Rat Cerebral Microvascular Endothelial Cells Research Articles

Hyperglycemia aggravates spinal cord injury through endoplasmic reticulum stress mediated neuronal apoptosis, gliosis and activation

BackgroundHyperglycemia has been shown to influence prognostic outcome of spinal cord injury (SCI). However, the corresponding mechanism is not very clear. AimThis study is expected to explore the role of endoplasmic reticulum (ER) stress in hyperglycemia aggravated SCI. MethodsHyperglycemia was established in rats by intraperitoneal (i.p.) injection of streptozotocin. SCI was performed at the T10 of spinal cord through weight dropping. ER stress was suppressed by oral gavage of 4-PBA. ER stress, histological change of the injured spinal cords, neuronal apoptosis, demyelination, glial proliferation, inflammatory factor production, blood-spinal cord barrier (BSCB) permeability, TJ (Occludin, Claudin5) and AJ (β-catenin, P120) protein degradation, and locomotor recovery were determined using western blotting, immunohistochemistry, HE staining, Evan’s Blue assay, BBB scores and inclined plane test, respectively. In vitro, rat spinal cord neurons cells (RSCNCs) and cerebral microvascular endothelial cells (RCMECs) were stimulated with high glucose (HG) and/or thapsigargin (TG). The effects of HG and/or TG on RSCNCs apoptosis, and AJ and TJ expression by RCMECs were evaluated with flow cytometry and western blotting, respectively. ResultsHyperglycemic rats exhibited enhanced ER stress, increased neuronal apoptosis, aggravated demyelination, increased glial proliferation and inflammatory factors secretion, more serious BSCB disruption and disturbed locomotor recovery. ER stress inhibition alleviated hyperglycemia induced adverse effect on neuronal apoptosis and BSCB permeability, whereas showed little influence on glial activation and inflammation. ConclusionER stress was aggravated in hyperglycemic rats after SCI, and subsequently promoted neuronal apoptosis and BSCB disruption in rats.

MiR-466b-1-3p regulates P-glycoprotein expression in rat cerebral microvascular endothelial cells

Epilepsy is one of the most common neurological disorders, and approximately one-third of epilepsy cases are resistant to treatment with anti-epileptic drug (AED). P-glycoprotein (P-gp) is a multi-drug transporter that is thought to play a pivotal role in multiple drug resistance (MDR) in epilepsy. The regulatory mechanism of P-gp remains largely unknown; however, recent studies have demonstrated that microRNAs (miRNAs) may regulate the chemo-resistance mediated by P-gp. This study investigated the effect of specific miRNAs that regulate P-gp expression in rat cerebral microvascular endothelial cells (RCMECs). Primary cultures of RCMECs were treated with phenobarbital (PB) at various concentrations to induce P-gp overexpression. MiRNA microarrays were used to investigate the expression profiles of miRNAs in the resistant RCMECs induced by PB and corresponding non-resistant cells. Our data demonstrated decreased miR-466b-1-3p expression in the resistant cells compared with the non-resistant cells. Moreover, the recombinant RNA of 466b-1-3p (mimic) and the artificial antisense RNA of miR-466b-1-3p (inhibitor) were constructed and transfected into resistant RCMECs. The expression and function of P-gp were measured by Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry using rhodamine efflux. The mRNA and protein levels of P-gp increased as the concentration of PB increased, whereas miR-466b-1-3p levels decreased with increasing PB concentrations (P<0.05). The miR-466b-1-3p mimic down-regulated P-gp expression, whereas the miR-466b-1-3p inhibitor up-regulated P-gp expression (P<0.05). These findings demonstrate that miR-466b-1-3p may regulate PB-induced P-gp expression in RCMECs.

