Neuroprotective mechanisms of OXCT1 via the SIRT3-SOD2 pathway after traumatic brain injury.

BACKGROUND: Ketones are not only utilized to produce energy but also play a neuroprotective role in many neurodegenerative diseases. However, whether this process has an impact on secondary brain damage after traumatic brain injury (TBI) remains unknown. OXCT1 (3-Oxoacid CoA-Transferase 1) is the rate-limiting enzyme in the intra-neuronal utilization of ketones. In this study, we investigated whether reduced expression of OXCT1 after TBI could impact neuroprotective mechanisms and exacerbate neurological dysfunction. MATERIALS AND METHODS: Experimental TBI was induced by a modified version of the weight drop model, it is a model of severe head trauma. Expression of OXCT1 in the injured hippocampus of mice was measured at different time points using immunoblotting assays. The release of abnormal mitochondrial cytochrome c from neurons of the mouse injured lateral hippocampus was measured 1 week after TBI using immunoblotting assays. Neuronal death was assessed by Nissl staining and the level of reactive oxygen species (ROS) within the neurons of the injured lateral hippocampus was assessed by Dihydroethidium staining. RESULTS: OXCT1 was overexpressed in hippocampal neurons by injection of adeno-associated virus into the lateral ventricle. OXCT1 expression levels decreased significantly 1 week post-TBI. After comparing the data obtained from different groups of mice, OXCT1 was found to significantly increase the expression of SIRT3 and reduce the proportion of acetylated SOD2, thus decreasing the production of ROS in the injured hippocampal neurons, reducing neuronal death, and improving cognitive function. CONCLUSIONS: OXCT1 has a critical previously unappreciated protective role in neurological impairment following TBI via the SIR3-SOD2 pathway. These findings highlight the potential of OXCT1 as a simple treatment for patients with TBI.

New molecular prognostic factors of adult diffuse lower-grade gliomas in post-2016 molecular era: a retrospective analysis from single center.

BACKGROUND AND OBJECTIVE: Several studies have examined the prognostic significance of IDH1/2 mutation, 1p/19q codeletion and MGMT promoter methylation in lower-grade gliomas but most of these used the 2007 fourth edition of the WHO classification. We evaluate prognostic significance of these indicators in the 2016 WHO updated fourth edition of CNS tumor classification. METHODS: A total of 180 intracranial glioma patients diagnosed according WHO 2016 edition between December 2016 and December 2018 Jinling Hospital (Nanjing, China) were reviewed retrospectively. We performed survival analysis on 109 patients with complete molecular pathology and follow-up data. RESULTS: Histologically, 52 were diagnosed as astrocytoma (WHO grade II and III), 17 as oligodendrogliomas (WHO grade II and III) and 40 as GBMs. At last follow-up, 50.5% patients had experienced tumor progression and 34.9% had died. Among grade II and III cases 36.2% experienced tumor progression and 27.5% died. In univariate Kaplan-Meier analysis, multifocal tumor, EGFR mutation or amplification, PIK3CA mutation and IDHwt/TERTpwt group were associated with shorter PFS (p < 0.001, p = 0.003, p = 0.005, p < 0.001, respectively) and OS (p = 0.010, p = 0.020, p = 0.018, p < 0.001, respectively) as were older age (≥55 years), multifocal tumor, IDH1/2 wild type, 1p/19q non-codeletion and negative methylation in the MGMT promoter region. A Cox proportional hazards model was created demonstrating that single tumor (HR = 0.180, p = 0.04), MGMTp methylation (HR = 0.095, p = 0.003) and chemoradiotherapy (HR = 0.006, p = 0.002) were independent prognostic factors for OS. CONCLUSIONS: Beyond histological classification as well as IDH1/2 mutation, 1p/19q codeletion status, we could incorporate IDH1/2mt combined with TERTpmt, EGFR mutation or amplification and PIK3CA mutation into the diagnostic criteria for DLGGs to supplement WHO 2016 pathological criteria.

