Simvastatin alters M1/M2 polarization of murine BV2 microglia via Notch signaling.

Microglia play a critical role in the regulation of CNS immune function, which can be greatly affected by M1/M2 polarization. The role of Notch signaling in Statins induced alteration of M1/M2 polarization in BV2 cells was assessed in this study. M1 markers in LPS and Jagged-1 treated group were significantly increased and such increase was attenuated by simvastatin; however, M2 markers were enhanced. Moreover, simvastatin enhance the expression of Notch signaling molecules, and its regulatory effects were blocked in Notch1 knocked down cells. In conclusion, these findings indicated that simvastatin alters M1/M2 polarization of murine BV2 microglia via Notch signaling.

Involvement of TLR2 and TLR9 in the anti-inflammatory effects of chlorogenic acid in HSV-1-infected microglia.

AIMS: There is no effective medication to date for herpes simplex virus encephalitis (HSE). In this study, we investigated the anti-inflammatory effect of chlorogenic acid (CGA) on herpes simplex virus (HSV)-1-induced responses in BV2 microglia. MAIN METHODS: The cellular model was established with BV2 cells stimulated by HSV-1 and then treated with CGA at different concentrations. Cell viability was assayed by the MTT assay. The mRNA expression of Toll-like receptor (TLR)-2, TLR9 and myeloid differentiation factor88 (Myd88) was assayed by real-time quantitative PCR, and the protein expression was assayed by flow cytometry or Western blotting. Tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were measured by ELISA as well as real-time quantitative PCR. Nuclear NF-κB p65 protein was assayed by Western blotting. KEY FINDINGS: The cell survival rate was significantly improved after CGA treatment, and CGA prevented increases in TLR2, TLR9 and Myd88 following HSV-1 challenge in BV2 cells both at the mRNA and protein levels. Moreover, CGA could attenuate HSV-induced TNF-α and IL-6 release into the supernatant. The mRNA levels of TNF-α and IL-6 were also significantly inhibited by CGA. The expression of NF-κB p65 increased significantly in the nucleus in HSV-1-stimulated microglia but could be reduced by CGA. SIGNIFICANCE: CGA inhibits the inflammatory reaction in HSE via the suppression of TLR2/TLR9-Myd88 signaling pathways. CGA may serve as an anti-inflammatory agent and provide a new strategy for treating HSE.

Corilagin Protects Against HSV1 Encephalitis Through Inhibiting the TLR2 Signaling Pathways In Vivo and In Vitro.

In this study, we tried to explore the molecular mechanism that Corilagin protected against herpes simplex virus-1 encephalitis through inhibiting the TLR2 signaling pathways in vivo and in vitro. As a result, Corilagin significantly prevented increase in the levels of TLR2 and its downstream mediators following Malp2 or HSV-1 challenge. On the other hand, in spite of TLR2 knockdown, Corilagin could still significantly suppress the expression of P38 and NEMO, phosphor-P38, and nuclear factor kappa B. The mRNA and protein expression of TLR2 and its downstream mediators in the brain tissue were also significantly lowered in mice treated with Corilagin. In addition, Corilagin inhibited expression of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 protein. In conclusion, Corilagin shows the potential to protect against HSV-1-induced encephalitis, and the beneficial effects may be mediated by inhibiting TLR2 signaling pathways.

Changes of c-fos, malondialdehyde and lactate in brain tissue after global cerebral ischemia under different brain temperatures.