Hydrogen-Rich Saline Attenuates Brain Injury Induced by Cardiopulmonary Bypass and Inhibits Microvascular Endothelial Cell Apoptosis Via the PI3K/Akt/GSK3β Signaling Pathway in Rats

Background/Aims: Cardiopulmonary bypass (CPB) is prone to inducing brain injury during open heart surgery. A hydrogen-rich solution (HRS) can prevent oxidation and apoptosis, and inhibit inflammation. This study investigated effects of HRS on brain injury induced by CPB and regulatory mechanisms of the PI3K/Akt/GSK3β signaling pathway. Methods: A rat CPB model and an in vitro cell hypoxia model were established. After HRS treatment, Rat behavior was measured using neurological deficit score; Evans blue (EB) was used to assess permeability of the blood-brain barrier (BBB); HE staining was used to observe pathological changes; Inflammatory factors and brain injury markers were detected by ELISA; the PI3K/Akt/GSK3β pathway-related proteins and apoptosis were assessed by western blot, immunohistochemistry and qRT –PCR analyses of brain tissue and neurons. Results: After CPB, brain tissue anatomy was disordered, and cell structure was abnormal. Brain tissue EB content increased. There was an increase in the number of apoptotic cells, an increase in expression of Bax and caspase-3, a decrease in expression of Bcl2, and increases in levels of Akt, GSK3β, P-Akt, and P-GSK3β in brain tissue. HRS treatment attenuated the inflammatory reaction ,brain tissue EB content was significantly reduced and significantly decreased expression levels of Bax, caspase-3, Akt, GSK3β, P-Akt, and P-GSK3β in the brain. After adding the PI3K signaling pathway inhibitor, LY294002, to rat cerebral microvascular endothelial cells (CMECs), HRS could reduce activated Akt expression and downstream regulatory gene phosphorylation of GSK3β expression, and inhibit CMEC apoptosis. Conclusion: The PI3K/Akt/GSK3β signaling pathway plays an important role in the mechanism of CPB-induced brain injury. HRS can reduce CPB-induced brain injury and inhibit CMEC apoptosis through the PI3K/Akt/GSK3β signaling pathway.

Iptakalim, an ATP-sensitive potassium channel opener, confers neuroprotection against cerebral ischemia/reperfusion injury in rats by protecting neurovascular unit cells

To investigate the role of iptakalim, an ATP-sensitive potassium channel opener, in transient cerebral ischemia/reperfusion (I/R) injury and its involved mechanisms. Intraluminal occlusion of middle cerebral artery (MCAO) in a rat model was used to investigate the effect of iptakalim at different time points. Infarct volume was measured by staining with 2,3,5-triphenyltetrazolium chloride, and immunohistochemistry was used to evaluate the expressions of Bcl-2 and Bax. In vitro, neurovascular unit (NVU) cells, including rat primary cortical neurons, astrocytes, and cerebral microvascular endothelial cells, were cultured and underwent oxygen-glucose deprivation (OGD). The protective effect of iptakalim on NVU cells was investigated by cell viability and injury assessments, which were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and release of lactate dehydrogenase. Caspase-3, Bcl-2 and Bax mRNA expressions were evaluated by real-time polymerase chain reaction (PCR). Administration of iptakalim 0 or 1 h after reperfusion significantly reduced infarct volumes, improved neurological scores, and attenuated brain edema after cerebral I/R injury. Iptakalim treatment (0 h after reperfusion) also reduced caspase-3 expression and increased the ratio of Bcl-2 to Bax by immunohistochemistry. Iptakalim inhibited OGD-induced cell death in cultured neurons and astrocytes, and lactate dehydrogenase release from cerebral microvascular endothelial cells. Iptakalim reduced mRNA expression of caspase-3 and increased the ratio of Bcl-2 to Bax in NVU cells. Iptakalim confers neuroprotection against cerebral I/R injury by protecting NVU cells via inhibiting of apoptosis.

Targeting the neurovascular unit: Development of a new model and consideration for novel strategy for Alzheimer's disease

Alzheimer's disease involves the complex and interconnected cascade of cellular and molecular events. Only a few treatments are available to slow the course of the disease at present. Recent studies suggest that neurovascular unit serves to maintain cerebral homeostasis, and pathological interactions between components of neurovascular unit lead to cerebral dysfunction. In present study, we established a functional unit trying to target major components of the neurovascular unit by the co-culture of rat cortical parenchymal culture and cerebral microvascular endothelial cells. This entity allowed the application of techniques such as immunofluorescent imaging and biological assays under defined conditions. The morphology of cell types, blood–brain barrier function and neuronal activation were investigated. The insight revealed that targeting components of the neurovascular unit, rather than just the neuron, might be a priority in Alzheimer's disease and more likely to provide cerebroprotection.

