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ABSTRACT: Recently, the role of inflammation has attracted great attention in the pathogenesis of mesial temporal lobe epilepsy (MTLE), and microRNAs start to emerge as promising new players in MTLE pathogenesis. In this study, we investigated the dynamic expression patterns of tumor necrosis factor alpha (TNF-α) and microRNA-155 (miR-155) in the hippocampi of an immature rat model of status epilepticus (SE) and children with MTLE. The expressions of TNF-α and miR-155 were significantly upregulated in the seizure-related acute and chronic stages of MTLE in the immature rat model and also in children with MTLE. Modulation of TNF-α expression, either by stimulation using myeloid-related protein (MRP8) or lipopolysaccharide or inhibition using lenalidomide on astrocytes, leads to similar dynamic changes in miR-155 expression. Our study is the first to focus on the dynamic expression pattern of miR-155 in the immature rat of SE lithium-pilocarpine model and children with MTLE and to detect their relationship at the astrocyte level. TNF-α and miR-155, having similar expression patterns in the three stages of MTLE development, and their relationship at the astrocyte level may suggest a direct interactive relationship during MTLE development. Therefore, modulation of the TNF-α/miR-155 axis may be a novel therapeutic target for the treatment of MTLE.
Journal of Molecular Neuroscience 05/2013; · 2.50 Impact Factor
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ABSTRACT: To observe the expression of dynamin-1 and phosphor-dynamin-1 in the hippocampus of children and rats with mesial temporal lobe epilepsy (MTLE) and to investigate the roles of dynamin-1 and phosphor-dynamin-1 in the development of MTLE.
Male Sprague-Dawley rats (aged 25 days) were randomly divided into acute control (AC), acute seizure (AS), latent control (LC), latent seizure (LS), chronic control (CC) and chronic spontaneous seizure (CS) groups. Lithium chloride-pilocarpine was used to induce a rat model of MTLE. The hippocampus samples of 5 children with a pathologically confirmed hippocampal sclerosis who received surgical operation were collected as a human model (HM) group, and the hippocampus samples of 4 dead children (without organic lesion of the hippocampus) were collected by autopsy as a human control (HC) group. The expression of dynamin-1 and phosphor-dynamin-1 in the hippocampus of children and rats with MTLE was measured by Western blot and immunohistochemistry.
The Western blot showed that the expression of phosphor-dynamin-1 was significantly lower in the AS and CS groups than in the corresponding control groups (AC and CC groups) (P<0.05). The expression of phosphor-dynamin-1 was significantly lower in the HM group than in the HC group (P<0.05). There were no significant differences in the expression of dynamin-1 among the AS, LS and CS groups and between the HM and HC groups (P>0.05). The immunohistochemical results showed that phosphor-dynamin-1 was highly expressed in the cytoplasm of hippocampal neurons of AC, CC and HC groups, but its expression was significantly reduced in the AS, CS and HM groups (P<0.05).
The expression of phosphor-dynamin-1, not dynamin-1, is downregulated in the hippocampus of children and rats with MTLE during seizures, which suggests that the phosphorylation/dephosphorylation of dynamin-1 may be involved in the development of MTLE.
Zhongguo dang dai er ke za zhi = Chinese journal of contemporary pediatrics 02/2013; 15(2):133-7.
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ABSTRACT: Mesial temporal lobe epilepsy (MTLE) is a particularly devastating form of human epilepsy with significant incidence of medical intractability. MicroRNAs (miRs) are small, noncoding RNAs that regulate the posttranscriptional expression of protein-coding mRNAs, which may have key roles in the pathogenesis of MTLE development. To study the dynamic expression patterns of brain-specific miR-124 and miR-134 and inflammation-related miR-132 and miR-21, we performed qPCR on the hippocampi of immature rats at 25 days of age. Expressions were monitored in the three stages of MTEL and in the control hippocampal tissues corresponding to the same timeframes. A similar expression method was applied to hippocampi obtained from children with MTLE and normal controls. The expression patterns of miR-124 and miR-134 nearly showed the same dynamics in the three stages of MTLE development. On the other hand, miR-132 and miR-21 showed significant upregulation in acute and chronic stages, while in the latent stage, miR-132 was upregulated and miR-21 was downregulated. The four miRs were upregulated in hippocampal tissues obtained from children with MTLE. The significant upregulation of miR-124 and miR-134 in the seizure-related stages and children suggested that both can be potential targets for anticonvulsant drugs in the epileptic developing brains, while the different expression patterns of miR-132 and miR-21 may suggest different functions in MTLE pathogenesis.