Restoration of Brain Angiotensin-Converting Enzyme 2 Alleviates Neurological Deficits after Severe Traumatic Brain Injury via Mitigation of Pyroptosis and Apoptosis.

Clinically, the renin-angiotensin-aldosterone system is activated intensely in patients with moderate to severe traumatic brain injury (TBI). Increased angiotensin II in circulatory blood after TBI can enter the brain through the disrupted blood-brain barrier. Angiotensin-converting enzyme 2 (ACE2) is an enzyme that metabolizes angiotensin II into angiotensin (1-7), which has been shown to have neuroprotective results. The expression and role of ACE2 in the brain after TBI remains elusive, however. We found that ACE2 protein abundance was downregulated around the contusional area in the brains of both humans and mice. Endogenous ACE2 was expressed in neurons, astrocytes, and microglia in the cortex of the mouse brain. Administration of recombinant human ACE2 intracerebroventricularly alleviated neurological defects after TBI in mice. Treatment of recombinant human ACE2 suppressed TBI-induced increase of angiotensin II and the decrease of angiotensin (1-7) in the brain, mitigated neural cell death, reduced the activation of NLRP3 and caspase3, decreased phosphorylation of mitogen-activated protein kinases, and nuclear factor kappa B, and reduced inflammatory cytokines tumor necrosis factor alpha and interleukin-1ß. Administration of ACE2 enzyme activator diminazene aceturate intraperitoneally rescued downregulation of ACE2 enzymatic activity and protein abundance in the brain. Diminazene aceturate treatment once per day in the acute stage after TBI alleviated long-term cognitive defects and neuronal loss in mice. Collectively, these results indicated that restoration of ACE2 alleviated neurological deficits after TBI by mitigation of pyroptosis and apoptosis.

Morphology parameters for rupture in middle cerebral artery mirror aneurysms.

OBJECTIVE: To identify the morphological parameters correlated with the rupture of middle cerebral artery (MCA) aneurysms. METHODS: We retrospectively analyzed the digital subtraction angiography (DSA) data of 48 patients with ruptured mirror MCA aneurysms. Morphological parameters included aneurysm with wall protrusion, maximum diameter (Dmax), height, neck width, aneurysm width, dome projection, parent artery average diameter (Dp), aspect ratio (AR), bottleneck factor (BNF), size ratio (SR), M1/M2 ratio, and height/width (H/W) ratio. These paired parameters were analyzed by conditional univariate and multivariate logistic regressions to screen out the independent risk factors. We established a score based on the independent risk factors. Receiver operating characteristics (ROC) were generated to estimate the prediction performance of the score in our large database of 763 aneurysms. RESULTS: In the univariate regressions, Dmax, height, aneurysm width, neck width, AR, BNF, H/W ratio, SR, anterior dome projection and aneurysm with wall protrusion were significant risk factors. Aneurysm width (OR 3.296, p=0.015), AR (OR 11.594, p=0.014) and anterior dome projection (OR 9.385, p=0.016) were independent risk factors in multivariate regression. The area under the curve (AUC) value of the score based on the three independent risk factors was 0.829. CONCLUSION: Aneurysm width, AR and anterior dome projection were independent risks factors of rupture.

N-acetylcysteine amide provides neuroprotection via Nrf2-ARE pathway in a mouse model of traumatic brain injury.