Apigenin Isolated from the Medicinal Plant Elsholtzia rugulosa Prevents β-Amyloid 25–35-Induces Toxicity in Rat Cerebral Microvascular Endothelial Cells

Endothelial cells of cerebral capillaries forming the blood-brain barrier play an important role in the pathogenesis and therapy of Alzheimer’s disease. Amyloid-β peptides are key pathological elements in the development of this disease. Apigenin (4’,5,7-tetrahydroxyflavone) is a plant flavonoid and pharmacologically active agent that can be isolated from several plant species. In the present study, effects of apigenin obtained from the medicinal plant Elsholtzia rugulosa (Labiatae) on primary cultured rat cerebral microvascular endothelial cells (CMECs) mediated by amyloid-β peptide 25–35 (Aβ25–35) were examined. Aβ25–35 showed toxic effects on CMECs, involving reduction of cell viability, release of lactate dehydrogenase (LDH), increase of nuclear condensation, over-production of intracellular reactive oxygen species (ROS), decrease of superoxide dismutase (SOD) activity, and breakage of the barrier integrity and function. Based on this model, we demonstrated that apigenin from the medicinal plant Elsholtzia rugulosa protected cultured rat CMECs by increasing cell viability, reducing LDH release, relieving nuclear condensation, alleviating intracellular ROS generation, increasing SOD activity, and strengthening the barrier integrity through the preservation of transendothelial electrical resistance, permeability property and characteristic enzymatic activity after being exposed to Aβ25–35. In conclusion, apigenin isolated from Elsholtzia rugulosa has the ability to protect rat CMECs against Aβ25–35-induced toxicity.

Pinocembrin attenuates blood–brain barrier injury induced by global cerebral ischemia–reperfusion in rats

Blood–brain barrier (BBB) disruption is a major consequence of cerebral ischemia/reperfusion. Several studies have reported the neuroprotection of pinocembrin on cerebral ischemia in vivo and in vitro, but the effects of pinocembrin on BBB and its underlying mechanisms are not clear. In this study, we investigated the effects of pinocembrin on BBB functions in the global cerebral ischemia/reperfusion (GCI/R) model in rats. Neurological scores and brain edema were evaluated. BBB permeability was assessed by detecting the concentrations of Evan's blue (EB) and fluorescein sodium (NaF) in brain tissue. The pathological changes of BBB ultrastructure were observed by transmission electron microscopy. Cerebral blood flow (CBF) was measured by laser Doppler flowmetery. The effects of pinocembrin on primary cultured rat cerebral microvascular endothelial cells (RCMECs) against oxygen–glucose deprivation/reoxygenation (OGD/R) were also investigated. The results showed pinocembrin decreased neurological score and lessened brain edema induced by GCI/R. Pinocembrin also reduced the concentrations of EB and NaF in brain tissue of the GCI/R rats. And pinocembrin alleviated the ultrastructural changes of cerebral microvessels, astrocyte end-feet and neurons, and improved CBF in the GCI/R rats. In addition, pinocembrin increased the viability and mitochondrial membrane potential of cultured RCMECs induced by OGD/R. In conclusion, these data demonstrate that pinocembrin alleviates blood–brain barrier injury induced by GCI/R in rats.