Journal of Molecular Neuroscience 01/2013; · 2.50 Impact Factor
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ABSTRACT: The pandemic of the childhood obesity represent a major public health problem all over the world. This leads to detection of many health conditions that were previously considered an adulthood diseases. The rise in the prevalence of the obesity and overweight among children means that the world will face an explosion in the prevalence of the metabolic syndrome (MS), which increases the risk of atherosclerotic disease and death in adulthood. The atherosclerotic process has proved to develop silently for decades during childhood and adolescence before the cardiovascular complications such as myocardial infarction and stroke occur. This means that obese children especially with MS could have heart attacks and suffer from heart disease in an age when they should be very healthy, but most of these data either derived from autopsy findings or studies that confirmed the presence of peripheral atherosclerosis. Very early detection of coronary atherosclerosis in obese children with metabolic syndrome through a non invasive method will be of great importance, allowing for early therapeutic intervention. The discovery of microRNAs (miRNAs) is considered a major scientific breakthrough in the last years; recent studies have suggested a potentially important role of miRNAs in the control of diversity aspects of cardiac functions in health and disease including coronary atherosclerosis. Moreover, circulating miRNAs profiles recently used as a non-invasive biomarker for diagnosis of multiple cardiovascular diseases. The identification of distinct circulating miRNA profiles may impact the development of specific miRNAs as biomarkers in pediatric cardiovascular diseases. Therefore, we postulate that some of these circulating miRNAs may be a potential biomarker for early non-invasive diagnosis of coronary atherosclerosis in very early asymptomatic stage in obese children with metabolic syndrome, giving an excellent chance to fight against the first killer in the adult population in childhood period.
Medical Hypotheses 10/2012; · 1.39 Impact Factor
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ABSTRACT: Escherichia coli endotoxin LPS regulates blood-brain barrier permeability by disrupting the tight junction (TJ) complex between brain endothelial cells. This study used Bend.3 cells to examine the signaling networks involved in the hyperpermeability of the brain endothelial barrier caused by LPS. The LPS-induced alterations in the brain endothelial barrier were associated with PKC (a, β, ζ) and RhoA, but were independent of PI3K and the tyrosine kinase pathway. Inhibition of PKC (a, β, ζ) and RhoA activity using shRNA and dominant negative mutants diminished the effects of LPS on the brain's endothelial TJs. The interactions between the PKC and Rho pathways were therefore examined. PKC-a and PKC-ζ, but not PKC-β interacted with RhoA in Bend.3 cells stimulated by LPS. PKC-a acted as the upstream molecule for Rho and PKC-ζ acted as the downstream target for Rho. Comparing the effect of double inhibition of "Rho and PKC" and single inhibition of "Rho" or "PKC" confirmed that this interaction is critical for LPS-induced brain endothelial cell hyperpermeability. Collectively these data are the first to suggest that LPS affects the brain's endothelial TJ barrier via PKC (a, β, ζ)- and RhoA, independent of the PI3K and tyrosine kinase pathways. In addition, PKC-a and PKC-ζ, respectively, act as the upstream and downstream regulator for RhoA in the process.
Biochemical and Biophysical Research Communications 07/2012; 425(2):182-8. · 2.48 Impact Factor
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ABSTRACT: The aim of this study was to investigate the signaling mechanisms surrounding changes in tight junction (TJ) and the permeability of human intestinal epithelial cell induced by tumor necrosis factor-alpha (TNF-α).
To confirm that TNF-α induces epithelial barrier hyperpermeability by disrupting tight junction, Caco-2 cells were exposed to TNF-α, and changes in epithelial permeability (via TER assay), F-actin dynamics (via Rhodamine-phalloidin staining) and tight junction protein expression (via western blot) were monitored. Moreover, to ensure that NF-κB participated in the regulatory mechanisms, Caco-2 cells were transfected with DNMu-IκBα or control plasmids, the above experiments were repeated and the activation effect of TNF-α on NF-κB was detected by luciferase reporter assays. Lastly, we took dominant negative plasmid and knockdown approaches to investigate the potential importance of the NF-κB/myosin light chain kinase (MLCK)/myosin light chain phosphorylation (pMLC) pathways in TNF-a-mediated damage.
