Hongtao Wu

Simvastatin-Mediated Upregulation of VEGF and BDNF, Activation of the PI3K/Akt Pathway, and Increase of Neurogenesis Are Associated with Therapeutic Improvement after Traumatic Brain Injury

This study was undertaken to evaluate the effect of simvastatin, a cholesterol-lowering agent, on the Akt-mediated signaling pathway and neurogenesis in the dentate gyrus (DG) of the hippocampus in rats after traumatic brain injury (TBI). Adult male Wistar rats were divided into three groups: (1) sham group (n = 8); (2) saline control group (n = 40); and (3) simvastatin-treated group (n = 40). Controlled cortical impact (CCI) injury was performed over the left parietal lobe. Simvastatin was administered orally at a dose of 1 mg/kg starting at day 1 after TBI and then daily for 14 days. Bromodeoxyuridine (BrdU) was injected intraperitoneally into rats. A modified Morris Water Maze (WM) task was performed between 31 and 35 days after treatment to test spatial memory (n = 8/group). Animals were sacrificed at 1, 3, 7, 14, and 35 days after treatment (n = 8/group/time point). Western blot was utilized to investigate the changes in the Akt-mediated signaling pathway. Enzyme-linked immunosorbent assay (ELISA) analyses were employed to measure vascular endothelial growth factor (VEGF) and brain-derived neurotrophin factor (BDNF) expression. Immunohistochemical and fluorescent staining were performed to detect the BrdU- and neuronal nuclei (NeuN)/BrdU-positive cells. Our data show that simvastatin treatment increases phosphorylation of v-akt murine thymoma viral oncogene homolog (Akt), glycogen synthase kinase-3beta (GSK-3beta), and cAMP response element-binding proteins (CREB); elevates the expression of BDNF and VEGF in the DG; increases cell proliferation and differentiation in the DG; and enhances the recovery of spatial learning. These data suggest that the neurorestorative effect of simvastatin may be mediated through activation of the Akt-mediated signaling pathway, subsequently upregulating expression of growth factors and inducing neurogenesis in the DG of the hippocampus, thereby leading to restoration of cognitive function after TBI in rats.

SIMVASTATIN ATTENUATES MICROGLIAL CELLS AND ASTROCYTE ACTIVATION AND DECREASES INTERLEUKIN-1B LEVEL AFTER TRAUMATIC BRAIN INJURY

OBJECTIVEOur 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. METHODSControlled cortical impact injury was performed in adult male Wistar rats. The rats were randomly divided into 3 groups: sham, saline control group, and simvastatin treatment group. Simvastatin was administered orally starting at day 1 after TBI until animals were killed at days 1, 3, 7, 14, and 35 after treatment. Functional outcome was measured using modified neurological severity scores. Enzyme-linked immunosorbent assay and immunohistochemical staining were used to measure the expression of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α and to identify activated microglial cells and astrocytes. RESULTSAt days 1 and 3 after simvastatin or saline treatment, cytokine levels in the lesion boundary zone were significantly higher in the simvastatin- and saline-treated rats compared with the sham group, peaking at day 3. Simvastatin only reduced the level of IL-1β but not IL-6 and tumor necrosis factor-α, compared with the saline group. Also, simvastatin significantly reduced the number of activated microglial cells and astrocytes compared with the saline control animals. There was also a trend toward improvement of modified neurological severity score, reaching statistical significance (P = 0.003) toward the end of the trial. CONCLUSIONOur data demonstrate that TBI causes inflammatory reaction, including increased levels of IL-1β, IL-6, and tumor necrosis factor-α, as well as activated microglial cells. Simvastatin selectively reduces IL-1β expression and inhibits the activation of microglial cells and astrocytes after TBI, which might be one of the mechanisms underlying the therapeutic benefits of simvastatin treatment of TBI.