Tumor Necrosis Factor α Primes Cerebral Endothelial Cells for Erythropoietin-Induced Angiogenesis

Erythropoietin (EPO) enhances angiogenesis in the ischemic brain. Stroke induces secretion of tumor necrosis factor α (TNF-α). We investigated the effect of TNF-α on EPO-induced in vitro angiogenesis in cerebral endothelial cells. Using a capillary-like tubular formation assay, we found that transient incubation of primary rat cerebral microvascular endothelial cells (RECs) with TNF-α substantially upregulated EPO receptor (EPOR) expression and addition of EPO into TNF-α-treated RECs significantly augmented the capillary-like tube formation. Blockage of TNF receptor 1 (TNFR1) suppressed TNF-α-upregulated EPOR expression and abolished EPO-induced tube formation. Attenuation of endogenous EPOR with small interfering RNA (siRNA) also inhibited EPO-enhanced tube formation. Treatment of RECs with EPO activated nuclear factor-kappa B (NF-κB) and Akt. Incubation of the TNF-α-treated endothelial cells with EPO activated vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), angiopoietin 1 (Ang1), and Tie2. Blockage of VEGFR2 and Tie2 resulted in reduction of EPO-augmented tube formation. These data indicate that interaction of TNF-α with TNFR1 sensitizes cerebral endothelial cells for EPO-induced angiogenesis by upregulation of EPOR, which amplifies the effect of EPO on activation of the VEGF/VEGFR2 and Ang1/Tie2 pathways. Our results provide the evidence for crosslink between TNF and EPOR to coordinate the onset of angiogenesis in cerebral endothelial cells.

Protective Effects of Luteolin against Amyloid β 25–35-induced Toxicity on Rat Cerebral Microvascular Endothelial Cells

Aim To evaluate the effects of luteolin against amyloid beta-peptide 25–35 (Aβ 25–35) on rat cerebral microvascular endothelial cells (CMECs). Methods CMECs were isolated from Wistar rats at 3-week-old, and randomly divided into 5 groups including control group, Aβ 25–35 group, 0.1, 1.0, and 10.0 μmol·L −1 luteolin groups. Cell viability was determined with MTS assay. Intracelluar ROS level and SOD activity were monitored by DCFH-DA and SOD inhibition assay, respectively. Transendothelial electrical resistance was measured using an EVOM resistance meter. γ-Glutamyl transpeptidase and alkaline phosphatase activities were detected using activity assay kits. Levels of TXA 2 and PGI 2 in culture medium were measured as their stable metabolites, TXB 2 and 6-keto-PGF 1α, by ELISA. Results Luteolin attenuated Aβ 25–35-induced toxicity at 0.1, 1.0, and 10 μmol·L −1, inhibited intracellular ROS generation, and increased SOD activity at 1.0 and 10 μmol·L −1. Luteolin was also found to preserve CMECs barrier function, involving the alleviation of TEER reduction, the increase of characteristic enzymatic activity, and regulation of TXA 2 and PGI 2 secretion. Conclusion Luteolin had the ability to protect rat CMECs against Aβ 25–35-induced toxicity.

Purification and identification of three novel antioxidant peptides from Cornu Bubali (water buffalo horn)

Cornu Bubali (water buffalo horn, WBH) has been used in Traditional Chinese Medicine (TCM) for thousands of years. In the present study, three peptides with antioxidant properties were purified from aqueous extract of Cornu Bubali (water buffalo horn, WBH) by consecutive chromatographic methods including gel filtration chromatography, ion-exchange chromatography and high performance liquid chromatography. The sequences of the three peptides were identified to be Gln-Tyr-Asp-Gln-Gly-Val (WBH-1, 708 Da), Tyr-Glu-Asp-Cys-Thr-Asp-Cys-Gly-Asn (WBH-2, 1018 Da) and Ala-Ala-Asp-Asn-Ala-Asn-Glu-Leu-Phe-Pro-Pro-Asn (WBH-3, 1271 Da) by matrix assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-LIFT-TOF/TOF MS). The antioxidant activity of these peptides was tested using 1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay directly. Methylthiazol tetrazolium (MTT) assay and lactate dehydrogenase (LDH) release assay were also used to evaluate the protection of peptides against hydrogen peroxide (H 2O 2) induced injury. The results showed that these peptides could reduce the DPPH radical and protect rat cerebral microvascular endothelial cells (rCMECs) against H 2O 2-induced injury, thus demonstrating that these peptides had antioxidant activity. These results suggest that WBH-1, WBH-2 and WBH-3, isolated from the aqueous extract of water buffalo horn are natural antioxidants and may contribute to the efficacy of WBH.