TNF-α could cause NF-κB activation, F-actin rearrangement, tight junction disruption and barrier dysfunction. These effects were alleviated by inhibiting NF-κB. TNF-α induced increase of MLCK transcription and MLC phosphorylation act later than NF-κB activation, which could be suppressed both by inactivating and deleting NF-κB.
TNF-α induces intestinal epithelial cell hyperpermeability by disrupting TJs, in part through MLCK upregulation, in which NF-κB is the positive upstream regulator for MLCK.
Cytokine 05/2012; 59(2):264-72. · 3.02 Impact Factor
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ABSTRACT: To study the influence of lipopolysaccharide (LPS) on the permeability of rat brain microvascular endothelial cells (BMECs) and possible molecular mechanism.
Monolayers of primary rat BMECs were separated and cultured, and then treated with (LPS group) or without LPS (control group). The barrier integrity was measured by transendothelial electrical resistance (TEER) assay. The degrees of RhoA activation were determined by Pull-down assay. The expression levels of p115RhoGEF, zonula occludens-1 (ZO-1), occludin and claudin-5 proteins were detected by Western blot analysis.
The average TEER values of rat BMECs in the LPS group were 108.3±4.2 Ω•cm2 and 85.4±2.5 Ω•cm2 respectively 3 and 12 hrs after LPS treatment, which were significantly lower than that in the control group (159.0±8.6 Ω•cm2). Compared with the control group, the activity of RhoA started to increase 5 minutes after LPS treatment, and the expression of p115RhoGEF protein started to increase 1 hr after LPS treatment and the cellular protein levels of ZO-1, occludin and claudin-5 decreased significantly 3 hrs after LPS treatment in the LPS group (P<0.05).
LPS may activate the p115RhoGEF/RhoA pathway and decrease protein expression of ZO-1, occludin and claudin-5, resulting in an increased permeability of rat BMECs.
Zhongguo dang dai er ke za zhi = Chinese journal of contemporary pediatrics 11/2011; 13(11):908-11.
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ABSTRACT: Hydrocephalus is a common complication of tuberculous meningitis (TBM) in children. The role of ventriculoperitoneal shunt (VPS) placement in grade IV patients is controversial. The aim of this study is to investigate the clinical value of VPS placement for patients with grade IV TBM with hydrocephalus (TBMH).
This study was carried out on children with grade IV TBMH from January 2006 to January 2011 in Xiangya Hospital, China. All patients were given VPS placement combined with medicine treatment. External ventricular drainage (EVD) was performed only in the presence of severe biochemical derangements or brainstem dysfunction requiring correction before shunt surgery. Outcomes were divided into normal, mild sequelae, severe sequelae, death, or vegetable status.
A total of 19 children with grade IV TBMH were recruited into the study. The average follow-up period was 29 months. Three of the 19 patients expired, four patients had a full recovery, eight had slight sequelae, and four had severe sequelae. Six complications related to the shunt surgery were seen among the patients.
This study demonstrates that direct ventriculoperitoneal shunt surgery could improve the outcome of grade IV TBMH. The response to EVD is not a reliable indication for selecting patients who would benefit from shunt surgery.
Child s Nervous System 09/2011; 28(2):209-15. · 1.54 Impact Factor
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ABSTRACT: To establish an animal model resembling human mesial temporal lobe epilepsy (MTLE); observe the dynamic changes of mossy fiber sprouting (MFS) and neuron loss in the hippocampus; and investigate the expression changes of Glial fibrillary acidic protein (GFAP) and Synapsin I in the hippocampus in immature rats.
MTLE models of immature rats were induced by lithium-pilocarpine. The surviving animals were continually monitored for 8 weeks. Nissl staining was used to observe the neuron loss and Timm staining was performed to evaluate MFS. Western blot (WB) and immunohistochemical methods were performed to detect the expression of GFAP and Synapsin I.
Status epilepticus (SE) was successfully induced in 94.1% of the rats with a high mortality of 68.8%; 75% of the survived rats were observed for spontaneous recurrent seizures (SRS) which resembles the features of human MTLE. Expression levels of glial fibrillary acidic protein and Synapsin I fluctuated in correspondence with the different stages of MTLE development.