Under global cerebral ischemia, the effect of different brain temperature on cerebral ischemic injury was studied. Male Sprague-Dawley rats were divided into normothermic (37-38°C) ischemia, mild hypothermic (31-32°C) ischemia, hyperthermic (41-42°C) ischemia and sham-operated groups. Global cerebral ischemia was established using the Pulsinelli four-vessel occlusion model and brain temperature was maintained at defined level for 60 min after 20-min ischemia. The expression of c-fos protein and the levels of malondialdehyde (MDA) and lactate in brain regions were detected by immunochemistry and spectrophotometrical methods, respectively. C-fos positive neurons were found in the hippocampus and cerebral cortex after cerebral ischemia reperfusion. Mild hypothermia increased the expression of c-fos protein in both areas, whereas hyperthermia decreased the expression of c-fos protein in the hippocampus at 24 h reperfusion, and the cerebral cortex at 48 h reperfusion when compared to normothermic conditions. In normothermic, mild hypothermic and hyperthermic ischemia groups, the levels of MDA and lactate in brain tissue were increased at 24, 48 and 72 h reperfusion following 20-min ischemia as compared with the sham-operated group (P<0.01). The levels of MDA and lactate in mild hypothermic group were significantly lower than those in normothermic group (P<0.01). It is suggested that brain temperature influences the translation of the immunoreactive protein product of c-fos after global cerebral ischemia, and MDA and lactate are also affected by hypothermia and hyperthermia.

Passive movement improves the learning and memory function of rats with cerebral infarction by inhibiting neuron cell apoptosis.

Passive movement has been found to improve evidently ischemic stroke patients' impaired capacity of learning and memory, but the optimal time window of initiating the therapy and the underlying mechanism are not fully understood. In this study, the effect of passive movement at different time windows on learning and memory of rats with cerebral infarction was detected. The results showed that the expression of caspase-3 and escape latency in the passive movement group were all considerably lower than those in the model group (P < 0.05), while the expression of Bcl-2 mRNA was significantly higher than those in the model group (P < 0.05). Moreover, we found that there were most significant changes of escape latency and expressions of Bcl-2 mRNA and caspase-3 when the therapy started at 24 h after focal cerebral infarction. These results suggest that passive movement is able to contribute to the recovery of learning and memory of rats with cerebral infarction, which is partially mediated by inhibiting neuron cell apoptosis, and the optimal therapeutic time is at 24 h after cerebral infarction.

Astrocyte-derived sonic hedgehog contributes to angiogenesis in brain microvascular endothelial cells via RhoA/ROCK pathway after oxygen-glucose deprivation.

The human adult brain possesses intriguing plasticity, including neurogenesis and angiogenesis, which may be mediated by the activated sonic hedgehog (Shh). By employing a coculture system, brain microvascular endothelial cells (BMECs) cocultured with astrocytes, which were incubated under oxygen-glucose deprivation (OGD) condition, we tested the hypothesis that Shh secreted by OGD-activated astrocytes promotes cerebral angiogenesis following ischemia. The results of this study demonstrated that Shh was mainly secreted by astrocytes and the secretion was significantly upregulated after OGD. The proliferation, migration, and tube formation of BMECs cocultured with astrocytes after OGD were significantly enhanced, but cyclopamine (a Shh antagonist) or 5E1 (an antibody of Shh) reversed the change. Furthermore, silencing Ras homolog gene family, member A (RhoA) of BMECs by RNAi and blocking Rho-dependent kinase (ROCK) by Y27632, a specific antagonist of ROCK, suppressed the upregulation of proliferation, migration, and tube formation of BMECs after OGD. These findings suggested that Shh derived from activated astrocytes stimulated RhoA/ROCK pathway in BMECs after OGD, which might be involved in angiogenesis in vitro.

Involvement of PI3K/Akt pathway in the neuroprotective effect of Sonic hedgehog on cortical neurons under oxidative stress.