Antipyretic and Antioxidant Activities of the Aqueous Extract of Cornu Bubali (Water Buffalo Horn)

Cornu Bubali (water buffalo horn, WBH) is an animal-derived product which is widely used in Traditional Chinese Medicine (TCM) for dispelling heat, relieving convulsions and cooling blood. The purpose of this study was to investigate the antipyretic activity of WBH aqueous extract and its potential mechanism. Two hyperthermia models, yeast-induced (infectious) and skimmed milk-induced (noninfectious) hyperthermia were employed to evaluate the antipyretic effect and the results showed that rectal temperature of hyperthermia animals was decreased significantly after oral administration of WBH extract. The production of tumor necrosis factor-alpha (TNF-alpha)-induced prostaglandin E(2) (PGE(2)) in rat cerebral microvascular endothelial cells (rCMECs) was inhibited by WBH extract in the concentrations of 10 microg/ml and 100 microg/ml. The WBH extract protected rCMECs survival from hydrogen peroxide (H(2)O(2))-induced toxicity and inhibited the H(2)O(2)-induced leakage of lactate dehydrogenase (LDH) enzyme release at a dose ranging from 5 microg/ml to 100 microg/ml. It could also increase the superoxide dismutase (SOD) and catalase (CAT) enzyme activities. The results suggest that Cornu Bubali exhibits antipyretic activity on both infectious and noninfectious hyperthermia. The antipyretic activity of WBH may be due to the effects on enhancing antioxidation enzyme activities, decreasing PGE(2) production, and protecting the rCMECs against H(2)O(2)-induced injury.

Cerebrovascular actions of insulin

Insulin resistance is a major risk factor for cerebrovascular and neurodegenerative diseases; however, cerebrovascular actions of insulin have not been studied. Therefore, we evaluated the vascular actions of insulin in isolated rat cerebral arteries (CAs). Diameter measurements of CAs showed a biphasic response to insulin; an initial constriction was followed by vasodilation. Endothelial denudation or large conductance calcium‐activated K+ channels (KCa) inhibition resulted in vasoconstriction to insulin. Inhibition of small/intermediate conductance KCa or inward‐rectifier or voltage gated or ATP dependent K+ channels reduced insulin induced vasodilation. Inhibition of nitric oxide (NO) synthase or cytochrome P450 reduced vasodilation while inhibition of cyclooxygenase enhanced vasodilation to insulin. Inhibition of endothelin type A receptors enhanced the vasodilation while type B blockade diminished vasodilation to insulin. Inhibition of reactive oxygen species (ROS) production or scavenging the ROS abolished the vasoconstriction to insulin. Fluorescence studies of cultured rat cerebral microvascular endothelial cells showed elevation of intracellular calcium and enhanced generation of NO and ROS in response to insulin. Thus, insulin induced vasodilation was dependent K+ channels and endothelial factors while vasoconstriction was mediated by ROS, prostanoids and endothelin.

Mitochondrial dysfunction in endothelium impairs vasodilation in Zucker obese rats with insulin resistance

Mitochondria affect cerebrovascular tone by activation of mitochondrial ATP‐sensitive K+ channels (mitoKATP). Insulin resistance (IR) causes mitochondrial dysfunction, therefore, we evaluated the effects of diazoxide, a mitoKATP activator, on cerebral arteries of Zucker obese (ZO) rats and lean (ZL) controls. Diameter studies showed reduced vasodilation to diazoxide in ZO compared to ZL. Endothelial denudation reduced vasodilation to diazoxide in all arteries implicating endothelial mitoKATP. Inhibition of nitric oxide synthase (NOS) reduced vasodilation in intact arteries from ZL but not ZO, indicating diminished NO effects. Paradoxically, NOS inhibition in endothelium denuded arteries enhanced vasodilation in ZO implying the expression and uncoupling of NOS in vascular smooth muscle (VSM). Inhibition of cyclooxygenase (COX) enhanced vasodilation in intact arteries from ZL but not ZO. In contrast, endothelial denudation enhanced vasodilation in ZO but not ZL. Thus, ZO arteries exhibit enhanced production of both vasoconstrictor and vasodilator prostanoids. Fluorescence studies of rat cerebral microvascular endothelial cells showed enhanced NO production in response to diazoxide and also confirmed reduced NO generation in ZO arteries. Thus, endothelial mitochondrial dysfunction in ZO arteries results in the impairment of NOS and COX activity. Support: NIH HL077731, HL030260, HL 065380.

