Extracellular cold-inducible RNA-binding protein mediated neuroinflammation and neuronal apoptosis after traumatic brain injury.

Background: Extracellular cold-inducible RNA-binding protein (eCIRP) plays a vital role in the inflammatory response during cerebral ischaemia. However, the potential role and regulatory mechanism of eCIRP in traumatic brain injury (TBI) remain unclear. Here, we explored the effect of eCIRP on the development of TBI using a neural-specific CIRP knockout (KO) mouse model to determine the contribution of eCIRP to TBI-induced neuronal injury and to discover novel therapeutic targets for TBI. Methods: TBI animal models were generated in mice using the fluid percussion injury method. Microglia or neuron lines were subjected to different drug interventions. Histological and functional changes were observed by immunofluorescence and neurobehavioural testing. Apoptosis was examined by a TdT-mediated dUTP nick end labelling assay in vivo or by an annexin-V assay in vitro. Ultrastructural alterations in the cells were examined via electron microscopy. Tissue acetylation alterations were identified by non-labelled quantitative acetylation via proteomics. Protein or mRNA expression in cells and tissues was determined by western blot analysis or real-time quantitative polymerase chain reaction. The levels of inflammatory cytokines and mediators in the serum and supernatants were measured via enzyme-linked immunoassay. Results: There were closely positive correlations between eCIRP and inflammatory mediators, and between eCIRP and TBI markers in human and mouse serum. Neural-specific eCIRP KO decreased hemispheric volume loss and neuronal apoptosis and alleviated glial cell activation and neurological function damage after TBI. In contrast, eCIRP treatment resulted in endoplasmic reticulum disruption and ER stress (ERS)-related death of neurons and enhanced inflammatory mediators by glial cells. Mechanistically, we noted that eCIRP-induced neural apoptosis was associated with the activation of the protein kinase RNA-like ER kinase-activating transcription factor 4 (ATF4)-C/EBP homologous protein signalling pathway, and that eCIRP-induced microglial inflammation was associated with histone H3 acetylation and the α7 nicotinic acetylcholine receptor. Conclusions: These results suggest that TBI obviously enhances the secretion of eCIRP, thereby resulting in neural damage and inflammation in TBI. eCIRP may be a biomarker of TBI that can mediate the apoptosis of neuronal cells through the ERS apoptotic pathway and regulate the inflammatory response of microglia via histone modification.

Cold-inducible RNA-binding protein inhibits neuron apoptosis through the suppression of mitochondrial apoptosis.

Cold-inducible RNA-binding protein (CIRP) is induced by mild hypothermia in several mammals, but the precise mechanism by which CIRP mediates hypothermia-induced neuroprotection remains unknown. We aimed to investigate the molecular mechanisms by which CIRP protects the nervous system during mild hypothermia. Rat cortical neurons were isolated and cultured in vitro under mild hypothermia (32°C). Apoptosis was measured by annexin V and propidium iodide staining, visualized by flow cytometry. Neuron ultrastructure was visualized by transmission electron microscopy. CIRP overexpression and knockdown were achieved via infection with pL/IRES/GFP-CIRP and pL/shRNA/F-CIRP-A lentivirus. RT(2) Profiler PCR Array Pathway Analysis and western blotting were used to evaluate the effects of CIRP overexpresion/knockdown on the neurons׳ transcriptome. Neuron late apoptosis was significantly reduced at day 7 of culture by 12h hypothermia, but neuron ultrastructure remained relatively intact. RT(2) Profiler PCR Array Pathway Analysis of 84 apoptosis pathway-associated factors revealed that mild hypothermia and CIRP overexpression induce similar gene expression profiles, specifically alterations of genes implicated in the mitochondrial apoptosis pathway. Mild hypothermia-treated neurons up-regulated 12 and down-regulated 38 apoptosis pathway-associated genes. CIRP-overexpressing neurons up-regulated 15 and down-regulated 46 genes. CIRP-knocked-down hypothermia-treated cells up-regulated 9 and down-regulated 40 genes. Similar results were obtained at the protein level. In conclusion, CIRP may inhibit neuron apoptosis through the suppression of the mitochondria apoptosis pathway during mild hypothermia.

