Abstract

This study examines the effects of combination therapy of collagen scaffolds and human marrow stromal cells (hMSCs) on the expression of tissue plasminogen activator (tPA) after traumatic brain injury (TBI) in rats. Adult male Wistar rats (n=48) were injured with controlled cortical impact and treated either with scaffolds suffused with hMSCs (3×106) or hMSCs (3×106) alone transplanted into the lesion cavity 1 week after TBI. A control group was treated with saline. Neurological function was assessed using the Morris Water Maze test (MWM) and modified Neurological Severity Scores (mNSS). The rats were sacrificed 14 days after TBI and brain samples were processed for immunohistochemical analysis and quantitative Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) studies. Enhanced functional improvement was observed on both the mNSS and MWM tests in the scaffold+hMSC-treated group compared to the other two groups. Immunostaining with anti-human mitochondrial antibody (E5204) showed more hMSCs in the injury zone of the scaffold+hMSC group compared to the hMSC-alone group. Triple staining showed that more neurons were tPA-positive in the scaffold+hMSC group compared to the other two groups (p<0.05). Western blot analysis and qRT-PCR showed that scaffold+hMSC and hMSC-alone treatment enhanced the expression of tPA compared to controls (p<0.05), but tPA expression was significantly greater in the scaffold+hMSC group. The induction of tPA by hMSCs after TBI may be one of the mechanisms involved in promoting functional improvement after TBI.

Background

Our previous studies demonstrated that simvastatin reduced neuronal death, increased neurogenesis, and promoted functional recovery after TBI. Objective: To investigate the effect of simvastatin on angiogenesis after TBI, and the related signaling pathways.

Methods

Saline or simvastatin (1 mg/kg) was administered orally to rats starting at day 1 after TBI or sham surgery and then daily for 14 days. Rats were sacrificed at 3 and 14 days after treatment. Brain sections and tissues were prepared for immunohistochemical staining, ELISA, and Western blot analysis, respectively. Cultured rat brain microvascular endothelial cells (RBMVECs) were subjected to oxygen-glucose deprivation (OGD) followed by immunocytochemical staining with phallotoxins and vascular endothelial growth factor receptor-2 (VEGFR-2). Western blot analysis was carried out to examine the simvastatin-induced activation of the v-akt murine thymoma viral oncogene homolog (Akt) signaling pathway. The expression of VEGFR-2 was detected by ELISA.

Results

Simvastatin significantly increased the length of vascular perimeter, promoted the proliferation of endothelial cells, and improved the sensorimotor function after TBI. Simvastatin stimulated endothelial cell tube formation after OGD in vitro. VEGFR-2 expression in both brain tissues and cultured RBMVECs was enhanced after simvastatin treatment, which may be modulated by activation of Akt. Akt-dependent endothelial nitric oxide synthase (eNOS) phosphorylation was also induced by simvastatin in vivo and in vitro.

Conclusion

Simvastatin augments TBI-induced angiogenesis in the lesion boundary zone and hippocampus and improves functional recovery. Simvastatin also promotes angiogenesis in vitro. These beneficial effects on angiogenesis may be related to simvastatin-induced activation of the VEGFR-2/Akt/eNOS signaling pathway.

We have previously demonstrated that human marrow stromal cells (hMSCs) embedded in collagen I scaffolds significantly enhance the restorative therapeutic effect of hMSCs after traumatic brain injury (TBI). In this study, we test the hypothesis that the collagen scaffold alters gene expression in hMSCs and that hMSCs impregnated into scaffolds increase the astrocytic expression of vascular endothelial growth factor (VEGF) in the injured brain. Following TBI induced by controlled cortical impact injury, scaffold with hMSCs (3.0 × 106), hMSCs-only and saline were implanted into the lesion cavity one week after brain injury (n = 8/each group). Morris water Maze and modified neurological severity scores were performed to evaluate the spatial learning and sensorimotor functions, respectively. Lesion volume and expression of VEGF were measured one week after different treatments. In vitro, total RNA from hMSCs was extracted one week after culture with or without collagen I scaffold for evaluation of gene microarrays. Furthermore, an RT-PCR study on a select subgroup of genes was performed to identify the changes of expression between the culturing hMSCs with collagen scaffolds and hMSCs only. The treatment of TBI with collagen scaffold impregnated with hMSCs significantly decreases the functional deficits from TBI within 7 days after treatment, and significantly enhances the VEGF expression of astrocytes in the injured brain compared to the hMSCs-only group. In vitro data indicate that collagen scaffolds stimulate hMSCs to express multiple factors which may contribute to hMSC survival, tissue repair and functional recovery after TBI.