Effects of Qingkailing effective components on nuclear factor-kappa B in an ischemia-reperfusion injury model of rat brain microvascular endothelial cells in vitro

To establish an ischemia-reperfusion injury model of rat cerebral microvascular endothelial cells (MVECs) in vitro, and to explore the relationship between nuclear factor-kappa B (NF-kappaB) and the protective effects of Qingkailing effective components (hyocholic acid, taurocholic acid, baicalin, jasminoidin, Pinctada martensii) on MVECs. Brain MVECs of male rats were digested with trypsin and subcultured, then the content of MVECs was adjusted to 1x10 (5)/mL and the MVECs were divided into normal control group, untreated group, hyocholic acid group, taurocholic acid group, baicalin group, jasminoidin group, Pinctada martensii group and nimodipine group, with six holes in each group. Except for the normal control group, the MVECs in the other groups were exposed in oxygen and glucose deprivation (OGD) circumstance in vitro to simulate ischemia-reperfusion injury. Immunocytochemical staining and image analysis system were used to observe the expression of NF-kappaB protein. Under a light microscope, the nuclei of MVECs in the normal control group were blank. Staining intensity of NF-kappaB protein in the nucleus in the untreated group was much deeper than that in the endochylema, with NF-kappaB shifted to nucleus after activation; a small quantity of NF-kappaB protein were expressed in the border of nucleus next to endochylema in groups of Qingkailing effective components, and the NF-kappaB protein expression was weaker than that in the untreated group. With the image analysis, we found that transmittance of nucleus and endochylema in the untreated group was significantly lower than that in the normal control group (P<0.01). Transmittance of nucleus and endochylema in the treated groups was higher than that in the untreated group (P<0.05, P<0.01). Qingkailing effective components have significant effect in inhibiting NF-kappaB protein transferring from endochylema to nucleus in vitro.

Cerebromicrovascular endothelial cells are resistant to l-glutamate

Cerebral microvascular endothelial cells (CMVECs) have recently been implicated as targets of excitotoxic injury by l-glutamate (l-glut) or N-methyl-d-aspartate (NMDA) in vitro. However, high levels of l-glut do not compromise the function of the blood-brain barrier in vivo. We sought to determine whether primary cultures of rat and piglet CMVECs or cerebral microvascular pericytes (CMVPCs) are indeed sensitive to l-glut or NMDA. Viability was unaffected by 8-h exposure to 1-10 mM l-glut or NMDA in CMVECs or CMVPCs isolated from both species. Furthermore, neither 1 mM l-glut nor NMDA augmented cell death induced by 12-h oxygen-glucose deprivation in rat CMVECs or by 8-h medium withdrawal in CMVPCs. Additionally, transendothelial electrical resistance of rat CMVEC-astrocyte cocultures or piglet CMVEC cultures were not compromised by up to 24-h exposure to 1 mM l-glut or NMDA. The Ca(2+) ionophore calcimycin (5 microM), but not l-glut (1 mM), increased intracellular Ca(2+) levels in rat CMVECs and CMVPCs assessed with fluo-4 AM fluorescence and confocal microscopy. CMVEC-dependent pial arteriolar vasodilation to hypercapnia and bradykinin was unaffected by intracarotid infusion of l-glut in anesthetized piglets by closed cranial window/intravital microscopy. We conclude that cerebral microvascular cells are insensitive and resistant to glutamatergic stimuli in accordance with their in vivo role as regulators of potentially neurotoxic amino acids across the blood-brain barrier.

