Suppression of inflammatory response by flurbiprofen following focal cerebral ischemia involves the NF-κB signaling pathway.

Some studies of animal models of middle cerebral artery occlusion indicate that inflammation plays a key role in the pathogenesis of cerebral ischemia and secondary damage. Flurbiprofen has been suggested to alleviate cerebral ischemia/reperfusion injury in both focal and global cerebral ischemia models, but the mechanisms underlying the protective action are still incompletely understood. In this study we want to investigate the protective effect of flurbiprofen after transient middle cerebral artery occlusion (MCAO) in rats and the role of the NF-κB signaling pathway on this neuroprotective effect. Male Wistar rats were subjected to transient middle cerebral artery occlusion for 2 h, followed by 24 h reperfusion. Flurbiprofen was administrated via tail-vein injection at the onset of reperfusion. HE staining and Immunohistochemistry were carried out to detect the morphological changes in ischemic penumbra cortex. The expression of inflammatory cytokines genes (IL-1ß, IL-6 and TNF-α) and the levels of p-NF-κB (p65) in ischemic penumbra cortex were measured by RT-PCR and western blot. Administration of flurbiprofen at the doses of 5 mg/kg and 10 mg/kg significantly attenuated cerebral ischemia/reperfusion injury, as shown by a reduction in the morphological changes and inhibition of pro-inflammatory cytokine expression in ischemic penumbra cortex. Moreover, our findings further demonstrated that the inhibition of NF-κB activity was involved in the neuroprotective effect of flurbiprofen on inflammatory responses. Flurbiprofen protects against cerebral injury by reducing expression of inflammatory cytokines genes and this effect may be partly due to the inhibition of NF-κB signaling pathway.

[Paeonol attenuates oxygen-glucose deprivation injury and inhibits NMDA receptor activation of cultured rat hippocampal neurons].

The purpose of this study is to determine if paeonol can protect hippocampal neurons against injury due to oxygen-glucose deprivation (OGD) injury. The rat neurons were cultured in an OGD environment and the model of OGD injury was established. Paeonol and MK-801, a positive control drug, were added before deprivation. Neuron viability was measured by the reduction of MTT; glutamate was analyzed by amino acid analyzer; binding activity of NMDA receptor was evaluated by liquid scintillation counting and the expression of NMDA receptor NR1 subunit mRNA was semiquantitatively determined by RT-PCR. Compared with OGD injury group, paeonol treatment obviously increased cell survival rate and reduced the binding activity of NMDA receptors and the release of glutamate; and down-regulating the expression of NR1 subunit. These results suggest that paeonol may exhibit its protective effect against OGD injury by the action on NMDA receptor of rats.

Protective effects of paeonol on cultured rat hippocampal neurons against oxygen-glucose deprivation-induced injury.

Mounting evidence has suggested that paeonol possesses plenty of pharmacologic actions. Our research is to determine if paeonol can protect cultured rat hippocampal neurons from oxygen-glucose deprivation(OGD)-induced injury and elucidate the underlying mechanism. We cultivated the rat hippocampal neurons as the object of study and then established the model of oxygen-glucose deprivation. Neuronal viability was measured by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), while intracellular Ca(2+) concentration was observed by fluorospectrophotometer. The binding force of N-methyl-D-aspartate (NMDA) receptor was evaluated by liquid scintillation counting. Compared with oxygen-glucose deprivation group, paeonol treatment obviously increased cell survival rate and reduced the activity of the binding force of NMDA receptors, reversing the overload of intracellular Ca(2+). These results demonstrate that paeonol protected rat neurons from oxygen-glucose deprivation-induced injury, resulting in alleviating the morphological damage and increasing neuron viability and suggest that paeonol may exhibit its protective effect against oxygen-glucose deprivation-induced injury by targeting on NMDA receptors.

[Influence of effective part of Zingiber officinal on expression of monocyte chemotactic protein-1 and adhesion molecules].