The Sonic hedgehog (SHH) signaling pathway plays a pivotal role in neurogenesis and brain damage repair. Our previous work demonstrated that the SHH signaling pathway was involved in the neuroprotection of cortical neurons against oxidative stress. The present study was aimed to further examine the underlying mechanism. The cortical neurons were obtained from one-day old Sprague-Dawley neonate rats. Hydrogen peroxide (H(2)O(2), 100 µmol/L) was used to treat neurons for 24 h to induce oxidative stress. Exogenous SHH (3 µg/mL) was employed to activate the SHH pathway, and cyclopamine (20 µmol/L), a specific SHH signal inhibitor, to block SHH pathway. LY294002 (20 µmol/L) were used to pre-treat the neurons 30 min before H(2)O(2) treatment and selectively inhibit the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. The cell viability was measured by MTT and apoptosis rate by flow cytometry analysis. The expression of p38, p-p38, ERK, p-ERK, Akt, p-Akt, Bcl-2, and Bax in neurons was detected by immunoblotting. The results showed that as compared with H(2)O(2) treatment, exogenous SHH could increase the expression of p-Akt by 20% and decrease the expression of p-ERK by 33%. SHH exerted no significant effect on p38 mitogen-activated protein kinase (p38 MAPK) pathway. Blockade of PI3K/Akt pathway by LY294002 decreased the cell viability by 17% and increased the cell apoptosis rate by 2-fold. LY294002 treatment could up-regulate the expression of the pro-apoptotic gene Bax by 12% and down-regulate the expression of the anti-apoptotic gene Bcl-2 by 54%. In conclusion, SHH pathway may activate PI3K/Akt pathway and inhibit the activation of the ERK pathway in neurons under oxidative stress. The PI3K/Akt pathway plays a key role in the neuroprotection of SHH. SHH/PI3K/Bcl-2 pathway may be implicated in the protection of neurons against H(2)O(2)-induced apoptosis.

Over-expression of VEGF in marrow stromal cells promotes angiogenesis in rats with cerebral infarction via the synergistic effects of VEGF and Ang-2.

bThis study explored whether the transplantation of modified marrow stromal cells (MSCs) has angiogenic effects in a left middle cerebral artery occlusion infarction/reperfusion (MCAO I/R) rat model and preliminarily examined the mechanism of angiogenesis following cerebral infarction. MSCs were isolated by using a direct adherent method and cultured. Vascular endothelial growth factor (VEGF) was transfected into MSCs by employing the liposome transfection. The transfection efficiency was measured by the optical density method. The protein expression of VEGF gene before and after transfection was measured by Western blotting. SD rat model of transient occlusion of the left middle cerebral artery was established by using an approach of intra-luminal occlusion. Tetrazolium (TTC) and HE staining were performed to observe the cerebral infarction. ELISAs were used to measure the levels of VEGF in the rat cerebral tissues. The expression patterns of angiopoietin-2 (Ang-2) and CD34 in cells surrounding the area of infarction were immunohistochemistrically observed. Ang-2 protein expression in the tissue surrounding the area of infarction was measured by Western blotting. VEGF expression in the MSCs increased after transfection at a rate of approximately 28%±3.4%. ELISA showed that the expression of VEGF in the cerebral tissue was significantly increased after induction of infarction, peaking on the 4th day and decreasing to the levels of the sham surgery group (normal) within 7 to 10 days. The VEGF level was significantly higher at each time point in the VEGF-MSC and MSC groups compared to the model group. Moreover, the VEGF level was higher in the VEGF-MSC group than in the MSC group and stayed relatively high until the 10th day. The immunohistochemical results showed that 10 days after the infarction, the number of Ang-2 and CD34-expressing cells in the area surrounding the infarction was significantly higher in the VEGF-MSC group and the MSC group compared to the model group. Moreover, the VEGF level was higher in the VEGF-MSC group than the MSC group. A similar trend in Ang-2 protein expression was revealed by Western blotting. In the MCAO rat model transfected with modified MSCs over-expressing VEGF, compared to the MSC transplantation group, the concentration of VEGF was significantly increased in the brain tissue after cerebral infarction. In addition, the level of Ang-2 was up-regulated, with angiogenesis promoted, the blood supply to the areas surrounding the cerebral infarction increased, and neurological function improved. We are led to speculate that the synergistic effects of VEGF and Ang-2 may be responsible for the angiogenesis following cerebral infarction.

1,2,3,4,6-penta-O-galloyl-ß-D-glucose protects PC12 Cells from MPP(+)-mediated cell death by inducing heme oxygenase-1 in an ERK- and Akt-dependent manner.