We established an animal model depicting the human MTLE by using immature rats. GFAP and Synapsin I expressions are involved in MTLE development. Neuron loss and mossy fiber sprouting may have a role in epileptogenesis.
The International journal of neuroscience 08/2011; 121(10):575-88. · 0.86 Impact Factor
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ABSTRACT: In endothelial cells, exposure to lipopolysaccharide (LPS) results in barrier dysfunction through a complex signaling mechanism. The RhoA/Rho-kinase pathway plays a significant role in endothelial cell permeability. p115RhoGEF, a specific guanine nucleotide exchange factors (GEFs) activates RhoA, triggering RhoA-dependent cytoskeletal remodeling. However, little is known about the role of p115RhoGEF in LPS-induced brain endothelial barrier breakdown. We hypothesized that suppression of p115RhoGEF may inhibit activation of RhoA and prevent LPS-induced brain microvascular endothelial cell hyperpermeability.
The cultured monolayer of bEnd.3 cells, an immortalized mouse brain endothelial cell line, was used in this study. bEnd.3 cells were pretreated with specific siRNA to knockdown p115RhoGEF or C3 transferase to inhibit RhoA activity, and then incubated with LPS (5μg/ml). The degree of RhoA activation was determined by a Rhotekin-based pull-down assay, and expression of p115RhoGEF, zonula occludens-1 (ZO-1), occludin and claudin-5 proteins were detected by Western blot analysis. The barrier function was measured by transendothelial electrical resistance (TEER). F-actin cytoskeleton was visualized by Rhodamine-phalloidin staining.
The expression level of p115RhoGEF protein was significantly increased in LPS-treated bEnd.3 cells. The activity of RhoA was enhanced after LPS stimulation and pretreatment with p115RhoGEF siRNA or exoenzyme C3 transferase reduced RhoA activation significantly as shown by the pull-down assay. Furthermore, depletion of p115RhoGEF partially prevented the LPS-induced decrease in TEER, stress fiber formation and tight junction proteins degradation.
These results suggest that p115RhoGEF is important for LPS signaling to RhoA and LPS-induced endothelial barrier dysfunction, providing new insight into the function of RhoGEFs in inflammation.
Brain research 02/2011; 1387:1-7. · 2.46 Impact Factor
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ABSTRACT: Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, is capable of activating the small GTPase RhoA, which in turn contributes to endothelial barrier dysfunction. However, the underlying signaling mechanisms remained undefined. Therefore, we aimed to determine the role of protein kinase C (PKC) isozymes in the mechanism of RhoA activation and in signaling TNF-α-induced mouse brain microvascular endothelial cell (BMEC) barrier dysfunction.
Bend.3 cells, an immortalized mouse brain endothelial cell line, were exposed to TNF-α (10 ng/mL). RhoA activity was assessed by pull down assay. PKC-α activity was measured using enzyme assasy. BMEC barrier function was measured by transendothelial electrical resistance (TER). p115RhoGEF phosphorylation was detected by autoradiography followed by western blotting. F-actin organization was observed by rhodamine-phalloidin staining. Both pharmacological inhibitors and knockdown approaches were employed to investigate the role of PKC and p115RhoGEF in TNF-α-induced RhoA activation and BMEC permeability.
We observed that TNF-α induces a rapid phosphorylation of p115RhoGEF, activation of PKC and RhoA in BMECs. Inhibition of conventional PKC by Gö6976 mitigated the TNF-α-induced p115RhoGEF phosphorylation and RhoA activation. Subsequently, we found that these events are regulated by PKC-α rather than PKC-β by using shRNA. In addition, P115-shRNA and n19RhoA (dominant negative mutant of RhoA) transfections had no effect on mediating TNF-α-induced PKC-α activation. These data suggest that PKC-α but not PKC-β acts as an upstream regulator of p115RhoGEF phosphorylation and RhoA activation in response to TNF-α. Moreover, depletion of PKC-α, of p115RhoGEF, and inhibition of RhoA activation also prevented TNF-α-induced stress fiber formation and a decrease in TER.
Taken together, our results show that PKC-α phosphorylation of p115RhoGEF mediates TNF-α signaling to RhoA, and that this plays a critical role in signaling F-actin rearrangement and barrier dysfunction in BMECs.