OBJECTIVE: To investigate the effect of effective parts of Zingiber officinal (EPZ) on the adhesion of ECV-304 cells with monocytes cultivated in vitro and on the expression of monocyte chemotactic protein-1 (MCP-1) and adhesion molecules. METHOD: The model of ECV-304 cell oxidative stress injury was established by hydrogen peroxide (H2O2). Then EPZ-contained blood serum was taken as experimental drug. The adherence of monocytes to endothelial cell were measured by method of rose Bengal. The total RNA of cells was extracted. The intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and MCP-1 mRNA expression in cells were detected by RT-PCR. MCP-1 protein expression were detected by ELISA. RESULT: EPZ could decrease the adhesion of monocytes with ECV-304 cells obviously. Meanwhile it could diminish the expression of ICAM-1, VCAM-1 and MCP-1 in injured ECV-304 cells. CONCLUSION: EPZ could inhibit H2O2-induced ICAM-1, VCAM-1 and MCP-1 expression in ECV-304 and could inhibit the adherence of monocytes to endothelial cell, which may result in the protect effect in endothelial cells.

Effect of compound EXP-2528 on angiotensin II-induced E-selectin and VCAM-1 expression in rat brain microvascular endothelial cells in vitro.

The aim of this study is to investigate the effect and mechanism of angiotensin (Ang) II on E-selectin and vascular cell adhesion molecule-1 (VCAM-1) expression in rat brain microvascular endothelial cells (BMEC) and evaluate the effect of compound EXP-2528, a novel Ang II type 1 (AT1) receptor antagonist. The experiment was performed in cultured BMEC of rat. The mRNA and protein expression of E-selectin and VCAM-1 in BMEC was analyzed by RT-PCR and Western blotting, respectively. The results showed that the mRNA and protein expression of E-selectin and VCAM-1 in BMEC were significantly upregulated by 4 h or 18 h exposure to 1 x 10(-7) mol x L(-1) Ang II. These effects were abolished by pretreatment with the selective AT1 receptor antagonists losartan and compound EXP-2528, but not with the AT2 selective antagonist PD123319. Combining losartan with PD123319 also significantly inhibited Ang II-induced E-selectin and VCAM-1 expression in BMEC, but there was no significant difference compared with losartan group. These findings indicated that Ang II upregulated E-selectin and VCAM-1 in BMEC by activating AT1 receptor and then involved in the development of cerebrovascular disease.

Hydroxyethylpuerarin attenuates focal cerebral ischemia-reperfusion injury in rats by decreasing TNF-alpha expression and NF-kappaB activity.

This study is to investigate the effect of hydroxyethylpuerarin on the expression of tumor necrosis factor-alpha (TNF-alpha) and activity of nuclear factor kappa B (NF-kappaB) after middle cerebral artery occlusion (MCAO) in rats. Rats were subjected to cerebral ischemia-reperfusion injury induced by MCAO. Hydroxyethylpuerarin (10, 20, 40 mg x kg(-1), iv) was administered just 30 min before occlusion and immediately after reperfusion. After a 24 h reperfusion following 2 h of MCAO, the number of viable neurons in hippocampal CA1 region was counted by hematoxylin and eosin (HE) staining. TNF-alpha protein and its mRNA expression were examined with radioimmunoassay (RIA) and reverse transcriptasepolymerase chain reaction (RT-PCR) respectively. NF-KB activity was observed by electrophoretic mobility shift assay (EMSA), and inhibition of NF-kappaB alpha (IkappaBalpha) protein expression was evaluated by Western blotting analysis. Animals treated with hydroxyethylpuerarin had a significant increase in neuronal survival in comparison with vehicle-treated group. Hydroxyethylpuerarin significantly reduced the protein and mRNA expression of TNF-alpha following 2 h of ischemia with 24 h of reperfusion. NF-kappaB DNA binding activity and the degradation of IkappaBalpha in the cytoplasm also decreased by hydroxyethylpuerarin treatment. The protective effects of hydroxyethylpuerarin against ischemia-reperfusion injury may be mediated by decreasing the expression of TNF-alpha and the activity of NF-kappaB in rats.