This study examined the ability of 1,2,3,4,6-penta-O-galloyl-ß-D-glucose (ß-PGG) to induce the expression of heme oxygenase-1 (HO-1) in the PC12 cells and its regulation in the PC12 cells. One week before treatment with the drug, nerve growth factor (NGF) was added to the cultures at a final concentration of 50 ng/mL to induce neuronal differentiation. After drug treatment, HO-1 gene transcription was analyzed by reverse transcription polymerase chain reaction (RT-PCR). Expression of HO-1 and NF-E2-related factor2 (Nrf2) and activation of extracellular signal-regulated kinase (ERK) and Akt were detected by Western blotting. The viability of the PC12 cells treated with different medicines was examined by MTT assay. The oxidative stress in the PC12 cells was evaluated qualitatively and quantitatively by DCFH-DA. The results showed that ß-PGG up-regulated HO-1 expression and this increased expression provided neuroprotection against MPP(+)-induced oxidative injury. Moreover, ß-PGG induced Nrf2 nuclear translocation, which was found to be upstream of ß-PGG-induced HO-1 expression, and the activation of ERK and Akt, a pathway that is involved in ß-PGG-induced Nrf2 nuclear translocation, HO-1 expression and neuroprotection. In conclusion, ß-PGG up-regulates HO-1 expression by stimulating Nrf2 nuclear translocation in an ERK- and Akt-dependent manner, and HO-1 expression by ß-PGG may provide the PC12 cells with an acquired antioxidant defense capacity to survive the oxidative stress.

Electro-acupuncture combined with transcranial magnetic stimulation improves learning and memory function of rats with cerebral infarction by inhibiting neuron cell apoptosis.

This study examined the effect of electro-acupuncture (EA) combined with transcranial magnetic stimulation (TMS) therapy at different time windows on learning and memory ability of rats with cerebral infarction and the underlying mechanism. Two hundred SD rats were randomly divided into four groups: normal group, sham-operated group, model group and EA+TMS group, and each group was then divided into five sub-groups in terms of the different time to start treatment post operation: 6, 12, 24, 48 and 72 h. Cerebral infarction models were established in the model and the EA+TMS groups by left middle cerebral artery occlusion/reperfusion (MCAO/R). After treatment for 14 d, the Morris water maze test was applied to examine the spatial learning and memory abilities of rats. In infarcted area, the expression of caspase-3 was immunohistochemically detected, and real-time fluorescent quantitative PCR was used to measure the expression of Bcl-2 mRNA. The results showed that in EA+TMS group compared with model group at the same treatment time windows, the escape latency was substantially shortened, the expression of caspase-3 was considerably decreased and the expression level of Bcl-2 mRNA significantly increased (P<0.05). In the EA+TMS sub-groups, the escape latency was shortest, the expression level of caspase-3 lowest, and the expression level of Bcl-2 mRNA highest at the treatment time window of 24 h. It was concluded that EA combined with TMS can promote neurological function of rats with cerebral infarction by increasing the expression level of Bcl-2 mRNA and decreasing the expression of caspase-3. The best time window is 24 h after perfusion treatment to ischemia.

Mitoxantrone exerts both cytotoxic and immunoregulatory effects on activated microglial cells.

Mitoxantrone (MX) is the most common immunosuppressive drug used in patients with rapidly worsening multiple sclerosis (MS), whose disease is not controlled by ß-interferon or glatiramer acetate. Although MX suppresses antigen-presenting cell (APC) and T-cell function in the periphery, its mechanism of action in the central nervous system (CNS) is not known. Given that MX can cross the disrupted blood-brain barrier, such as in MS patients, we in the present study have tested our hypothesis that MX in the CNS exerts cytotoxic and immunomodulatory effects on microglia, the major CNS-resident APCs that play a crucial role in MS pathogenesis. The cytotoxic effect of MX on microglial cells was determined by MTT and flow cytometry test, whereas the regulatory function was tested with enzyme-linked immunosorbent assay (ELISA) method. Indeed, we have found that MX induced microglial cell death in a dose-dependent manner, and the cell death was mainly from late apoptosis and necrosis. Further, MX induced significantly increased levels of interleukin (IL)-10 production of microglia, whereas IL-23p19 production/expression was significantly suppressed. Thus, our study for the first time demonstrates the immunosuppressive/regulatory effect of MX on microglia, which represents an important mechanism underlying the therapeutic effect of this drug on MS patients.