Journal of Neuroinflammation 01/2011; 8:28. · 3.83 Impact Factor
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ABSTRACT: To investigate the molecular mechanism for change in permeability in brain microvascular endothelial cells (bEnd.3) induced by lipopolysaccharide (LPS).
Monolayers of bEnd.3 were exposed to LPS, in the presence or absence of exoenzyme C3 transferase. We monitored the monolayer barrier integrity by transendothelial electrical resistance assay (TEER), activity of RhoA by pull down assay, NF-κB by luciferase reporter assay, and F-actin dynamic structure by Rhodamine-phalloidin staining.
Incubation of monolayers with LPS caused substantial barrier hyperpermeability. Under the normal condition, the average TEER of bEnd.3 was (82.33±3.11) ω.cm², while it decreased apparently to (53.67±2.01) ω.cm² and (37.67±3.05) ω.cm² when the cells had been treated for 3 and 12 h with LPS (P<0.05). Such effects could be inhibited partly by pretreatment of RhoA inhibitor exoenzyme C3 transferase. LPS activated RhoA and NF-κB at 0.5 h. The C3 transferase could significantly reverse the NF-κB activation (P<0.05). The F-actin rearrangements displayed in a time-dependent manner and occurred originally after the stimulation of LPS for 3 h, which could be diluted by the pretreatment of C3 transferase as well.
LPS induces the disruption of F-actin cytoskeleton and brain microvascular endothelial barrier integrity, in part, through RhoA and NF-κB activation. The mechanism underlying this pathophysiological effect of RhoA is to influence the disruption of the F-actin cytoskeleton by regulating NF-κB activities.
Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences 11/2010; 35(11):1129-37.
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ABSTRACT: Gram-negative bacteria-induced multiple organ failure/dysfunction syndrome (MOF/MODS) is one of the leading causes of death through the world. The member of immunoglobulin family programmed death-1 (PD-1) is a negative immune regulator. This study investigated the protective effect of PD-1 as well as the underlying mechanism in LPS-induced endotoxemia.
Ten PD-1(+/+) and ten PD-1 knockout (PD-1(-/-)) mice were injected peritoneally with LPS (10 mg/kg), and the survival was observed within 72 hrs after LPS injection. The other 40 PD-1(+/+) and 40 PD-1(-/-) mice were injected peritoneally with LPS (5 mg/kg). Blood samples were collected before injection and 1.5, 3 and 6 hrs after LPS injection (n=10 each time point). Serum levels of various inflammatory mediators were measured using ELISA.
The survival rate in PD-1(-/-) mice was noticeably lower than that in PD-1(+/+) mice after 10 mg/kg LPS injection. Serum levels of inflammatory mediators TNF-α, IL-1β, IL-12 and IL-17 in PD-1/mice were higher than those in PD-1(+/+) mice after 5 mg/kg LPS injection.
PD-1 can protect mice from LPS-induced endotoxemia probably through its regulation on inflammatory mediator production.
Zhongguo dang dai er ke za zhi = Chinese journal of contemporary pediatrics 10/2010; 12(10):812-5.
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ABSTRACT: The purpose of this study was to assess weather the immortalized mouse brain endothelial cell line Bend.3 displays the comparative barrier characteristics as the primary brain microvascular endothelial cells (BEMC).
Immortalized mouse brain endothelial cell line, Bend.3 cells were cultured in transwell inserts and their restrictive characteristics were assessed by transendothelial electrical resistance (TEER) and horseradish peroxidase (HRP) permeability assays. Western blot and direct fluorescent staining methods were used to detect the tight junction protein expression and F-actin distribution.
The TEER in Bend.3 cells increased with the prolonged culture time and increased to 82.3+/-6.0 Omega cm2 10 days after culture, which was significantly higher than that 3 days after culture (37.3+/-3.1 Omega cm2; P<0.05). There were significant differences in the permeability rates for HRP 3 and 10 days after culture (4.3+/-0.20)% vs (2.2+/-0.05)% (P<0.05). Western blot indicated high level expression of tight junction proteins occludin and ZO-1 in Bend.3 cells 10 days after culture. F-actin was visualized around the cell membrane and presented scrobiculate linear fluorescence 10 days after culture.