BACKGROUND: Increasing evidence demonstrate N-acetylcysteine amide (NACA) provides neuroprotection and attenuated oxidative stress in rats following traumatic brain injury (TBI). The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) signal pathway is activated after TBI and provides a protective effect against TBI. However, the function and mechanism of NACA in mice after TBI remain unknown. This study was to evaluate the neuroprotection of NACA and the potential action of the Nrf2-ARE pathway in a weight-drop mouse model of TBI. MATERIALS AND METHODS: Four groups of animals were randomly divided into sham, TBI, TBI+vehicle, and TBI+NACA (100 mg/kg, administered intraperitoneally). The protein levels of Nrf2, heme oxygenase-1 (HO-1), NAD(P)H: quinine oxidoreductase-1 (NQO1), cleaved caspase-3 and the mRNA levels of HO-1 and NQO1 were detected. The neurobehavior, neuronal degeneration, apoptosis and oxidative stress were also assessed. RESULTS: Treatment with NACA significantly improved neurologic status at days 1 and 3 following TBI. Moreover, NACA promoted Nrf2 activation a day after TBI. The protein and mRNA levels of HO-1 and NQO1 were upregulated by NACA. Meanwhile, NACA treatment significantly reduced the level of malondialdehyde (MDA) and enhanced the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx), which indicated NACA attenuated oxidative stress following TBI. NACA prominently reduced the protein level of cleaved caspase-3 and TUNEL-positive cells, indicating its antiapoptotic effect. Additionally, Fluoro-Jade C staining showed NACA alleviated neuronal degeneration a day after TBI. CONCLUSIONS: Our study reveals that NACA potentially provides neuroprotection via the activation of the Nrf2-ARE signaling pathway after TBI in mice.

GSH depletion and consequent AKT inhibition contribute to the Nrf2 knockdown-induced decrease in proliferation in glioblastoma U251 cells.

Nuclear factor erythroid 2-related factor 2 (Nrf2), a pivotal transcription regulator that controls the expression of numerous antioxidant and cytoprotective genes, was recently defined as a proto-oncogene. However, the role and mechanism of Nrf2 in glioma pathoetiology remain unclear. In the present study, we first evaluated the expression patterns of Nrf2 in normal human astrocytes and 3 glioblastoma (GBM) cell lines (U251, U87 and A172) and found that all 3 GBM cell lines overexpressed Nrf2, with the highest level observed in the U251 cells. We further assessed the biological effects of Nrf2 in U251 cells by specific knockdown of Nrf2 using lentivirus­mediated RNA interference. We discovered that Nrf2 deficiency led to a decrease in U251 cell proliferation and caused intracellular redox imbalance [diminished glutathione (GSH) levels and increased reactive oxygen species (ROS) levels]. Both N-acetylcysteine and glutathione monoethyl ester (GMEE) supplementation completely eliminated the increased levels of ROS that were present in the Nrf2­deficient U251 cells. However, only GMEE supplementation both reversed Nrf2 deficiency-induced cell growth arrest and restored intracellular GSH levels. Moreover, AKT and ERK1/2 signaling were both impaired in the Nrf2-knockdown U251 cells, but GMEE supplementation restored AKT signaling but not ERK1/2 signaling, and blocking AKT signaling with an AKT-specific inhibitor greatly diminished the GMEE-induced Nrf2-deficient cell proliferation. In conclusion, our findings revealed novel functions for Nrf2 in the regulation of redox status and cell proliferation, and that intracellular GSH levels and AKT signaling are required for this process, a new viewpoint by which to comprehend the role and underlying mechanism of Nrf2 in tumorigenesis.

Flat-detector computed tomography PBV map in the evaluation of presurgical embolization for hypervascular brain tumors.