Objective

Our previous studies demonstrated that simvastatin treatment promotes neuronal survival and reduces inflammatory cytokine release from astrocytes after traumatic brain injury (TBI) in rats. Since reactive astrocytes produce inflammation mediators, in the current study we investigated the effect of simvastatin on astrocyte activation after TBI and its underlying signaling mechanisms.

Methods

Saline or simvastatin (1 mg/kg) was orally administered to rats starting at Day 1 after TBI and then daily for 14 days. Rats were sacrificed at 1, 3, 7, 14 days after treatment. Brain sections and tissues were prepared for immunohistochemical staining and Western blot analysis, respectively. Cultured astrocytes were subjected to oxygen-glucose deprivation (OGD) and followed by immunocytochemical staining with GFAP/caveolin-1 and Western blot analysis. Lipid rafts were isolated from the cell lysate and Western blot was carried out to detect the changes in epidermal growth factor receptor (EGFR) expression and phosphorylation in the lipid rafts.

Results

Simvastatin significantly promoted neuronal survival after TBI and attenuated activation of astrocytes. Simvastatin modified the caveolin-1 expression in lipid rafts in astrocyte cell membrane, suppressed the phosphorylation of EGFR in lipid rafts of astrocytes after OGD, and inhibited the OGD-induced interleukin-1 (IL-1) production.

Conclusions

These data suggest that simvastatin reduces reactive astrogliosis and rescues neuronal cells after TBI. These beneficial effects of simvastatin may be mediated by inhibiting astrocyte activation after TBI through modifying the caveolin-1 expression in lipid rafts and the subsequent modulation of EGFR phosphorylation in lipid rafts.

Objective

Our previous studies demonstrated that simvastatin promotes neurological functional recovery after traumatic brain injury (TBI) in rat; however, the underlying mechanisms remain poorly understood. The purpose of this study was to investigate the anti-inflammatory effect of simvastatin by measuring the level of cytokines and activation of glial cells.

Methods

Controlled cortical impact injury was performed in adult male Wistar rats. The rats were randomly divided into three groups: sham, saline control group and simvastatin treatment group. Simvastatin was administered orally starting at day 1 after TBI until sacrifice. Animals were sacrificed at 1, 3, 7, 14, and 35 days after treatment. Functional outcome was measured using modified neurological severity scores (mNSS). ELISA and immunohistochemical staining were employed to measure the expression of IL-1β, IL-6 and TNF-α, and to identify activated microglia and astrocytes.

Results

At days 1 and 3 after simvastatin or saline treatment, cytokine levels in the lesion boundary zone were significantly higher in the simvastatin-treated rats and saline-treated rats compared to the sham group, peaking at day 3. Simvastatin only reduced the level of IL-1 β but not IL-6 and TNF-α compared with the saline group. Also, simvastatin reduced significantly the number of activated microglia and astrocytes compared to the saline control animals. There was also a trend towards improvement of mNSS score, reaching statistical significance (P=0.003) towards the end of the trial.

Conclusion

Our data demonstrate that TBI causes inflammatory reaction, including increased levels of IL-1β, IL-6 and TNF-α, as well as activated microglia. Simvastatin selectively reduces IL-1β expression and inhibits the activation of microglia and astrocytes after TBI, which may be one of the mechanisms underlying the therapeutic benefits of simvastatin treatment of TBI.