Effects of acetylpuerarin on hippocampal neurons and intracellular free calcium subjected to oxygen-glucose deprivation/reperfusion in primary culture.

OBJECTIVES: This study was undertaken to find out the effects of acetylpuerarin on hippocampal neurons and intracellular free calcium in primary culture subjected to oxygen-glucose deprivation/reperfusion. METHODS: According to different reperfusion time (1 h, 6 h, 12 h, 24 h), three concentrations (1.6 micromol l(-1), 0.4 micromol l(-1), 0.1 micromol l(-1)) of acetylpuerarin, and MK-801 (10 micromol l(-1)), a positive control drug, neurons were randomly divided into 21 groups. Each group was observed by inverted phase contrast microscope; neuron viability was measured by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); intracellular Ca(2+) was observed by Fura-2/AM ester through fluorospectrophotometer. RESULTS: The injured neurons were protected and degeneration and necrosis were alleviated in treatment groups of acetylpuerarin and MK-801. Acetylpuerarin increased the neuron viability at high, middle and low concentrations. Fluorescence detection results showed that the calcium concentration in the group treated with acetylpuerarin and MK-801 was lowered in each reperfusion time. CONCLUSION: Our results demonstrated that acetylpuerarin could protect the hippocampal neurons from ischemia-reperfusion injury in rats by alleviating the morphological damage, increasing neuron viability and decreasing calcium concentration in neuron.

Effects of scutellarin on apoptosis induced by cobalt chloride in PC12 cells.

The present study investigated the protective effects of scutellarin on cobalt chloride (CoCl2)-induced apoptosis in PC12 cells. Incubation of PC12 cells with 500 microM CoCl2 for 24 h resulted in significant apoptosis as evaluated by the crystal violet, electron microscopy and flow cytometry assays. The increase of caspase-3 activity, decrease of Bcl-XL expression, phosphorylation of p38 mitogen-activated protein kinase (MAPK) and accumulation of intracellular reactive oxygen species (ROS) were also seen in CoCl2-treated PC12 cells. Scutellarin at 0.1, 1 and 10 microM significantly protected against the apoptotic cell death induced by CoCl2. Scutellarin decreased caspase-3 activity, increased Bcl-XL expression, inhibited p38 phosphorylation and attenuated ROS production. These results demonstrate that scutellarin can protect PC12 cells from cobalt chloride induced apoptosis by scavenging ROS, inhibiting p38 phosphorylation, up-regulating Bcl-XL expression and decreasing caspase-3 activity, and may reduce the cellular damage in pathological conditions associated with hypoxia-mediated neuronal injury.

Neuroprotective effects of hydroxyethylpuerarin against focal cerebral ischemia-reperfusion in rats.

Our present study was performed to investigate whether hydroxyethylpuerarin (HEP) has a neuroprotective effect on brain injury after focal cerebral ischemia/reperfusion by middle cerebral artery occlusion (MCAO) in adult male Wistar rats. Animals were subjected to one hour of middle cerebral artery occlusion and 48 hours of reperfusion with the pretreatment of drugs (HEP 15, 30, 60 mg/ kg or nimodipine 0.4 mg/kg i.v.) or vehicle. The behavioral tests were used to evaluate the damage to central nervous system. The percentage of brain infarct area was assessed in the brain slices stained with 2% solution of 2, 3, 5-triphenyl tetrazolium chloride (TTC). The pathologic histological changes were observed by H&E staining and the occurrence of apoptosis was determined by flow cytometry. The results showed that pretreatment with HEP at doses of 15, 30, 60 mg/kg exhibited significant neuroprotective effects on rats against focal cerebral ischemia-reperfusion injury by markedly decreasing neurological deficit scores and the percentage of infarct area, reducing necrosis and apoptosis of neurons. All these findings suggest that HEP might provide neuroprotection against focal cerebral ischemia/reperfusion injury probably through its antioxidant and anti-inflammatory property.