Increase in phosphorylation of Akt and its downstream signaling targets and suppression of apoptosis by simvastatin after traumatic brain injury

OBJECT In their previous studies, the authors found that simvastatin treatment of traumatic brain injury (TBI) in rats had beneficial effects on spatial learning functions. In the current study they wanted to determine whether simvastatin suppressed neuronal cell apoptosis after TBI, and if so, they wanted to examine the underlying mechanisms of this process. METHODS Saline or simvastatin (1 mg/kg) was administered orally to rats starting on Day 1 after TBI and then daily for 14 days. Modified Neurological Severity Scores were used to evaluate the sensory motor functional recovery. Rats were killed at 1, 3, 7, 14, and 35 days after treatment, and brain tissue was harvested for terminal deoxynucleotidyl nick-end labeling (TUNEL) staining, caspase-3 activity assay, and Western blot analysis. RESULTS Simvastatin significantly decreased the modified Neurological Severity Scores from Days 7 to 35 after TBI, significantly reduced the number of TUNEL-positive cells at Day 3, suppressed the caspase-3 activity at Days 1 and 3 after TBI, and increased phosphorylation of Akt as well as Forkhead transcription factor 1, inhibitory-kappaB, and endothelial nitric oxide synthase, which are the downstream targets of the prosurvival Akt signaling protein. CONCLUSIONS These data suggested that simvastatin reduces the apoptosis in neuronal cells and improves the sensory motor function recovery after TBI. These beneficial effects of simvastatin may be mediated through activation of Akt, Forkhead transcription factor 1 and nuclear factor-kappaB signaling pathways, which suppress the activation of caspase-3 and apoptotic cell death, and thereby, lead to neuronal function recovery after TBI.

Effect of Simvastatin on Glioma Cell Proliferation, Migration and Apoptosis

OBJECTIVEIn this study, we investigated the effects of simvastatin on proliferation, migration, and apoptosis in human U251 and U87 glioma cells and the underlying molecular mechanism. METHODSWe used colony formation assay to test the cell proliferation, in vitro scratch assay to examine the cell migration, and caspase-3 activity assay, annexin V staining, and cytochrome C release to evaluate the cell apoptosis. Lipid raft fractions were isolated from glioma cells. Total cholesterol content assay was used to test the change of cholesterol level in lipid raft fractions. Immunocytochemistry staining was performed to detect the changes of lipid rafts in cell membranes. Western blotting analysis was performed to examine the signal transduction both in cells and in lipid raft fractions. RESULTSSimvastatin inhibited proliferation and migration of U251 and U87 cells dose dependently. Simvastatin induced an increase of caspase-3 activity and annexin V staining, and down-regulated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Simvastatin also decreased cholesterol content in lipid raft fractions, suppressed caveolin-1 expression in the lipid rafts, and induced Fas translocation into lipid rafts, suggesting that simvastatin may inhibit the prosurvival PI3K/Akt pathway and trigger caspase-3–dependent apoptotic cell death through the modulation of lipid rafts. CONCLUSIONThese results suggest that modulation of lipid rafts, Fas translocation, and PI3K/Akt/caspase-3 pathway are involved in the antitumor effect of simvastatin and may have a potential role in cancer prevention and treatment.

Combination treatment with resveratrol and sulforaphane induces apoptosis in human U251 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.

Effects of treating traumatic brain injury with collagen scaffolds and human bone marrow stromal cells on sprouting of corticospinal tract axons into the denervated side of the spinal cord