BACKGROUND: Preoperative embolization of hypervascular brain tumors is frequently used to minimize intraoperative bleeding. OBJECTIVE: To explore the efficacy of embolization using flat-detector CT (FDCT) parenchymal blood volume (PBV) maps before and after the intervention. MATERIALS AND METHODS: Twenty-five patients with hypervascular brain tumors prospectively received pre- and postprocedural FDCT PBV scans using a biplane system under a protocol approved by the institutional research ethics committee. Semiquantitative analysis, based on region of interest measurements of the pre- and post-embolization PBV maps, operating time, and blood loss, was performed to assess the feasibility of PBV maps in detecting the perfusion deficit and to evaluate the efficacy of embolization. RESULTS: Preoperative embolization was successful in 18 patients. The relative PBV decreased significantly from 3.98±1.41 before embolization to 2.10±2.00 after embolization. Seventeen patients underwent surgical removal of tumors 24 hours after embolization. The post-embolic tumor perfusion index correlated significantly with blood loss (ρ=0.55) and operating time (ρ=0.60). CONCLUSIONS: FDCT PBV mapping is a useful method for evaluating the perfusion of hypervascular brain tumors and the efficacy of embolization. It can be used as a supplement to CT perfusion, MRI, and DSA in the evaluation of tumor embolization.

Deletion of Nrf2 Exacerbates Oxidative Stress After Traumatic Brain Injury in Mice.

Traumatic brain injury (TBI) is a worldwide public health and medical problem. Oxidative stress is recognized as an important contributing factor in the pathogenesis of TBI. The present study was designed to explore the anti-oxidative effect of Nuclear factor erythroid 2-related factor 2 (Nrf2) on brain damage induced by traumatic injury in a mouse model. Moderate weight-drop impact head injury was induced in adult male mice. The mice were randomly divided into four groups: Nrf2(+/+) sham-operation, Nrf2(-/-) sham-operation, Nrf2(+/+) TBI, and Nrf2(-/-) TBI group. Neurological scores were evaluated 24 h after TBI, followed by collection of the brain specimens. Brain edema was detected by the wet-dry ratio method. The expression of NOX2 protein in the brain specimen was investigated using Western Blot analysis and immunohistochemical staining. In addition, malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were evaluated in the brain tissues. Twenty-four hours after TBI, our results showed Nrf2(+/+) TBI mice have more severe neurological deficits and brain edema than Nrf2(+/+) sham group. On the other hand, the Nrf2(-/-) TBI mice were found to have significantly increased neurological deficits and brain edema, compared to Nrf2(+/+) TBI mice (P < 0.05). At the same time, we found that the expression of NOX2 protein, MDA level were significantly increased in Nrf2(-/-) mice, while SOD activity was considerably decreased after TBI compared to Nrf2(+/+) mice (P < 0.05). We demonstrated that deletion of Nrf2 exacerbates brain injury after TBI in mice, suggesting that Nrf2 may play an important role in protecting brain injury after TBI, possibly by modulating oxidative stress.

Differential expression of microRNAs in postoperative radiotherapy sensitive and resistant patients with glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most malignant primary brain tumor and more resistant to radiotherapy. However, hetero-radiosensitivity occurs in different patients. MicroRNAs (miRNAs) play important roles in the initiation and progression of a multitude of tumors. The study aims to examine the different microRNAs expression profiles of postoperative radiotherapy sensitive and resistant patients with GBM, to make an inquiry about their potential role and discover a certain set of radio-sensitivity markers. Three paired samples from six GBM patients who had only been treated with postoperative radiotherapy were selected, and then, they were divided into radiotherapy sensitive group and resistant group according to their overall survivals, local recurrence rates, and Karnofsky Performance Scale scores. Expression profiles of miRNAs in these two groups were determined by the method of microarray assay. Comparing with resistant patients, 13 miRNAs were significantly upregulated and 10 miRNAs were greatly downregulated in sensitive group. Among them, four miRNAs were validated by quantitative RT-PCR. The differentially expressed miRNAs and their putative target genes were revealed by bioformatic analysis to play a role in cell signaling, proliferation, aging, and death. High-enrichment pathway analysis identified that some classical pathways participated in numerous metabolic processes, especially in cell cycle regulation, such as mTOR, MAPK, TGF-beta, and PI3K-Akt signaling pathways. Our research will contribute to identifying clinical diagnostic markers and therapeutic targets in the treatment of GBM by postoperative radiotherapy.