Effects of immediate and delayed mild hypothermia on endogenous antioxidant enzymes and energy metabolites following global cerebral ischemia.

BACKGROUND: The optimal time window for the administration of hypothermia following cerebral ischemia has been studied for decades, with disparity outcomes. In this study, the efficacy of mild brain hypothermia beginning at different time intervals on brain endogenous antioxidant enzyme and energy metabolites was investigated in a model of global cerebral ischemia. METHODS: Forty-eight male Sprague-Dawley rats were divided into a sham-operated group, a normothermia (37°C - 38°C) ischemic group and a mild hypothermic (31°C - 32°C) ischemia groups. Rats in the last group were subdivided into four groups: 240 minutes of hypothermia, 30 minutes of normothermia plus 210 minutes of hypothermia, 60 minutes of normothermia plus 180 minutes of hypothermia and 90 minutes of normothermia plus 150 minutes of hypothermia (n = 8). Global cerebral ischemia was established using the Pulsinelli four-vessel occlusion model for 20 minutes and mild hypothermia was applied after 20 minutes of ischemia. Brain tissue was collected following 20 minutes of cerebral ischemia and 240 minutes of reperfusion, and used to measure the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), reduced glutathione (GSH) and adenosine triphosphate (ATP). RESULTS: Mild hypothermia that was started within 0 to 60 minutes delayed the consumption of SOD, GSH-Px, GSH, and ATP (P < 0.05 or P < 0.01) in ischemic tissue, as compared to a normothermic ischemia group. In contrast, mild hypothermia beginning at 90 minutes had little effect on the levels of SOD, GSH-Px, GSH, and ATP (P > 0.05). CONCLUSIONS: Postischemic mild brain hypothermia can significantly delay the consumption of endogenous antioxidant enzymes and energy metabolites, which are critical to the process of cerebral protection by mild hypothermia. These results show that mild hypothermia limits ischemic injury if started within 60 minutes, but loses its protective effects when delayed until 90 minutes following cerebral ischemia.

Sonic hedgehog protects cortical neurons against oxidative stress.

Oxidative stress is one of the most important pathological mechanisms in neurodegenerative diseases and ischemia. Recent studies have indicated that the sonic hedgehog (SHH) signaling pathway is involved in these diseases, but the underlying mechanisms remains elusive. Here we report that the SHH pathway was activated in primary cultured cortical neurons after exposure to hydrogen peroxide (H2O2). H2O2 treatment decreased the cell viability of neurons, and inhibition of endogenous SHH signaling exacerbated its neurotoxicity. Activation of SHH signaling protected neurons from H2O2-induced apoptosis and increased the cell viability while those effects were partially reversed by blocking SHH signals. Exogenous SHH increased the activities of Superoxide dismutase (SOD) and Glutathione peroxidase (GSH-PX) in H2O2-treated neurons and decreased production of Malondialdehyde (MDA). It also promoted expression of the anti-apoptotic gene Bcl-2 and inhibited expression of pro-apoptotic gene Bax. Activation of SHH signals upregulated both Neurotrophic factors vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF). Pretreatment with SHH inhibited the activation of ERK (extracellular signal-regulated kinases) signals induced by H2O2. Our findings demonstrate that activation of SHH signaling protects cortical neurons against oxidative stress and suggest a potential role of SHH for the clinic treatments of brain ischemia and neurodegenerative disorders.

S-methylisothiourea induces apoptosis of herpes simplex virus-1-infected microglial cells.