OBJECT This study was designed to investigate how transplantation into injured brain of human bone marrow stromal cells (hMSCs) impregnated in collagen scaffolds affects axonal sprouting in the spinal cord after traumatic brain injury (TBI) in rats. Also investigated was the relationship of axonal sprouting to sensorimotor functional recovery after treatment. METHODS Adult male Wistar rats (n = 24) underwent a controlled cortical impact injury and were divided into three equal groups (8 rats/group). The two treatment groups received either hMSCs (3 × 10(6)) alone or hMSC (3 × 10(6))-impregnated collagen scaffolds transplanted into the lesion cavity. In the control group, saline was injected into the lesion cavity. All treatments were performed 7 days after TBI. On Day 21 after TBI, a 10% solution of biotinylated dextran amine (10,000 MW) was stereotactically injected into the contralateral motor cortex to label the corticospinal tract (CST) originating from this area. Sensorimotor function was tested using the modified neurological severity score (mNSS) and foot-fault tests performed on Days 1, 7, 14, 21, 28, and 35 after TBI. Spatial learning was tested with Morris water maze test on Days 31-35 after TBI. All rats were sacrificed on Day 35 after TBI, and brain and spinal cord (cervical and lumbar) sections were stained immunohistochemically for histological analysis. RESULTS Few biotinylated dextran amine-labeled CST fibers crossing over the midline were found in the contralateral spinal cord transverse sections at both cervical and lumbar levels in saline-treated (control) rats. However, hMSC-alone treatment significantly increased axonal sprouting from the intact CST into the denervated side of the gray matter of both cervical and lumbar levels of the spinal cord (p < 0.05). Also, this axonal sprouting was significantly more in the scaffold+hMSC group compared with the hMSC-alone group (p < 0.05). Sensorimotor functional analysis showed significant improvement of mNSS (p < 0.05) and foot-fault tests (p < 0.05) in hMSC-alone and scaffold+hMSC-treated rats compared with controls (p < 0.05). Functional improvement, however, was significantly greater in the scaffold+hMSC group compared with the hMSC-alone group (p < 0.05). Morris water maze testing also showed significant improvement in spatial learning in scaffold+hMSC and hMSC-alone groups compared with the control group (p < 0.05), with rats in the scaffold+hMSC group performing significantly better than those in the hMSC-alone group (p < 0.05). Pearson correlation data showed significant correlation between the number of crossing CST fibers detected and sensorimotor recovery (p < 0.05). CONCLUSIONS Axonal plasticity plays an important role in neurorestoration after TBI. Transplanting hMSCs with scaffolds enhances the effect of hMSCs on axonal sprouting of CST fibers from the contralateral intact cortex into the denervated side of spinal cord after TBI. This enhanced axonal regeneration may at least partially contribute to the therapeutic benefits of treating TBI with hMSCs.

The Treatment of TBI with Human Marrow Stromal Cells Impregnated into Collagen Scaffold: Functional Outcome and Gene Expression Profile

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 (3.0×10(6)), 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 7days 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.

Attenuation of astrogliosis and modulation of endothelial growth factor receptor in lipid rafts by simvastatin after traumatic brain injury

Traumatic brain injury (TBI) is a leading cause of serious, long-term disability worldwide. Widely distributed neuronal damage has been well documented in the cerebral cortex, hippocampus, and dentate gyrus of the brain during the first few days after experimental TBI in rats.13 TBI results in a rapid response from resident astrocytes, a process often referred to as reactive astrogliosis which plays a key role in the process of neuro-inflammation.8 Although its role after neuronal injury is still controversial, reactive astrogliosis is believed to be detrimental to the injured neurons in the early phase after TBI.18 Our previous studies have demonstrated the beneficial effects of statins on functional recovery and neuronal survival after TBI in rats.28 Although the mechanism of the neuroprotective effects of statins is still not clear, previous studies suggest that simvastatin has anti-inflammatory effects after TBI, including reducing the secretion of inflammatory mediators IL-1β, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α).2,9 Since the cytokines are mostly secreted by reactive astrocytes, inhibiting reactive astrocytes has become an appealing approach to attenuate neuro-inflammation and thereby protect neurons after TBI. Targeting the astrogliosis pathways hours or even days after the initial injury may prevent the progression of neuronal damage and be a promising therapeutic strategy.16 The epidermal growth factor receptor (EGFR) pathway is a key signaling pathway in the activation of astrocytes after brain injury.11 Activation of EGFR triggers astrocytes into a visibly active phenotype with significantly increased cell motility, which is one of the characteristics of reactive astrocytes. Blocking EGFR activation in adult astrocytes promotes the survival of neurons after glaucomatous optic neuronal injury.10 In all cell types examined so far, EGFR selectively partitions into the cholesterol-rich lipid rafts. The presence within lipid rafts of a variety of membrane proteins involved in cell signaling, including EGFR, suggests that these lipid domains play important roles in the process of signal transduction.19 Lipid rafts may function as platforms that allow local accumulation of raft-associated proteins, which promote the interaction of protein complexes, and modulate neurotransmitter signaling.14 Simvastatin, a clinically widely used cholesterol synthesis inhibitor, lowers raft cholesterol content in various types of cells, reduces association of N-methyl-D-aspartate (NMDA) receptors to lipid rafts22 and affects raft-dependent signaling such as the Akt-mediated signaling pathway.29 Therefore simvastatin may affect the functionality of proteins associated with lipid rafts (e.g., EGFR). The purpose of this study is to determine whether simvastatin protects neurons by attenuating reactive astrogliosis, thereby exerting beneficial effects after TBI. We hypothesize that simvastatin promotes neuronal survival by inhibiting the activation of astrocytes after brain injury through the modification of lipid rafts, and regulation of EGFR phosphorylation in the astrocyte membrane.