Restoration of tissue damage, and never activity after hypoxia-ischemia by implantation of peripheral blood mononuclear cells.

Hypoxia-ischemia (HI) encephalopathy is a frequent cause of disability and mortality with limited therapeutic options. Here, we collected peripheral blood mononuclear cells (PB-MNCs) from healthy donors and labeled them with CM-DiI before implanting these cells by tail-vein injection into rats at day 3 after hypoxia-ischemia (HI). For immune-suppression the animals received daily injections of cyclosporine throughout the experiment, commencing 24h before cell transplantation. Then we observed the PB-MNCs by fluorescent microscopy, examined motor function of rats by rotarod and cylinder tests, measured the lesion volume using image-pro plus software, and analyzed the apoptosis of neural cells in HI rats by tunnel assay. The results showed PB-MNCs could survive in the brain of hosts, migrate to the damage area and express neural marker. In addition, The HI rats that received PB-MNCs showed a reduction in motor function impairment, lesion volume and neural cell apoptosis. To better understand the mechanism of cell migration, PB-MNCs were also injected into normal rats via tail-vein. The expression of stromal cell-derived factor-1 (SDF-1) in the brain of normal and HI rats was measured by RT- PCR and western-blot, while the response of PB-MNCs in vitro to HI or normal brain extracts were measured by cell migration assay. Collectively these data suggest that the migration of PB-MNCs is directed to the damaged brain through an SDF-1-dependent pathway. Our results suggest that intravenous transplantation of PB-MNCs may be a feasible candidate for HI therapy.

Differential expression of the RNA-binding motif protein 3 in human astrocytoma.

BACKGROUND: The RNA-binding motif protein 3 (RBM3), which is transcriptionally induced by low temperature and hypoxia, has recently been found to be upregulated in human tumors. However, its expression status in human astrocytoma is not well defned. This article focuses on the differential expression of RBM3 in human astrocytomas of different grades and normal brain tissues. METHODS: RBM3 was detected in astrocytomas and normal brain tissues by quantitative real-time PCR, immunohistochemistry, and Western blotting. Analysis of variance was performed on the data from quantitative real-time PCR. The Fisher's exact test was used to analyze the immunohistochemistry results. A P-value of less than 0.05 indicates a statistically significant difference. RESULTS: On one hand, the mRNA expression levels of three X-chromosome-related RBM genes (RBMX, RBM3, and RBM10) were detected by quantitative real-time PCR. The results showed that there were no significant differences in RBMX and RBM10 mRNA expression levels in human astrocytomas of different grades and normal brain tissues. However, RBM3 mRNA expression levels were elevated in high-grade (World Health Organization (WHO) Grade III-IV) astrocytomas versus low-grade (WHO Grade I-II) astrocytomas (5.06 ± 0.66 vs. 1.60 ± 0.58; P < 0.05) or normal controls (5.06 ± 0.66 vs. 1.03 ± 0.22; P < 0.05) as determined by quantitative real-time PCR analysis. On the other hand, immunohistochemistry showed an increased RBM3 labeling index in astrocytomas of different grades and normal brain tissues (positive staining rate: astrocytoma Grade IV, 92.9%; astrocytoma Grade III, 81.8%; astrocytoma Grade I-II, 50%; normal brain tissues, 37.5%; high-grade astrocytoma versus normal brain tissues, P < 0.05; high-grade astrocytoma versus low-grade astrocytoma, P < 0.05). The higher protein levels of RBM3 were also validated in high-grade astrocytomas and low-grade astrocytomas compared with normal brain tissues by Western blotting. CONCLUSIONS: These data suggest that the overexpression of RBM3 may serve as an important molecular mechanism underlying astrocytic carcinogenesis. Moreover, RBM3 may have proliferative and/or proto-oncogenic functions in human astrocytomas.