in rats.

Chen JH, Kuo HC, Lee KF, Tsai TH - Int J Mol Sci (2015)

Bottom Line: It mediated the effects of SAM administration on the apoptotic and ER stress pathways.Our results demonstrate that the ischemic injury of neuronal cells increased cell cytoxicity and apoptosis, which were accompanied by sustained activation of the IRE1-alpha/TRAF2, JNK1/2, and p38 MAPK pathways.Based on these results, we also provide the molecular evidence supporting the ischemia-reperfusion-related neuronal injury.

View Article: PubMed Central - PubMed

Affiliation: Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei 112, Taiwan. chenjiannhwa@yahoo.com.tw.

ABSTRACT
Ischemia-reperfusion injury resulting from arterial occlusion or hypotension in patients leads to tissue hypoxia with glucose deprivation, which causes endoplasmic reticulum (ER) stress and neuronal death. A proteomic approach was used to identify the differentially expressed proteins in the brain of rats following a global ischemic stroke. The mechanisms involved the action in apoptotic and ER stress pathways. Rats were treated with ischemia-reperfusion brain injuries by the bilateral occlusion of the common carotid artery. The cortical neuron proteins from the stroke animal model (SAM) and the control rats were separated using two-dimensional gel electrophoresis (2-DE) to purify and identify the protein profiles. Our results demonstrated that the SAM rats experienced brain cell death in the ischemic core. Fifteen proteins were expressed differentially between the SAM rats and control rats, which were assayed and validated in vivo and in vitro. Interestingly, the set of differentially expressed, down-regulated proteins included catechol O-methyltransferase (COMT) and cathepsin D (CATD), which are implicated in oxidative stress, inflammatory response and apoptosis. After an ischemic stroke, one protein spot, namely the calretinin (CALB2) protein, showed increased expression. It mediated the effects of SAM administration on the apoptotic and ER stress pathways. Our results demonstrate that the ischemic injury of neuronal cells increased cell cytoxicity and apoptosis, which were accompanied by sustained activation of the IRE1-alpha/TRAF2, JNK1/2, and p38 MAPK pathways. Proteomic analysis suggested that the differential expression of CALB2 during a global ischemic stroke could be involved in the mechanisms of ER stress-induced neuronal cell apoptosis, which occurred via IRE1-alpha/TRAF2 complex formation, with activation of JNK1/2 and p38 MAPK. Based on these results, we also provide the molecular evidence supporting the ischemia-reperfusion-related neuronal injury.

No MeSH data available.


Related in: MedlinePlus

Immunohistochemistry staining for indicated proteins in rat brains. Validation of the identified differential proteins and oxygen-glucose deprivation (OGD)-associated molecules between untreated (Control) and treated (OGD) in N2a cells. (A) Data presented in western blots are derived from a representative study, and comparisons of protein expression are calculated from three replicate experiments. The association of TRAF2 with IRE1-a was determined by immunoprecipitation with TRAF2 followed by western blot with anti-IRE1-alpha antibody; (B) Effects of the kinase inhibitors blocking OGD-induced expression of ER stress related proteins. Kinase inhibitors were treated with or without OGD after 1 h. Total cell lysates were prepared and subjected to western blot analysis. Protein levels of phosphorylated JNK1/2 and p38 MAPK and the association of TRAF2 with IRE1-alpha and β-actin were detected with the indicated antibodies. * p < 0.05, compared with control group.
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ijms-16-11873-f006: Immunohistochemistry staining for indicated proteins in rat brains. Validation of the identified differential proteins and oxygen-glucose deprivation (OGD)-associated molecules between untreated (Control) and treated (OGD) in N2a cells. (A) Data presented in western blots are derived from a representative study, and comparisons of protein expression are calculated from three replicate experiments. The association of TRAF2 with IRE1-a was determined by immunoprecipitation with TRAF2 followed by western blot with anti-IRE1-alpha antibody; (B) Effects of the kinase inhibitors blocking OGD-induced expression of ER stress related proteins. Kinase inhibitors were treated with or without OGD after 1 h. Total cell lysates were prepared and subjected to western blot analysis. Protein levels of phosphorylated JNK1/2 and p38 MAPK and the association of TRAF2 with IRE1-alpha and β-actin were detected with the indicated antibodies. * p < 0.05, compared with control group.