Induction of angiogenesis and modulation of vascular endothelial growth factor receptor-2 by simvastatin after traumatic brain injury.

BACKGROUND:Our previous studies demonstrated that simvastatin reduced neuronal death, increased neurogenesis, and promoted functional recovery after traumatic brain injury (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, enzyme-linked immunosorbent assay, and Western blot analysis. Cultured rat brain microvascular endothelial cells were subjected to oxygen-glucose deprivation 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 enzyme-linked immunosorbent assay. 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 oxygen-glucose deprivation in vitro. VEGFR-2 expression in both brain tissues and cultured rat brain microvascular endothelial cells was enhanced after simvastatin treatment, which may be modulated by activation of Akt. Akt-dependent endothelial nitric oxide synthase 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/endothelial nitric oxide synthase signaling pathway.

Simvastatin attenuates axonal injury after experimental traumatic brain injury and promotes neurite outgrowth of primary cortical neurons.

The beneficial effects of simvastatin on experimental traumatic brain injury (TBI) have been demonstrated in previous studies. In this study, we investigated the effects of simvastatin on axonal injury and neurite outgrowth after experimental TBI and explored the underlying mechanisms. Wistar rats were subjected to controlled cortical impact or sham surgery. Saline or simvastatin was administered for 14 days. A modified neurological severity score (mNSS) test was performed to evaluate functional recovery. Immunohistochemistry studies using synaptophysin, neurofilament H (NF-H) and amyloid-β precursor protein (APP) were performed to examine synaptogenesis and axonal injury. Primary cortical neurons (PCNs) were subjected to oxygen glucose deprivation (OGD) followed by various treatments. Western blot analysis was utilized to assess the activation of phosphatidylinositol-3 kinase (PI-3K)/Akt/mammalian target of rapamycin (mTOR) and glycogen synthase kinase 3β (GSK-3β)/adenomatous polyposis coli (APC) pathways. Simvastatin decreased the density of APP-positive profiles and increased the density of NF-H -positive profiles. Simvastatin reduced mNSS, which was correlated with the increase of axonal density. Simvastatin treatment stimulated the neurite outgrowth of PCNs after OGD, which was attenuated by LY294002 and enhanced by lithium chloride (LiCl). Simvastatin activated Akt and mTOR, inactivated GSK-3β and dephosphorylated APC in the injured PCNs. Our data suggest that simvastatin reduces axonal injury, enhances neurite outgrowth and promotes neurological functional recovery after experimental TBI. The beneficial effects of simvastatin on neurite outgrowth may be mediated through manipulation of the PI-3K/Akt/mTOR and PI-3K/GSK-3β/APC pathways.