NFAT5 protects astrocytes against oxygen-glucose-serum deprivation/restoration damage via the SIRT1/Nrf2 pathway.

Nuclear factor of activated T cells (NFAT) is a multifunctional cytokine family. NFAT5 was recently reported to be involved in many neuronal functions, but its specific function remains unclear. In this study, our aim is to investigate whether NFAT5 overexpression can protect astrocytes against oxygen-glucose-serum deprivation/restoration (OGSD/R) damage. In vivo, rats were subjected to ischemia-reperfusion injury, resulting in increased water content, infarct volume, and expression of NFAT5 protein in rat spinal cord. After primary culture for spinal cord astrocytes, the in vitro OGSD/R model was established. The results of the CCK8 assay and flow cytometry showed that, in the OGSD/R group, astrocyte cell viability was downregulated, but astrocyte apoptosis increased. Caspase 3 activity increased as well. Levels of NFAT5, as detected by real-time quantitative PCR and western blot, decreased under OGSD/R, as did SIRT1. Commercial kits for activity assays were used to show that OGSD/R inhibited SIRT1 activation but accelerated SOD activation after OGSD/R. Next, pcDNA-NFAT5 or NFAT5 siRNA was transfected into astrocytes. Overexpression of NFAT5 not only promoted the survival of the astrocytes and SIRT1 activation under OGSD/R but also inhibited cell apoptosis and SOD activation. Moreover, overexpression of NFAT5 apparently diminished histone acetylation and promoted the nuclear transport of Nrf2. Our results show that NFAT5 protects spinal astrocytes in a manner that depends on activation of the SIRT1/Nrf2 pathway. These findings present a novel potential molecular mechanism for NFAT5 therapy in the context of spinal cord injury.

Impact of Heat Shock Protein A 12B Overexpression on Spinal Astrocyte Survival Against Oxygen-Glucose-Serum Deprivation/Restoration in Primary Cultured Astrocytes.

Heat shock protein A 12B (HSPA12B) is a newly discovered member of the heat shock protein 70 family. Preclinical evidence indicates that HSPA12B helps protect the brain from ischemic injury, although its specific function remains unclear. The aim of this study is to investigate whether HSPA12B overexpression can protect astrocytes from oxygen-glucose-serum deprivation/restoration (OGD/R) injury. We analyzed the effects of HSPA12B overexpression on spinal cord ischemia-reperfusion injury and spinal astrocyte survival. After ischemia-reperfusion injury, we found that HSPA12B overexpression decreased spinal cord water content and infarct volume. MTT assay showed that HSPA12B overexpression increased astrocyte survival after OGD/R treatment. Flow cytometry results showed a marked inhibition of OGD/R-induced astrocyte apoptosis. Western blot assay showed that HSPA12B overexpression significantly increased regulatory protein B-cell lymphocyte 2 (Bcl-2) levels, whereas it decreased expression of the Bax protein, which forms a heterodimer with Bcl-2. Measurements of the level of activation of caspase-3 by Caspase-Glo®3/7 Assay kit showed that HSPA12B overexpression markedly inhibited caspase-3 activation. Notably, we demonstrated that the effects of HSPA12B on spinal astrocyte survival depended on activation of the PI3K/Akt signal pathway. These findings indicate that HSPA12B protects against spinal cord ischemia-reperfusion injury and may represent a potential treatment target.

Analyzing time-series microarray data reveals key genes in spinal cord injury.

Although many scholars have utilized high-throughput microarrays to delineate gene expression patterns after spinal cord injury (SCI), no study has evaluated gene changes in raphe magnus (RM) and somatomotor cortex (SMTC), two areas in brain primarily affected by SCI. In present study, we aimed to analyze the differentially expressed genes (DEGs) of RM and SMTC between SCI model and sham injured control at 4, 24 h, 7, 14, 28 days, and 3 months using microarray dataset GSE2270 downloaded from gene expression omnibus and unpaired significance analysis of microarray method. Protein-protein interaction (PPI) network was constructed for DEGs at crucial time points and significant biological functions were enriched using DAVID. The results indicated that more DEGs were identified at 14 days in RM and at 4 h/3 months in SMTC after SCI. In the PPI network for DEGs at 14 days in RM, interleukin 6, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), FBJ murine osteosarcoma viral oncogene homolog (FOS), tumor necrosis factor, and nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) were the top 5 hub genes; In the PPI network for DEGs at 3 months in SMTC, the top 5 hub genes were ubiquitin B, Ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1), FOS, Janus kinase 2 and vascular endothelial growth factor A. Hedgehog and Wnt signaling pathways were the top 2 significant pathways in RM. These hub DEGs and pathways may be underlying therapeutic targets for SCI.

Prognostic value of coexistence of abnormal expression of micro-RNA-200b and cyclic adenosine monophosphate-responsive element-binding protein 1 in human astrocytoma.

Our aim was to investigate the expression of micro-RNA-200b (miR-200b) and cAMP-responsive element-binding protein 1 (CREB-1) in astrocytoma and its efficacy for predicting outcome. Both miR-200b and CREB-1 messenger RNA expression was measured in 122 astrocytomas and 30 nonneoplastic brain specimens by quantitative real-time polymerase chain reaction. Expression of miR-200b was significantly lower in astrocytoma than in nonneoplastic brain (P < .001), whereas CREB-1 messenger RNA expression was significantly elevated in the tumors (P < .001). Both miR-200b down-regulation and CREB-1 up-regulation were significantly associated with advanced pathologic grade (P = .002 and P = .006, respectively). Low miR-200b expression correlated negatively with Karnofsky performance score (P = .03), and high CREB-1 expression correlated positively with mean tumor diameter (P = .03). By Kaplan-Meier analysis, low miR-200b, high CREB-1, and coexistence of abnormal miR-200b and CREB-1 expression (low miR-200b/high CREB-1) were predictive of shorter progression-free survival and overall survival in both grade III and grade IV astrocytoma. By multivariate analysis, only low miR-200b/high CREB-1 expression was an independent prognostic factor for poor prognosis in astrocytoma of advanced grade. Both miR-200b and CREB-1 may play important cooperative roles in the progression of human astrocytoma. The efficacy of miR-200b and CREB-1 together as a predictor of prognosis in astrocytoma patients is shown for the first time.