Our study showed that S-methylisothiourea (SMT) had anti-inflammatory effects in treating herpes simplex encephalitis in mice, and SMT also induced apoptosis of herpes simplex virus (HSV-1)-infected microglial cells. Both animal and cell models were employed in this study. Both models included the following five groups: a normal control group, a virus group (HSV-1 infected), an SMT group (HSV-1-infected + SMT (0.1 mg/10 g)), a dexamethasone group (HSV-1 infected + dexamethasone (2 µg/10 g)), and an APS group (HSV-1-infected + APS (0.8 mg/10 g)). ELISA was used to measure tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-10, and Greiss method was used for measuring nitric oxide (NO) secretion. HE staining was performed for detecting changes in mice brain. Flow cytometry assay for caspase-3, caspase-8, caspase-9, and caspase-12 expressions was also carried out to assess apoptosis. Expressions of TNF-α, IL-1ß, and NO were significantly elevated after stimulation of microglial cells with HSV-1. Following SMT intervention, TNF-α, IL-1ß, and NO levels were significantly decreased. The inflammatory changes in HSV-1-infected murine brain tissues were also reduced. SMT induction of apoptosis of HSV-stimulated microglia seemed to be through three pathways: the death receptor, mitochondrially gated, and endoplasmic reticulum. SMT can reduce HSV-induced inflammatory insult to the brain. Its mechanism of action is most probably due to the induction of microglial cell apoptosis.

Overexpression of aldehyde dehydrogenase-2 attenuates neurotoxicity induced by 4-hydroxynonenal in cultured primary hippocampal neurons.

4-Hydroxynonenal (4HNE) is a toxic aldehyde which can accumulate during neurodegenerative diseases, such as AD. 4HNE-induced neuronal cytotoxicity includes the damage of neurite growth as well as a potential threat leading to the neuronal cell death. This study was designed to examine whether overexpression of aldehyde dehydrogenase-2 (ALDH2) affects 4HNE-induced neurite outgrowth blockage and neuronal death in primary hippocampal neurons in vitro. Plasmid-encoding rat ALDH2 was constructed and transfected into cultured rat hippocampal neurons. In vitro-cultured rat hippocampal neurons with the transfection of ALDH2 gene were showing resistance to 4HNE-induced neurite damage. Overexpression of ALDH2 in cultured rat hippocampal neurons blocked 4HNE-induced (3.2 µM for 24 h) reduction of neurite outgrowth and branching. In addition to the effect on neurite growth, ALDH2 overexpression also can protect neurons from 4HNE-evoked (10 µM for 24 h) neuronal death. Furthermore, we found that overexpression of ALDH2 can decrease the caspase-3 protein expression level; at the same time, it can decrease the reactive oxygen species (ROS) level and the disruption of mitochondrial transmembrane potential in cultured hippocampal neurons. Our data suggested that overexpressed ALDH2 gene may moderate 4HNE-induced neuronal death by regulating caspase-3 protein and ROS level in cultured hippocampal neurons. Based on these findings, ALDH2 gene can be a potential therapeutic target for treatment of neurodegenerative diseases, such as AD.

Mild hypothermia reduces ischemic neuron death via altering the expression of p53 and bcl-2.

