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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

Effects of oxygen-glucose deprivation (OGD) on morphological characteristics, and assessment of cell death in Neuro-2a (N2a) cells. (A) Changes in nuclei by DAPI staining. N2a cells were treated with OGD, and stained with DAPI. Apoptotic cells were measured under fluorescence microscopy as described in Materials and methods. Apoptotic cells indicate condensed and fragmented nuclei (red arrows). Magnification, ×200; (B) After an indicated treatment for 24 h, the cells were stained with FITC-conjugated Annexin-V and PI for flow cytometry analysis as described in Materials and Methods. The percentages presented in each frame depicted the apoptotic cells.
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ijms-16-11873-f005: Effects of oxygen-glucose deprivation (OGD) on morphological characteristics, and assessment of cell death in Neuro-2a (N2a) cells. (A) Changes in nuclei by DAPI staining. N2a cells were treated with OGD, and stained with DAPI. Apoptotic cells were measured under fluorescence microscopy as described in Materials and methods. Apoptotic cells indicate condensed and fragmented nuclei (red arrows). Magnification, ×200; (B) After an indicated treatment for 24 h, the cells were stained with FITC-conjugated Annexin-V and PI for flow cytometry analysis as described in Materials and Methods. The percentages presented in each frame depicted the apoptotic cells.

Mentions: It was reported in a previous study that the deprivation of oxygen and glucose (OGD) in the ischemic brain eventually leads to cell death. Accumulation of unfolded protein response and oxidative stress were believed to be the main contributors to neuron death and ER stress-inducing involvement of the signaling pathway by apoptosis [14,16]. It was necessary to determine whether the OGD model of neuronal ischemia is cytotoxic to human N2a cells and to the molecular mechanisms underlying the OGD-mediated activation of IRE1-alpha inducing Jun N-terminal kinase (JNK1/2) and p38 MAP Kinase (MAPK). The MTT assays were performed after N2a cells were exposed to an ischemic environment for 24 h to examine the protein levels under regulation of ER stress. As shown in Figure 5, exposure to OGD for 24 h caused N2a cells to show the characteristic features of cell shrinking, rounding, and apoptotic body formation. The extent of apoptosis of OGD induction was quantified as a percentage of annexin V-positive cells and was shown as 18%. OGD treatment of human N2a cells also resulted in decreased levels of COMT and CATD, while OGD induced CALB2 expression (Figure 5). We therefore found that, compared to the control group, elicitation with OGD significantly increased total lysate protein CALB2 and decreased COMT and CATD. In addition, endogenous IRE1-alpha co-immunoprecipitated with TRAF2 in N2a cells induced after two and three hours of OGD doses. Taken together, previous results showed that increased ROS production induced by OGD [14] is often ER stress-associated with the up-regulation of IRE1-alpha/TRAF2, JNK1/2, and p38 MAPK pathways as a mechanism increasing ischemic injury [17,18]. We detected one protein, CALB2, a calcium-binding protein, which previously had been coordinated in the endoplasmic reticulum-mitochondria and the intrinsic apoptotic pathway [19]. To further examine OGD-induced damage in N2a cells, we studied the effects of kinase inhibitor as it gave resistance to OGD-induced apoptosis and oxidative damage. As shown in Table 2, N2a cells were followed to OGD and then co-treated with inhibitors, p38/MAPK inhibitor SB203580, JNK1/2 MAPK inhibitor SP600125 as well as the ROS scavenger NAC groups. This treatment significantly reversed the OGD-induced apoptosis to 21%, 10%, 8% and 5%, respectively (p < 0.01) and then increased SOD activity of neuron cells to 35%, 77%, 80% and 88%, respectively (p < 0.01); however, ERK1/2 MAPK inhibitor had no effect.


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)

Effects of oxygen-glucose deprivation (OGD) on morphological characteristics, and assessment of cell death in Neuro-2a (N2a) cells. (A) Changes in nuclei by DAPI staining. N2a cells were treated with OGD, and stained with DAPI. Apoptotic cells were measured under fluorescence microscopy as described in Materials and methods. Apoptotic cells indicate condensed and fragmented nuclei (red arrows). Magnification, ×200; (B) After an indicated treatment for 24 h, the cells were stained with FITC-conjugated Annexin-V and PI for flow cytometry analysis as described in Materials and Methods. The percentages presented in each frame depicted the apoptotic cells.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-11873-f005: Effects of oxygen-glucose deprivation (OGD) on morphological characteristics, and assessment of cell death in Neuro-2a (N2a) cells. (A) Changes in nuclei by DAPI staining. N2a cells were treated with OGD, and stained with DAPI. Apoptotic cells were measured under fluorescence microscopy as described in Materials and methods. Apoptotic cells indicate condensed and fragmented nuclei (red arrows). Magnification, ×200; (B) After an indicated treatment for 24 h, the cells were stained with FITC-conjugated Annexin-V and PI for flow cytometry analysis as described in Materials and Methods. The percentages presented in each frame depicted the apoptotic cells.
Mentions: It was reported in a previous study that the deprivation of oxygen and glucose (OGD) in the ischemic brain eventually leads to cell death. Accumulation of unfolded protein response and oxidative stress were believed to be the main contributors to neuron death and ER stress-inducing involvement of the signaling pathway by apoptosis [14,16]. It was necessary to determine whether the OGD model of neuronal ischemia is cytotoxic to human N2a cells and to the molecular mechanisms underlying the OGD-mediated activation of IRE1-alpha inducing Jun N-terminal kinase (JNK1/2) and p38 MAP Kinase (MAPK). The MTT assays were performed after N2a cells were exposed to an ischemic environment for 24 h to examine the protein levels under regulation of ER stress. As shown in Figure 5, exposure to OGD for 24 h caused N2a cells to show the characteristic features of cell shrinking, rounding, and apoptotic body formation. The extent of apoptosis of OGD induction was quantified as a percentage of annexin V-positive cells and was shown as 18%. OGD treatment of human N2a cells also resulted in decreased levels of COMT and CATD, while OGD induced CALB2 expression (Figure 5). We therefore found that, compared to the control group, elicitation with OGD significantly increased total lysate protein CALB2 and decreased COMT and CATD. In addition, endogenous IRE1-alpha co-immunoprecipitated with TRAF2 in N2a cells induced after two and three hours of OGD doses. Taken together, previous results showed that increased ROS production induced by OGD [14] is often ER stress-associated with the up-regulation of IRE1-alpha/TRAF2, JNK1/2, and p38 MAPK pathways as a mechanism increasing ischemic injury [17,18]. We detected one protein, CALB2, a calcium-binding protein, which previously had been coordinated in the endoplasmic reticulum-mitochondria and the intrinsic apoptotic pathway [19]. To further examine OGD-induced damage in N2a cells, we studied the effects of kinase inhibitor as it gave resistance to OGD-induced apoptosis and oxidative damage. As shown in Table 2, N2a cells were followed to OGD and then co-treated with inhibitors, p38/MAPK inhibitor SB203580, JNK1/2 MAPK inhibitor SP600125 as well as the ROS scavenger NAC groups. This treatment significantly reversed the OGD-induced apoptosis to 21%, 10%, 8% and 5%, respectively (p < 0.01) and then increased SOD activity of neuron cells to 35%, 77%, 80% and 88%, respectively (p < 0.01); however, ERK1/2 MAPK inhibitor had no effect.

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