Quantitative anatomic comparison of the extended pterional transtemporal transtentorial approach and the subtemporal transtentorial approach to the petroclival region.

AIM: The anatomic characters and applicability of the extended pterional transtemporal transtentorial (EPTT) approach versus the subtemporal transtentorial (ST) approach for surgical treatment of petroclival tumors were evaluated. MATERIAL AND METHODS: Ten sides from five adult Chinese injected cadavers were manipulated using both two approaches. Four deep bony anatomic landmarks were specified in the skull base to create two adjoining triangles that were respectively located in the anterior and posterior petroclival region. The real, projected area and the percentage of the projected area were determined and calculated to compare the deep exposure from the two approaches. RESULTS: There was no difference regarding the percentage of the projected area was calculated in the anterior triangles (EPTT, 21.5±12.5%; ST, 28.8±14.9%; p=0.1948), but a significant difference was present in the posterior triangles (EPTT, 74.0±4.5%; ST, 51.5±4.3%; p < 0.01). Compared with the ST approach, the EPTT approach provides an equivalent percentage of projected area in the middle cranial fossa and a wider exposed area in the posterior cranial fossa. CONCLUSION: Through anatomic comparative analysis the EPTT approach provides better exposure and is more appropriate than the ST approach for large and giant petroclival tumors predominantly in the posterior cranial fossa with extensive invasion to parasellar structures and the cavernous sinus.

γ-secretase inhibitor DAPT sensitizes t-AUCB-induced apoptosis of human glioblastoma cells in vitro via blocking the p38 MAPK/MAPKAPK2/Hsp27 pathway.

AIM: Trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) is a soluble epoxide hydrolase inhibitor that suppresses glioblastoma cell growth in vitro. The aim of this study was to examine whether the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) could sensitize glioma cells to t-AUCB-induced apoptosis. METHODS: Both U251 and U87 human glioblastoma cell lines were tested. Cell growth was assessed using the cell counting kit-8. Cell apoptosis was detected with caspase-3 activity assay kits and flow cytometry. The protein levels in the p38 MAPK/MAPKAPK2/Hsp27 pathway in the cells were analyzed using Western blots. RESULTS: Pretreatment with DAPT (2 µmol/L) substantially potentiated the growth inhibition caused by t-AUCB (200 µmol/L) in U251 and U87 cells. Furthermore, pretreatment with DAPT markedly increased t-AUCB-induced apoptosis of U251 and U87 cells. T-AUCB alone did not significant affect caspase-3 activity in the cells, but t-AUCB plus DAPT pretreatment caused significant increase of caspase-3 activity. Furthermore, pretreatment with DAPT completely blocked t-AUCB-induced phosphorylation of p38 MAPK, MAPKAPK2 and Hsp27 in the cells. CONCLUSION: The γ-secretase inhibitor DAPT sensitizes t-AUCB-induced apoptosis of human glioblastoma cells in vitro via blocking the p38 MAPK/MAPKAPK2/Hsp27 pathway, suggesting that the combination of t-AUCB and DAPT may be a potentially effective strategy for the treatment of glioblastoma.