Mentions: Our results clearly demonstrated that OGD resulted in neuron cell death and that oxidative damage was relative to the expression of CALB2 and activation of ER stress-signaling pathways. Recent studies have indicated that regulation of excess calcium release results from oxidative stress, and many calcium-binding proteins, such as CALB2, may function downstream of the ER calcium release to modulate apoptosis in response to neuronal damage [20]. To investigate the role of CALB2 in OGD-mediated oxidative impairment and ER stress signaling, a basic study was conducted to demonstrate the novel role of CALB2 by a brief event of OGD. As shown in Figure 6, NAC almost blocked the OGD-elicited expression of CALB2 as well as the phosphorylation of JNK1/2 and p38 MAPK and the interaction of IRE1-alpha/TRAF2 complex, as compared to the OGD model of 1 h. In addition, OGD co-treated with p38/MAPK inhibitor SB203580 resulted in the inhibition of the OGD-induced expression of the CALB2 and IRE1-alpha/TRAF2 complex. At the same time, JNK1/2 MAPK inhibitor SP600125 reduced the effects on the IRE1-alpha/TRAF2 association (Figure 6). Taken together, the results showed that up-regulation of CALB2 was essentially involved in oxidative stress-signaling OGD-influenced cell death with both IRE1-alpha/TRAF2 and JNK1/2 and p38 MAPK-related pathways. The results confirmed that OGD-treated ischemic injury was associated with the IRE1-alpha/TRAF2, JNK1/2, and p38 MAPK-dependent signal transduction and increases in CALB2 expression.


Global proteomic analysis of brain tissues in transient ischemia brain damage in rats.

Chen JH, Kuo HC, Lee KF, Tsai TH - Int J Mol Sci (2015)

Immunohistochemistry staining for indicated proteins in rat brains. Validation of the identified differential proteins and oxygen-glucose deprivation (OGD)-associated molecules between untreated (Control) and treated (OGD) in N2a cells. (A) Data presented in western blots are derived from a representative study, and comparisons of protein expression are calculated from three replicate experiments. The association of TRAF2 with IRE1-a was determined by immunoprecipitation with TRAF2 followed by western blot with anti-IRE1-alpha antibody; (B) Effects of the kinase inhibitors blocking OGD-induced expression of ER stress related proteins. Kinase inhibitors were treated with or without OGD after 1 h. Total cell lysates were prepared and subjected to western blot analysis. Protein levels of phosphorylated JNK1/2 and p38 MAPK and the association of TRAF2 with IRE1-alpha and β-actin were detected with the indicated antibodies. * p < 0.05, compared with control group.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490420&req=5