Bend.3 cells have similar barrier characteristics to BEMC, and their barrier function may reach to the best effect 10 days after culture.
Zhongguo dang dai er ke za zhi = Chinese journal of contemporary pediatrics 06/2010; 12(6):474-8.
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ABSTRACT: The present study was conducted to investigate the effect of hepatocyte growth factor (HGF) on cortical neurons exposed to oxygen-glucose deprivation/reperfusion (OGD/R). Primary cultured cerebral cortical neurons were prepared from Sprague-Dawley rats. The cells were used for experiments after culture for 12 d in vitro. To initiate OGD/R, the culture medium was replaced by glucose-free medium, and cells were transferred to a humidified incubation chamber flushed by a gas mixture of 95% N(2) and 5% CO(2) at 37 °C for 2 h. Following this treatment, neurons were fed with glucose-supplemented (25 mmol/L) medium, and returned to the incubator under normoxic condition for 0-24 h. The cell viability was assessed by MTT assay, and cell injury was evaluated by lactate dehydrogenase (LDH) leakage rate. The percentage of apoptotic cells was analyzed by flow cytometry and Hoechst 33258 staining. The expressions of c-Met mRNA and protein were detected by RT-PCR and Western blot analysis, respectively. Oxygen-glucose deprivation for 2 h decreased the cell viability and increased LDH leakage rate in cultured cerebral cortical neurons. The cell viability declined and LDH leakage rate increased with the reperfusion time going on (0-24 h). To explore the influence of HGF on neurons under oxygen-glucose deprivation for 2 h/reperfusion for 24 h (OGD(2)/R(24)) condition, the cultures were pretreated with HGF at different concentrations (5-120 ng/mL) 2 h prior to OGD(2)/R(24). The results showed that OGD(2)/R(24) treatment significantly decreased the cell viability, increased LDH leakage rate and the percentage of apopototic cells. Pretreatment with HGF at 5 ng/mL and 10 ng/mL did not affect the decrease in cell viability resulting from OGD(2)/R(24). In the presence of 20 ng/mL HGF, the increase in cell viability in cortical neurons exposed to OGD(2)/R(24) began to appear, and 80 ng/mL of HGF exhibited the maximal effect. HGF at 5, 10 and 20 ng/mL did not affect the increase in LDH leakage rate in cortical neurons exposed to OGD(2)/R(24). In the presence of 40 ng/mL HGF, the decrease in LDH leakage rate in cortical neurons subjected to OGD(2)/R(24) began to appear, and 80 ng/mL of HGF displayed the maximal effect. In addition, HGF at 80 ng/mL significantly attenuated cell apoptosis resulting from OGD(2)/R(24). As detected by semi-quantitative RT-PCR and Western blot analysis, c-Met mRNA and protein were expressed in cerebral cortical neurons cultured for 12 d in vitro. c-Met mRNA and protein expressions in cortical neurons exposed to OGD(2)/R(24) were significantly upregulated and were not affected by pretreatment of HGF at 80 ng/mL. Treatment with c-Met inhibitor SU11274 (5 μmol/L) completely eliminated HGF-mediated protection of cortical neurons subjected to OGD(2)/R(24). The results suggest that HGF directly protects cortical neurons against OGD/R-induced cell injury in a dose-dependent manner, and HGF has a potent anti-apoptotic action on neurons exposed to OGD/R.
Sheng li xue bao: [Acta physiologica Sinica] 05/2008; 60(2):235-42.
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ABSTRACT: Hepatocyte growth factor (HGF) has been revealed to exert multipotent activities on a variety of cells. In this study, we investigated whether HGF had a direct neuroprotection on cultured cerebral cortical neurons subjected to hypoxia/reoxygenation (H/R) and explored the intracellular signalings mediated the effects. The decrease in cell viability and increase in number of apoptotic cells resulting from H/R were significantly prevented by HGF pre-treatment. HGF stimulated both ERK1/2 and Akt activities in cortical neurons. Inhibition of ERK activation completely abolished the protective effects of HGF, and inhibition of Akt activation reduced, but did not completely eliminate the HGF mediated neuroprotection. It is suggested that the neuroprotection of HGF depend on ERK1/2 pathway, and, to a lesser extent, PI-3K/Akt pathway. In addition, we found that pre-treatment with HGF remarkably attenuated the decrease in expression of Bcl-2 and Bcl-xL induced by H/R, but failed to affect the amount of Bax. It is likely that Bcl-2 and Bcl-xL contribute to the protective effects of HGF.