Pretreatment with tert-butylhydroquinone attenuates cerebral oxidative stress in mice after traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a worldwide health problem, identified as a major cause of death and disability. Increasing evidence has shown that oxidative stress plays an important role in TBI pathogenesis. The antioxidant transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), is a known mediator in protection against TBI-induced brain damage. The objective of this study was to test whether tert-butylhydroquinone (tBHQ), a novel Nrf2 activator, can protect against TBI-induced oxidative stress. METHODS: Adult male imprinting control region mice were randomly divided into three groups: (1) sham + vehicle group; (2) TBI + vehicle group; and (3) TBI + tBHQ group. Closed-head brain injury was applied using the Feeney weight-drop method. We accessed the neurologic outcome of mice at 24 h after TBI, and subsequently measured protein levels of Nrf2 and the NOX2 subunit of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase), the concentration of malondialdehyde, superoxide dismutase activity, and brain edema. RESULT: The NOX2 protein level was increased fivefold in the TBI + vehicle group, whereas pretreatment with tBHQ markedly attenuated the NOX2 protein expression relative to that in the TBI + vehicle group. TBI increased Nrf2 formation by 5% compared with the sham group, whereas treatment with tBHQ further upregulated the Nrf2 protein level by 12% compared with the sham group. The level of the oxidative damage marker malondialdehyde was reduced by 29% in the TBI + tBHQ group compared with the TBI + vehicle group, Moreover, pretreatment with tBHQ significantly increased the antioxidant enzyme superoxide dismutase activity. Administration of tBHQ also significantly decreased TBI-induced brain edema and neurologic deficits. CONCLUSIONS: Pretreatment with tBHQ effectively attenuated markers of cerebral oxidative stress after TBI, thus supporting the testing of tBHQ as a potential neuroprotectant and adjunct therapy for TBI patients.

Temozolomide and irradiation combined treatment-induced Nrf2 activation increases chemoradiation sensitivity in human glioblastoma cells.

Resistance to chemoradiotherapy is a major obstacle to successful treatment of glioblastoma. Recently, the role of NF-E2-related factor 2 (Nrf2) in enhancing chemoradiation sensitivity has been reported in several types of cancers. Here, we investigated whether temozolomide (TMZ) and irradiation (IR) combined treatment induced Nrf2 activation in human glioblastoma cells. And we further performed a preliminary study about the effect of Nrf2 on chemoradiation sensitivity. Immunohistochemical staining for Nrf2 in paired clinical specimens showed that TMZ and IR combined treatment increased the expression and nuclear localization of Nrf2 in human glioblastoma tissues. Moreover, we found nuclear Nrf2 expression in the glioblastoma tissues obtained from the patients undergoing TMZ and IR combined treatment was associated with the time to tumor recurrence. In vitro, we further verified these findings. First, we detected increased nuclear localization of Nrf2 following treatment with TMZ+IR in human glioblastoma cell lines. Second, we demonstrated TMZ+IR increased the levels of Nrf2 protein in both nuclear and cytoplasmic fractions of U251 cells and induced Nrf2 target genes expression. Finally, downregulating Nrf2 expression increased TMZ+IR-induced cell death in the U251 cells. These findings suggest TMZ+IR combined treatment induces Nrf2 activation in human glioblastoma cells. The activation of Nrf2 may be associate with enhancing chemoradiation sensitivity in human glioblastoma cell. Blocking Nrf2 activation may be a promising method enhancing chemoradiation sensitivity of glioblastoma cells.

Puerarin ameliorates oxidative stress in a rodent model of traumatic brain injury.

BACKGROUND: A wealth of evidence has suggested that oxidative stress is involved in the secondary brain injury after traumatic brain injury (TBI). Recently, numerous in vivo and in vitro studies were reported that puerarin could inhibit oxidative stress through the activation of phosphatidylinositol 3-kinase (PI3K)-Akt pathway. It is unknown, however, whether puerarin can provide neuroprotection and reduce oxidative stress after TBI. The present study investigated the effects of puerarin on the TBI-induced neurodegeneration, oxidative stress, and the possible role of PI3K-Akt pathway in the neuroprotection of puerarin, in a rat model of TBI. MATERIALS AND METHODS: Rats were randomly distributed into various subgroups undergoing the sham surgery or TBI procedures. Puerarin (200 mg/kg) was given intraperitoneally at 10 min before injury and PI3K-Akt pathway inhibitor LY294002 was also administered intracerebroventricular in one subgroup. All rats were killed at 24 h after TBI for examination. RESULTS: Our data indicated that puerarin could significantly reduce TBI-induced neuronal degeneration, accompanied by the partial restoration of the redox disturbance and enhanced expression of phospho-Akt in the pericontusional cortex after TBI. Moreover, PI3K-Akt pathway inhibitor LY294002 could partially abrogate the neuroprotection of puerarin in rats with TBI. CONCLUSIONS: These results indicate that puerarin can ameliorate oxidative neurodegeneration after TBI, at least in part, through the activation of PI3K-Akt pathway.