Resveratrol (trans-3,4’, 5-trihydroxystilbene) is a naturally occurring polyphenolic compound that has anti-inflammatory, antioxidant, neuroprotective properties and acts as a chemopreventive agent. Resveratrol causes cell cycle arrest and induces apoptotic cell death in various types of cancer cells. In the current studies, the effect of resveratrol on phosphoinositide kinase-3 (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway was examined in human U251 glioma cells. Resveratrol decreased both the expression and phosphorylation of Akt. Inhibitors of PI3K (LY294002) and Akt (SH-6) enhanced resveratrol-induced LDH release and caspase-3 activation. Resveratrol reduced phosphorylation of ribosomal protein S6 and the mTOR inhibitor rapamycin further enhanced resveratrol-induced cell death. These results suggest that the downregulation of PI3K/Akt/mTOR signaling pathways may be an important mediator in resveratrol-induced apoptosis in glioma cells.

Resveratrol is a naturally occurring polyphenolic compound highly enriched in grapes, peanuts, red wine, and a variety of food sources. Sulforaphane belongs to the family of isothiocyanates and is highly enriched in cruciferous vegetables. Our previous study showed that resveratrol, when used at high concentrations, inhibited cell proliferation, caused the cell cycle arrest and induced apoptotic cell death in glioma cells. In the current study, we tested the effect of combination treatment with resveratrol and sulforaphane, when both were used at low concentrations, on cell proliferation, migration and death in human U251 glioma cells. Our study shows that combination treatment with resveratrol and sulforaphane inhibits cell proliferation and migration, reduces cell viability, induces lactate dehydrogenase release, decreases pro-survival Akt phosphorylation and increases caspase-3 activation. The use of combination of bioactive food components, such as resveratrol and sulforaphane, may be a viable approach for the treatment of glioma.

Motivation: In this work, we aim to develop a computational approach for predicting DNA-binding sites in proteins from amino acid sequences. To avoid overfitting with this method, all available DNA-binding proteins from the Protein Data Bank (PDB) are used to construct the models. The random forest (RF) algorithm is used because it is fast and has robust performance for different parameter values. A novel hybrid feature is presented which incorporates evolutionary information of the amino acid sequence, secondary structure (SS) information and orthogonal binary vector (OBV) information which reflects the characteristics of 20 kinds of amino acids for two physical–chemical properties (dipoles and volumes of the side chains). The numbers of binding and non-binding residues in proteins are highly unbalanced, so a novel scheme is proposed to deal with the problem of imbalanced datasets by downsizing the majority class.

Results: The results show that the RF model achieves 91.41% overall accuracy with Matthew's correlation coefficient of 0.70 and an area under the receiver operating characteristic curve (AUC) of 0.913. To our knowledge, the RF method using the hybrid feature is currently the computationally optimal approach for predicting DNA-binding sites in proteins from amino acid sequences without using three-dimensional (3D) structural information. We have demonstrated that the prediction results are useful for understanding protein–DNA interactions.

Availability: DBindR web server implementation is freely available at http://www.cbi.seu.edu.cn/DBindR/DBindR.htm.

Contact: xsun@seu.edu.cn

Supplementary information: Supplementary data are available at Bioinformatics online.

We have sequenced the genome of Shigella flexneri serotype 2a, the most prevalent species and serotype that causes bacillary dysentery or shigellosis in man. The whole genome is composed of a 4 607 203 bp chromosome and a 221 618 bp virulence plasmid, designated pCP301. While the plasmid shows minor divergence from that sequenced in serotype 5a, striking characteristics of the chromosome have been revealed. The S.flexneri chromosome has, astonishingly, 314 IS elements, more than 7-fold over those possessed by its close relatives, the non-pathogenic K12 strain and enterohemorrhagic O157:H7 strain of Escherichia coli. There are 13 translocations and inversions compared with the E.coli sequences, all involve a segment larger than 5 kb, and most are associated with deletions or acquired DNA sequences, of which several are likely to be bacteriophage-transmitted pathogenicity islands. Furthermore, S.flexneri, resembling another human-restricted enteric pathogen, Salmonella typhi, also has hundreds of pseudogenes compared with the E.coli strains. All of these could be subjected to investigations towards novel preventative and treatment strategies against shigellosis.