Salvianolic Acid B Inhibits Hydrogen Peroxide-Induced Endothelial Cell Apoptosis through Regulating PI3K/Akt Signaling

BackgroundSalvianolic acid B (Sal B) is one of the most bioactive components of Salvia miltiorrhiza, a traditional Chinese herbal medicine that has been commonly used for prevention and treatment of cerebrovascular disorders. However, the mechanism responsible for such protective effects remains largely unknown. It has been considered that cerebral endothelium apoptosis caused by reactive oxygen species including hydrogen peroxide (H2O2) is implicated in the pathogenesis of cerebrovascular disorders.Methodology and Principal FindingsBy examining the effect of Sal B on H2O2-induced apoptosis in rat cerebral microvascular endothelial cells (rCMECs), we found that Sal B pretreatment significantly attenuated H2O2-induced apoptosis in rCMECs. We next examined the signaling cascade(s) involved in Sal B-mediated anti-apoptotic effects. We showed that H2O2 induces rCMECs apoptosis mainly through the PI3K/ERK pathway, since a PI3K inhibitor (LY294002) blocked ERK activation caused by H2O2 and a specific inhibitor of MEK (U0126) protected cells from apoptosis. On the other hand, blockage of the PI3K/Akt pathway abrogated the protective effect conferred by Sal B and potentated H2O2-induced apoptosis, suggesting that Sal B prevents H2O2-induced apoptosis predominantly through the PI3K/Akt (upstream of ERK) pathway.SignificanceOur findings provide the first evidence that H2O2 induces rCMECs apoptosis via the PI3K/MEK/ERK pathway and that Sal B protects rCMECs against H2O2-induced apoptosis through the PI3K/Akt/Raf/MEK/ERK pathway.

Conditioned mediums of different rat cerebral microvascular endothelial cells against damage of ischemia and ischemia/reperfusion neurons

Using the method of lactate dehydrogenase (LDH) assay, to observe the activities of rat cerebral microvascular endothelial cells (CMECs) intervened by Tongluo Jiunao Injection (TLJNI), a traditional Chinese compound drug removing toxin to dredge brain collaterals, and then further study the effects of different kinds of conditioned mediums (CMECs-CM) of cerebral microvascular endothelial cells on ischemia and ischemia/reperfusion cerebral cortex cells, and to probe into the drug pharmacological mechanisms of CMECs in modulating the neurons. Three kinds of CMECs (normal, ischemic and ischemic/reperfusional) were all treated by TLJNI previously, and then the three pairs of CMECs-CM without serum were collected respectively for LDH assay. Rat cerebral cortex neurons were also primarily cultured and then divided into similar three groups (normal, ischemic and ischemic/reperfusional). The neuron responses caused by CMECs-CM at different concentrations were observed by using LDH transudation rate assay. The LDH release values of ischemic and ischemic/reperfusional CMECs with TLJNI treatment were obviously reduced (P<0.01) compared with the same kinds of CMECs untreated. For ischemic neurons, both conditioned medium of ischemic CMECs (Is-CM) and conditioned medium of ischemic CMECs with drug treatment (IsT-CM) in high concentration of 100% increased the LDH transudation rate (P<0.01), while in low concentration of 10%, IsT-CM reduced the transudation rate (P<0.05). For ischemia/reperfusion neurons, all kinds of CMECs-CM reduced the transudation rate respectively (P<0.05 or P<0.01). As far as each group concentration was concerned, 10% or 50% showed relatively stronger effects, and both conditioned medium of normal CMECs (N-CM) group and conditioned medium of ischemic/reperfusional CMECs (Rp-CM) group had statistical significance (P<0.05 or P<0.01). For normal neurons, all kinds of CMECs-CM increased the transudation rate respectively (P<0.05 or P<0.01). As far as each group concentration was concerned, only conditioned medium of normal CMECs (N-CM) had statistical significance (P<0.05 or P<0.01). The study shows that TLJNI is capable of preventing the damage of CMECs from both ischemia and ischemia/reperfusion states. Chinese drug can restrain the brain ischemia and ischemia/reperfusion damage by the media that CMECs modulate the neurons, demonstrating the pharmacological mechanisms of TLJNI. This work also indicates that there exist some active substances against ischemia/reperfusion injury secreted from CMECs-CM with TLJNI treatment.