FTY720 for cancer therapy (Review).

2-Amino-2-[2-(4-octylphenyl)]-1,3-propanediol hydrochloride (FTY720) is a potent immunosuppressant which has been approved by the Food and Drug Administration (FDA) as a new treatment for multiple sclerosis. As an immunosuppressant, it displays its anti-multiple sclerosis, immunosuppressive effects by activating sphingosine-1-phosphate receptors (S1PRs). In addition to the immunosuppressive effects, FTY720 also shows preclinical antitumor efficacy in several cancer models. In most cases, phosphorylation of FTY720 is not required for its cytotoxic effect, indicating the involvement of S1PR-independent mechanisms which are starkly different from the immunosuppressive property of FTY720. In the present study, we reviewed the rapidly advancing field of FTY720 in cancer therapy as well as some molecular targets of the unphosphorylated form of FTY720.

Knockdown of NF-E2-related factor 2 inhibits the proliferation and growth of U251MG human glioma cells in a mouse xenograft model.

NF-E2-related factor 2 (Nrf2) is a pivotal transcription factor of cellular responses to oxidative stress and recent evidence suggests that Nrf2 plays an important role in cancer pathobiology. However, the underlying mechanism has yet to be elucidated, particularly in glioma. In the present study, we investigated the role of Nrf2 in the clinical prognosis, cell proliferation and tumor growth of human glioblastoma multiforme (GBM). We detected overexpression of Nrf2 protein levels in GBM compared to normal brain tissues. Notably, higher protein levels of Nrf2 were significantly associated with poorer overall survival and 1-year survival for GBM patients. Furthermore, we constructed the plasmid Si-Nrf2 and transduced it into U251MG cells to downregulate the expression of Nrf2 and established stable Nrf2 knockdown cells. The downregulation of Nrf2 suppressed cell proliferation in vitro and tumor growth in mouse xenograft models. We performed immunohistochemistry staining to detect the protein levels of Nrf2, Ki-67, caspase-3 and CD31 in the xenograft tumors and found that the expression levels of Nrf2 and Ki-67 were much lower in the Si-Nrf2 group compared to the Si-control group. In addition, the number of caspase-3-positive cells was significantly increased in the Si-Nrf2 group. By analysis of microvessel density (MVD) assessed by CD31, the MVD value in the Si-Nrf2 group decreased significantly compared to the Si-control group. These findings indicate that the knockdown of Nrf2 may suppress tumor growth by inhibiting cell proliferation, increasing cell apoptosis and inhibiting angiogenesis. These results highlight the potential of Nrf2 as a candidate molecular target to control GBM cell proliferation and tumor growth.

ERK and PI3K signaling cascades induce Nrf2 activation and regulate cell viability partly through Nrf2 in human glioblastoma cells.

The ERK and PI3K signaling cascades are aberrantly activated in human glioblastoma cells, resulting in the dysregulation of numerous downstream transcription factors. The dark side of the transcription factor, NF-E2-related factor 2 (Nrf2) in human cancer has been revealed. It has been accepted that high levels of Nrf2 promote tumor progression. In the present study, we investigated the effect of the ERK and PI3K signaling cascades on Nrf2 in human glioblastoma cells. Immunohistochemical staining for Nrf2 in clinical specimens showed that the expression and nuclear localization of Nrf2 were increased in human glioblastoma tissues when compared to peritumoral normal tissues. In addition, we detected decreased nuclear localization of Nrf2 following combined treatment with ERK and PI3K inhibitors in three human glioblastoma cell lines and selected the cell line (U251) most sensitive to the inhibitors for further study. Our data demonstrated that inhibition of ERK and PI3K not only suppressed the nuclear accumulation of Nrf2 protein but also decreased the expression of the Nrf2 protein. In addition, combined inhibition of ERK and PI3K also decreased the mRNA levels of Nrf2 target genes. Finally, we found that Nrf2 overexpression partly reversed the ERK and PI3K inhibitor-induced inhibition of cell viability. Therefore, the ERK and PI3K signaling cascades regulate the expression and activation of Nrf2 and control cell viability partly through Nrf2 in U251 human glioblastoma cells. Thus, targeting the ERK and PI3K signaling cascades for Nrf2 activation may provide new methods for the treatment of glioblastoma.