OBJECTIVE: Studies exploring roles of p53 and bcl-2 in neuroprotection by hypothermia in focal cerebral ischemia have not provided consistent results. In the present study, we determined whether p53 and bcl-2 are involved in the hypothermia-induced neuroprotection. METHODS: Male Sprague-Dawley rats were divided into four groups: normothermic (37-38 degrees C) ischemia, hypothermic (31-32 degrees C) ischemia, hyperthermic (41-42 degrees C) ischemia and sham-operated group. Global cerebral ischemia was established for 20 minutes using the Pulsinelli four-vessel occlusion model and the brain temperature was maintained at defined levels for 60 minutes following the 20 min ischemia. The mortality in rats was evaluated at 72 hour and 168 hour reperfusion. The expression of p53 and bcl-2 proteins was detected at 24, 48 and 72 hours after reperfusion. At the same intervals, neuron necrosis and apoptosis in brain regions was also detected using hematoxylin and eosin (HE) staining and terminal deoxynucleotldyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL). RESULTS: The mortalities of rats in normothemia, hypothermia and hyperthermia groups was 33.3, 16.7 and 50% at 72 hour reperfusion. At 168 hours of reperfusion, the mortality in the three groups was 58.3, 25 and 100%, respectively. In light microscopy studies, necrotic neurons and apoptotic neurons were found in the hippocampus after global cerebral ischemia. Surviving neurons in hippocampus was increased in mild hypothermic ischemia group (p<0.05) and decreased in hyperthermia ischemia group (p<0.01) at 24, 48 and 72 hour reperfusion. TUNEL-positive neurons in hippocampus decreased in hypothermic ischemia group (p<0.05 or p<0.01) and increased in hyperthermic ischemia group (p<0.01) at 24, 48 and 72 hour reperfusion. The expression of p53 and bcl-2 proteins was found in the neurons of cerebral cortex after global cerebral ischemia. P53 decreased and bcl-2 increased in hypothermia group. CONCLUSION: Hypothermia reduces ischemic neuronal necrosis and apoptosis by reducing p53 and increasing bcl-2 expression. Hyperthermia accelerated ischemic neuronal injury by increasing p53 and reducing bcl-2 expression.

Favorable effects of MMP-9 knockdown in murine herpes simplex encephalitis using small interfering RNA.

BACKGROUND AND PURPOSE: The prognosis of herpes simplex encephalitis (HSE) remains poor despite available antiviral treatment. Matrix metalloproteinase-9 (MMP-9) is currently considered to play a major role in promoting cerebrovascular complications which contribute to the high mortality and morbidity of HSE. We hypothesize that temporally knockdown MMP-9 expression in early phase of HSE might be an effective treatment strategy. METHODS: The animal models of herpes simplex encephalitis were established by intracerebrally inoculated herpes simplex virus type 1 (HSV-1) in mice. Mice were inoculated intracerebrally with MMP-9 targeting siRNA (MMP-9 siRNA). MMP-9 expression was assessed by RT-PCR and western blot analysis at 3 and 7 days after HSV-1 infected. The blood-brain barrier (BBB) permeability was quantitated by Evans blue dye extravasations and brain water content. Immunohistochemistry method was adopted to analyse the expression of AQP4 protein. Quantitative real-time PCR analysis was used to detect cytokines expression. Neurological score was quantified using an established neurological scale at 7 days after HSE. RESULTS: Using synthetic small interfering RNA, we found a single intracerebral injection of siRNA targeting murine MMP-9 mRNA (MMP-9 siRNA) silenced MMP-9 expression and reduced it to normal level at day 7 post-infection. The improvement in neurological function and increased cumulative survival reflected the functional consequence of this therapy. MMP-9 knockdown mice also displayed less uptake of Evans blue and reduced brain water content compared with control siRNA-treated group. Also the HSV-1-induced upregulation of proinflammatory cytokines was significantly diminished in MMP-9 siRNA-treated mice. In addition, aquaporin-4 expression in perivascular decreased in MMP-9 siRNA-treated mice and might contribute to the protection of blood-brain barrier. DISCUSSION: This compelling evidence suggests that MMP-9 is a key pathogenic factor within HSE, and local injection of synthetic siRNA in the brain could knock down MMP-9 expression in acute phase of HSE, reduce brain edema and improves mice neurological function and increase cumulative survival.

Effect of Corilagin on anti-inflammation in HSV-1 encephalitis and HSV-1 infected microglias.