Cinnamaldehyde reduces IL-1β-induced cyclooxygenase-2 activity in rat cerebral microvascular endothelial cells

Cinnamaldehyde is a principle compound isolated from Guizhi-Tang, which is a famous traditional Chinese medical formula used to treat influenza, common cold and other pyretic conditions. The aim of the present study was to investigate the effects of cinnamaldehyde on expression and activity of cyclooxygenase (COX) and prostaglandin E 2 (PGE 2) in rat cerebral microvascular endothelial cells (RCMEC). RCMEC were cultured, and identified by immunohistochemistry for von Willebrand factor in cytoplasm of the cells. Then cells were incubated in M199 medium containing interleukin (IL)-1β in the presence or absence of cinnamaldehyde. After incubation, the medium was collected and the amount of PGE 2 was measured by enzyme-linked immunosorbent assay (ELISA). The cells were harvested, mRNA expression and activity of COX were analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR) with SYBR Green dye and ELISA respectively. Positive immunostaining for von Willebrand factor was present diffusely in the cytoplasm of > 95% RCMEC. IL-1β increased the mRNA expression and activity of COX-2, and production of PGE 2 in a dose- and time-dependent manner in RCMEC, while mRNA and activity of COX-1 were not significantly altered. Cinnamaldehyde significantly decreased IL-1β-induced COX-2 activity and PGE 2 production in a dose-dependent manner, while it showed no inhibitory effect on IL-1β-induced COX-2 mRNA expression in cultured RCMEC. In conclusion, cinnamaldehyde reduces IL-1β-induced COX-2 activity, but not IL-1β-induced COX-2 mRNA expression, and consequently inhibits production of PGE 2 in cultured RCMEC.

Effect of 3-Phenyl-2-Propene-1-ol on PGE2Release from Rat Cerebral Microvascular Endothelial Cells Stimulated by IL-1β

Fever, an elevation in body temperature, is thought to be terminally mediated by prostaglandin E(2) (PGE(2)). Both Guizhi Tang (GZT) and its active fraction A (Fr.A) showed an antipyretic effect in rats. 3-Phenyl-2-propene-1-ol was one of the active compounds isolated from Fr.A. In the present study, we examined the influence of interleukin-1beta (IL-1beta) on prostaglandin E(2) (PGE(2)) release, and the effect of 3-phenyl-2-propene-1-ol on IL-1beta-induced PGE(2) release from rat cerebral endothelial cells (rCMEC). Cultured rCMEC were used in the study. In vitro, cells express typical phenotypic markers of brain endothelium. Using a monoclonal antibody against von Willebrand factor, immunocytochemical analysis revealed positive immunoreactivity in the cytoplasm of cultured cells. rCMEC were incubated in M199 medium containing IL-1beta in the presence or absence of 3-phenyl-2-propene-1-ol. After incubation, the conditioned media were collected and the amount of PGE(2) was measured by enzyme-linked immunosorbent assay (ELISA). IL-1beta increased the production of PGE(2) in a dose- and time-dependent manner. 3-Phenyl-2-propene-1-ol significantly decreased IL-1beta-induced PGE(2) release in a dose-dependent manner. Our results indicate that 3-phenyl-2-propene-1-ol inhibits the PGE(2) release from rCMEC stimulated by IL-1beta, and may have an antipyretic effect.

Effects of Naoxintong-containing serum on NO and CGRP in rat cerebral microvascular endothelial cells

Effects of Naoxintong (NXT, a formula of Chinese Materia Medica)-containing serum on Nitrogen monoxide (NO) and calcitonin gene related peptide (CGRP) in rat cerebral microvascular endothelial cells (rCMEC) was investigated, rCMEC was injured in vitro by incubating for 4 hours at 100% NO in a hypoxia chamber. The results indicated that NXT could antagonize the reduction of NO and CGRP secreted by rCMEC during hypoxia, the effect of which was dose-dependent. After treated with NXT-containing serum at dosage of 5.0 - 30 and 50 -1.1 g/kg/U respectively, the amount of NO and CGRP secreted by rCMEC were remarkably increased during hypoxia in vitro.