Activation of metabotropic glutamate receptor 5 reduces the secondary brain injury after traumatic brain injury in rats.

A wealth of evidence has shown that microglia-associated neuro-inflammation is involved in the secondary brain injury contributed to the poor outcome after traumatic brain injury (TBI). In vitro studies were reported that activation of metabotropic glutamate receptor 5 (mGluR5) could inhibit the microglia-associated inflammation in response to lipopolysaccharide and our previous study indicated that mGluR5 was expressed in activated microglia following TBI. However, there is little known about whether mGluR5 activation can provide neuro-protection and reduce microglia-associated neuro-inflammation in rats after TBI. The goal of the present study was to investigate the effects of mGluR5 activation with selective agonist CHPG, on cerebral edema, neuronal degeneration, microglia activation and the releasing of pro-inflammatory cytokines, in a rat model of TBI. Rats were randomly distributed into various subgroups undergoing the sham surgery or TBI procedures, and 250 nmol of CHPG or equal volume vehicle was given through intracerebroventricular injection at 30 min post-TBI. All rats were sacrificed at 24 h after TBI for the further measurements. Our data indicated that post-TBI treatment with CHPG could significantly reduce the secondary brain injury characterized by the cerebral edema and neuronal degeneration, lead to the inhibition of microglia activation and decrease the expression of pro-inflammatory cytokines in both mRNA transcription and protein synthesis. These results provide the substantial evidence that activation of mGluR5 reduces the secondary brain injury after TBI, in part, through modulating microglia-associated neuro-inflammation.

Knockdown of Nrf2 enhances autophagy induced by temozolomide in U251 human glioma cell line.

Glioblastoma multiforme (GBM) and oxidative stress are closely linked. Oxidative stress affects many signaling pathways and may cause the induction of autophagy. The NF-E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) signaling pathway is the main pathway responsible for cell defense against oxidative stress and Nrf2 is a critical transcription factor related with cancer multidrug resistance. However, the relation between Nrf2 and regulation of autophagy is not well understood. In this study, we used temozolomide (TMZ), which inhibited the viability of GBM cells mainly by inducing autophagic cell death and explored the role of Nrf2 downregulation on autophagy induced by TMZ in GBM cells. In U251-Si-Nrf2 48 h after transfection the protein levels of Nrf2 were significantly downregulated, while the protein levels of LC3B-II increased by western blot analysis. Knockdown of Nrf2 also led to a significant increase of autophagic vacuoles and acidic vesicular organelles (AVOs), revealed by trans-mission electron microscopy (TEM) and acridine orange (AO) staining using flow cytometry. Collectively, these findings demonstrate that knockdown of Nrf2 can enhance the basal level of autophagy in the U251 glioma cell line. Furthermore, after the treatment with TMZ (100 µM) for 3 days, the U251-Si-Nrf2 transfected cells showed less viability rate by cell counting kit-8 (CCK-8) assay and the levels of autophagy increased obviously through analysis of western blot and AO staining using flow cytometry. Taken together, our results suggest that knockdown of Nrf2 may enhance autophagy induced by TMZ in the U251 glioma cell line, which should be further evaluated for novel anticancer activity.