The aim of this explore is to study the anti-inflammatory effect of Corilagin in herpes simplex virus (HSV)-1 infected microglial cells and HSV-1 infected mouse brain. The cellular model was set with microglial cells stimulated by HSV-1 and divided respectively, into virus, astragalus polysaccharides (APS), Dexamethasone and Corilagin group. A normal control group consisting of uninfected microglial cells was also included. ELISA for measuring TNF-alpha, IL-1beta and IL-10 and Greiss method for detecting NO secretion in supernatant, flow cytometry assay for examining apoptosis rate, expression of caspase-3, caspase-8, caspase-9 and caspase-12, and western-blot for measuring protein expression of cytochrome c were performed. The animal model was set up using Balb/c male mice that were intracranially inoculated with HSV-1. Animals were then divided in groups as described for the cellular model. Here, too a normal control group was included. HE staining was used to assay pathological changes in brain. As results, after Corilagin intervention, the release of TNF-alpha, IL-1beta and NO from HSV-stimulated migroglia cells was significantly inhibited. Furthermore, Corilagin induced apoptosis of HSV-stimulated microglia through all the 3 known apoptotic pathways. The animal model treated with Corilagin also displayed significant decrease of herpes simplex encephalitis induced brain pathological changes. In conclusion, Corilagin has the potential to reduce HSV-1-induced inflammatory insult to the brain, and its mode of action is through the induction of apoptosis of microglias and reduction of cytokines production.

Activation of sonic hedgehog signaling pathway in olfactory neuroblastoma.

OBJECTIVES: Sonic hedgehog (Shh) signaling pathway is associated with tumor development; however, the role of Shh signaling in the development of olfactory neuroblastoma (ONB) is unknown. This study aimed to investigate the relationship between the regulation of Shh signaling and the pathogenesis of ONB. METHODS: The expression of Shh signaling components was characterized by immunohistochemistry in human non-tumor olfactory epithelium and ONB specimens, and by RT-PCR and immunoblotting in human ONB cell lines. The impact of the treatment with cyclopamine (a selective inhibitor of the Shh pathway) and/or exogenous Shh on ONB cell proliferation, cycle and apoptosis was examined by MTT, soft agar colony formation and flow cytometry assays, respectively. The influence of Shh signaling on the expression of Shh signaling components and cell cycle-related regulators was determined by immunoblotting and quantitative RT-PCR, respectively. RESULTS: The expression of Pacthed1, Gli1 and Gli2 was detected in 70, 70, and 65% of human ONB specimens, respectively, and in proportion of ONB cell lines, but not in non-tumor olfactory epithelium. Treatment with cyclopamine inhibited the proliferation and colony formation of ONB cells, induced ONB cell cycle arrest and apoptosis, and down-regulated the expression of Pacthed1, Gli1 and cyclin D1, but up-regulated p21 expression in vitro. These regulatory effects of cyclopamine were partially or completely erased by exogenous Shh. CONCLUSION: These data suggest that the Shh signaling pathway is crucial for the growth of ONB.

A critical role of Sonic Hedgehog signaling in maintaining the tumorigenicity of neuroblastoma cells.

Accumulated evidence suggests a major role for the activation of the Sonic Hedgehog (SHH) signaling pathway in the development of neural crest stem cells that give rise to the sympathetic nervous system. We therefore investigated the involvement of SHH signaling in the pathogenesis of neuroblastoma, a common childhood malignant tumor of the sympathetic nervous system. Human neuroblastoma cell lines and a majority of primary neuroblastoma specimens showed high-level expression of the pathway targets and components, indicating persistent activation of the SHH pathway. All of the neuroblastoma cell lines we examined expressed significant levels of SHH ligand, suggesting an autocrine, ligand-dependent activation of the SHH pathway in neuroblastoma cells. Inhibition of SHH signaling by cyclopamine induced apoptosis and blocked proliferation in all major types of neuroblastoma cells, and abrogated the tumorigenicity of neuroblastoma cells. Moreover, the knockdown of GLI2 in neuroblastoma BE (2)-C and SK-N-DZ cell lines resulted in the inhibition of colony formation. Our study has revealed a molecular mechanism for the persistent activation of the SHH pathway which promotes the development of neuroblastoma, and suggests a new approach for the treatment of this childhood malignant tumor. (Cancer Sci 2009; 100: 1848-1855).