Zinc finger protein 667 expression is upregulated by cerebral ischemic preconditioning and protects cells from oxidative stress

Abstract

Brain ischemic injury is associated with clinical emergencies such as acute ischemic and hemorrhagic stroke, head trauma, prolonged severe hypotension and cardiac arrest. Ischemic preconditioning (IPC) is the most powerful endogenous mechanism against ischemic injury. However, the majority of IPC treatments are invasive and thus impractical in the clinical setting. Identifying the endogenous neuroprotective mechanism induced by IPC is important for developing new strategies to reduce stroke severity. Zinc finger protein 667 (ZNF667) is a novel zinc finger protein that is upregulated by myocardial IPC. However, its functional role in neuronal ischemia has not been elucidated. In this study, the changes of ZNF667 expression on cerebral IPC and its potential neuroprotective function were investigated. The cerebral ischemia model was established by ameliorated four-vessel occlusion in rats. The northern blot results demonstrated that ZNF667 expression was increased in the hippocampus and cortex at 12 and 24 h after cerebral ischemic pretreatment. To investigate the neuroprotective function of ZNF667, enhanced green fluorescent protein (EGFP)-ZNF667 fusion protein was expressed in C2C12 and brain astrocytoma cells and its subcellular localization was detected by confocal microscopy. EGFP-ZNF667 fusion proteins were localized in the nucleus of C2C12 and brain astrocytoma cells, indicating that ZNF667 may act as a transcription factor in neural cells. To mimic oxidative stress associated with ischemia/reperfusion injury, hydrogen peroxide (H₂O₂) was used to treat cells. Cell viability was measured by the lactate dehydrogenase (LDH) and WST-1 assays. A decrease in viability was detected in C2C12 and astrocytoma cells following H₂O₂ treatment, whereas ZNF667 gene overexpression significantly improved cell viability following H₂O₂ treatment. These results suggested that ZNF667 plays a neuroprotective role by acting as a transcription factor in cerebral IPC.

Keywords: cell protection; cerebral ischemic preconditioning; gene expression; oxidative stress; zinc finger protein 667.

Figure 1.

Cerebral ischemic pretreatment (IPC) upregulates zinc finger protein 667 (ZNF667) mRNA levels in rat brains. Northern blot analyses of ZNF667 mRNA expression levels in rat brain (A) hippocampus and (B) cortex at various time points following cerebral IPC. The band intensity of ZNF667 mRNA was normalized to that of 18S RNA bands. A significant increase in ZNF667 mRNA expression was oberved in the hippocampus and cortex 12 and 24 h after IPC treatment. ^*P<0.05 vs. sham operation group (#).

Figure 2.

Exogenously expressed zinc finger protein 667 (ZNF667) is localized in the nucleus of C2C12 and brain astrocytoma cells. Confocal microscopy images of (A) C2C12 cells and (B) brain astrocytoma cells transfected with pEGFP-N1 (control) and pEGFP-N1/ZNF667 constructs. Green fluorescent protein (GFP) fluorescence was observed under confocal microscopy after 24 h of transfection. Control enhanced GFP (EGFP) fluorescence was shown to be localized in the cytoplasm and nucleus, whereas EGFP-ZNF667 fusion protein was localized only in the nucleus.

Figure 3.

Overexpression of zinc finger protein 667 (ZNF667) reduces lactate dehydrogenase (LDH) release from C2C12 and brain astrocytoma cells induced by H₂O₂ treatment. (A) C2C12 cells and (B) brain astrocytoma cells were transfected with pcDNA3.1 vector (pcDNA3.1) and pcDNA3/ZNF667 (ZNF667) constructs. Following transfection, cells were treated with 0.5 mmol/l H₂O₂ for 3 h. After exposure of the cells to oxidative stress, cell damage was assessed by the LDH release assay. An increase of LDH release was detected in C2C12 and astrocytoma control cells after H₂O₂ treatment, whereas ZNF667 gene overexpression significantly reduced LDH release induced by H₂O₂ (^*P<0.01 vs. pcDNA3.1 control (ctrl), n=6; ^#P<0.01 vs. pcDNA3.1 H₂O₂, n=6).

Figure 4.

Overexpression of zinc finger protein 667 (ZNF667) increased the viability of C2C12 and brain astrocytoma cells treated with H₂O₂. (A) C2C12 cells and (B) brain astrocytoma cells transfected with pcDNA3.1 vector (pcDNA3.1) and pcDNA3/ZNF667 (ZNF667) were treated with 0.5 mmol/l H₂O₂ for 24 h. Cell viability was then determined by the WST-1 assay. A decrease in cell viability was detected in C2C12 and astrocytoma control cells after H₂O₂ treatment, whereas ZNF667 gene overexpression significantly increased cell viability after H₂O₂ treatment (^*P<0.01 vs. pcDNA3.1 control (ctrl), n=6; ^#P<0.01 vs. pcDNA3.1 H₂O₂, n=6).