ijms-16-11873-f006: Immunohistochemistry staining for indicated proteins in rat brains. Validation of the identified differential proteins and oxygen-glucose deprivation (OGD)-associated molecules between untreated (Control) and treated (OGD) in N2a cells. (A) Data presented in western blots are derived from a representative study, and comparisons of protein expression are calculated from three replicate experiments. The association of TRAF2 with IRE1-a was determined by immunoprecipitation with TRAF2 followed by western blot with anti-IRE1-alpha antibody; (B) Effects of the kinase inhibitors blocking OGD-induced expression of ER stress related proteins. Kinase inhibitors were treated with or without OGD after 1 h. Total cell lysates were prepared and subjected to western blot analysis. Protein levels of phosphorylated JNK1/2 and p38 MAPK and the association of TRAF2 with IRE1-alpha and β-actin were detected with the indicated antibodies. * p < 0.05, compared with control group.
Mentions: Our results clearly demonstrated that OGD resulted in neuron cell death and that oxidative damage was relative to the expression of CALB2 and activation of ER stress-signaling pathways. Recent studies have indicated that regulation of excess calcium release results from oxidative stress, and many calcium-binding proteins, such as CALB2, may function downstream of the ER calcium release to modulate apoptosis in response to neuronal damage [20]. To investigate the role of CALB2 in OGD-mediated oxidative impairment and ER stress signaling, a basic study was conducted to demonstrate the novel role of CALB2 by a brief event of OGD. As shown in Figure 6, NAC almost blocked the OGD-elicited expression of CALB2 as well as the phosphorylation of JNK1/2 and p38 MAPK and the interaction of IRE1-alpha/TRAF2 complex, as compared to the OGD model of 1 h. In addition, OGD co-treated with p38/MAPK inhibitor SB203580 resulted in the inhibition of the OGD-induced expression of the CALB2 and IRE1-alpha/TRAF2 complex. At the same time, JNK1/2 MAPK inhibitor SP600125 reduced the effects on the IRE1-alpha/TRAF2 association (Figure 6). Taken together, the results showed that up-regulation of CALB2 was essentially involved in oxidative stress-signaling OGD-influenced cell death with both IRE1-alpha/TRAF2 and JNK1/2 and p38 MAPK-related pathways. The results confirmed that OGD-treated ischemic injury was associated with the IRE1-alpha/TRAF2, JNK1/2, and p38 MAPK-dependent signal transduction and increases in CALB2 expression.

Bottom Line: It mediated the effects of SAM administration on the apoptotic and ER stress pathways.Our results demonstrate that the ischemic injury of neuronal cells increased cell cytoxicity and apoptosis, which were accompanied by sustained activation of the IRE1-alpha/TRAF2, JNK1/2, and p38 MAPK pathways.Based on these results, we also provide the molecular evidence supporting the ischemia-reperfusion-related neuronal injury.

View Article: PubMed Central - PubMed

Affiliation: Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei 112, Taiwan. chenjiannhwa@yahoo.com.tw.

ABSTRACT
Ischemia-reperfusion injury resulting from arterial occlusion or hypotension in patients leads to tissue hypoxia with glucose deprivation, which causes endoplasmic reticulum (ER) stress and neuronal death. A proteomic approach was used to identify the differentially expressed proteins in the brain of rats following a global ischemic stroke. The mechanisms involved the action in apoptotic and ER stress pathways. Rats were treated with ischemia-reperfusion brain injuries by the bilateral occlusion of the common carotid artery. The cortical neuron proteins from the stroke animal model (SAM) and the control rats were separated using two-dimensional gel electrophoresis (2-DE) to purify and identify the protein profiles. Our results demonstrated that the SAM rats experienced brain cell death in the ischemic core. Fifteen proteins were expressed differentially between the SAM rats and control rats, which were assayed and validated in vivo and in vitro. Interestingly, the set of differentially expressed, down-regulated proteins included catechol O-methyltransferase (COMT) and cathepsin D (CATD), which are implicated in oxidative stress, inflammatory response and apoptosis. After an ischemic stroke, one protein spot, namely the calretinin (CALB2) protein, showed increased expression. It mediated the effects of SAM administration on the apoptotic and ER stress pathways. Our results demonstrate that the ischemic injury of neuronal cells increased cell cytoxicity and apoptosis, which were accompanied by sustained activation of the IRE1-alpha/TRAF2, JNK1/2, and p38 MAPK pathways. Proteomic analysis suggested that the differential expression of CALB2 during a global ischemic stroke could be involved in the mechanisms of ER stress-induced neuronal cell apoptosis, which occurred via IRE1-alpha/TRAF2 complex formation, with activation of JNK1/2 and p38 MAPK. Based on these results, we also provide the molecular evidence supporting the ischemia-reperfusion-related neuronal injury.

No MeSH data available.


Related in: MedlinePlus