Colloids and Surfaces B Biointerfaces 03/2008; 61(2):290-7. · 3.46 Impact Factor
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ABSTRACT: To explore the mechanisms of hypoxic preconditioning on protecting cultured astrocytes from hypoxia injury.
Cultured astrocytes were divided randomly into several groups: control(C), hypoxia(H) and hypoxic preconditioning (HP). Cells MTT metabolic activity, qualitation of apoptosis and modality to explore the protection effects of hypoxic preconditioning. Immunocytochemistry of Bcl-2 and Bax to explore the mechanisms of hypoxic preconditioning on protecting astrocytes from hypoxia.
Compared with H group there was marked increase of MTT metabolic activity in HP48 and HP72 groups. Immunocytochemistry of Bcl-2 and Bax showed that compared with H group, expression of Bcl-2 was increased in HP group, while expression of Bax was decreased in HP group.
Hypoxic preconditioning can protect astrocytes from hypoxia. One possible mechanism maybe concerned with inhibition of Bax and maintain of Bcl-2 to depress apoptosis procedure.
Zhongguo ying yong sheng li xue za zhi = Zhongguo yingyong shenglixue zazhi = Chinese journal of applied physiology 02/2008; 24(1):30-4.
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Cell Biology International - CELL BIOL INT. 01/2008; 32(3).
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ABSTRACT: To observe the effect of thrombin on the cytotoxicity of astrocytes injured by hypoxia/reoxygenation(H/R) and to explore its relationship with inducible nitric oxide synthase (iNOS).
Primary astrocytes were cultured in DMEM with 10% approximately 15% calf serum and divided into 6 groups: a control group, a Tm control group, an H/R group, a Tm+H/R group, a hirudin (HR) control group, and a Tm+HR+ H/R group. The cell damage and viability were detected by the 3-(4, 5-dimethylthazol-2-yl)-2, 5 diphenyl tetrazo liumbromide (MTT) conversion method. The NO level in the cultured cell supernatant was assayed by Griess reagent. The flow cytometry was performed to evaluate the apoptosis rate of astrocytes. The iNOS mRNA was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Immunocytochemistry was used to observe the expression of iNOS protein.
The cell viability injured by H/R was lower than that of the control group, the NO production and apoptosis rate in the cell of H/R group were higher than those of the control group. Incubation of H/R cell with 10kU/L Tm enhanced the cytotoxicity of H/R stimulation compared with the cells injured by H/R. Hirudin can reverse the effect of thrombin. RT-PCR and immuneocytochemistry analysis demonstrated that the levels of iNOS mRNA and iNOS protein increased in the cells treated by H/R. Tm enhanced the expression of iNOS mRNA and iNOS protein in the cells treated by H/R. Hirudin blocked the effect of Tm.
Increasing the level of iNOS and enhancing the production of NO may be the mechanism of thrombinos cytotoxicity in astrocytes injured by H/R.
Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences 11/2007; 32(5):831-5.
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ABSTRACT: To explore the effect of thrombin precondition (TPC) on the rat cerebral astrocytes(As) cultured in oxygen-glucose deprivation (OGD).
Astrocytes were pretreated with thrombin (TB) at various concentrations (0.005 approximately 5.000 kU/L), and then insulted by OGD. The cell damage and viability were evaluated by the lactate dehydrogenase (LDH) effusion rate and the 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) conversion method. Detection of apoptotic cells was determined by the flow cytometry technique. The glutamate uptake of astrocytes was studied with [3H]-glutamate incorporation.
OGD increased the LDH, decreased the cell viability, increased the number of apoptotic astrocytes, and decreased the glutamate uptake (P<0.01). While preconditioned with thrombin at the same condition, the LDH decreased, the cell viability increased, the percentage of apoptotic cells decreased, and the glutamate uptake increased (P<0.05). The maximum protective effect of thrombin was observed at 0.1 kU/L.
Low concentration of thrombin precondition (TPC) can protect the astrocytes from oxygen-glucose deprived injury, and attenuate its apoptosis in a dose-dependent manner.
Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences 10/2007; 32(5):845-9.