[Protective effect of effective parts of Zingiber Offecinal on vascular endothelium of the experimental hyperlipidemic rats].

OBJECTIVE: To observe the influence of effective parts of Zingiber Officinale on serum IL-6, TNF-alpha in oroler to investigate the protective effects of the effective parts of Zingiber Officinal (EPZ) on endothelium of the experimental hyperlipidemic rats and the mechanism of its effects. METHODS: The hyperlipidemia model of rats was constructed by feeding high-fat forage and filled with the effective parts of Zingiber Officinale 200 mg/kg, 400 mg/kg, 800 mg/kg every day for 13 weeks. Blood was drawn to determine both the level of serum IL-6 and TNF-alpha. All the aortaes were taken to oberserve morphologic change and the intima-media thickness were detected. RESULTS: The effective parts of Zingiber Officinale could markedly decrease intima-media thickness, but had no marked influence in the level of serum IL-6 and TNF-alpha. CONCLUSION: The Effect Parts of Zingiber Officinale has the effect of protection of the endothelia of hyperlipidemia rats, which has nothing with the level of serum IL-6 and TNF-alpha.

[Protective effects of hydroxyethylpuerarin against brain astrocytes injury induced by hydrogen peroxide].

AIM: To study the protective effects of hydroxyethylpuerarin against the injury of astrocytes induced by hydrogen peroxide (H2O2). METHODS: Experiments were performed with cells from passage 4. Plasma membrane integrity was measured by lactate dehydrogenase (LDH) release. The occurrence of apoptosis was measured by flow cytometry. The glutamate uptake of astrocytes was studied with [3H]-glutamate incorporation. Intracellular superoxide dismutase (SOD) activity and malondialdehyde (MDA) level were assessed by automatic biochemistry analyzer. RESULTS: Compared with H2O2 injured group, the occurrence of apoptosis, levels of LDH release and intracellular MDA of astrocytes reduced in hydroxyethylpuerarin pre-treated groups, but the glutamate uptake and intracellular SOD activity of astrocytes increased. CONCLUSION: Hydroxyethylpuerarin could reduce the occurrence of apoptosis and improve neurotrophic function of astrocytes, which may be related with its antioxidant effects during oxidative stress.

Angiotensin II stimulates intercellular adhesion molecule-1 via an AT1 receptor/nuclear factor-kappaB pathway in brain microvascular endothelial cells.

Microvascular changes in the brain are significant causes of cerebral edema and ischemia injury. A number of studies suggest that angiotensin (Ang) II may be involved in the initiation and regulation of processes occurring in brain ischemia. We recently reported that Ang II injures brain microvascular endothelial cells (BMEC) partially via stimulating intercellular adhesion molecule-1 (ICAM-1) expression. However, the signaling cascade leading to Ang II-induced ICAM-1 expression in BMEC was unclear. The present study tested the hypothesis that Ang II induces ICAM-1 expression via an AT1 receptor/nuclear factor-kappaB (NF-kappaB) pathway in BMEC. Ang II directly stimulated the expression of ICAM-1 mRNA and protein in primary cultured BMEC. Ang II treatment also resulted in the degradation of IkappaBalpha and increase of NF-kappaB p65 subunit in the nucleus as well as the DNA binding activity of nuclear NF-kappaB. These effects were abolished by pretreatment with the selective AT1 receptor antagonists, losartan and compound EXP-2528, or losartan plus the AT2 receptor antagonist PD123319, but not by PD123319 alone. Moreover, there were no significant differences between the losartan and losartan plus PD123319 groups. These findings indicate that Ang II-induced ICAM-1 upregulation in brain microvascular endothelial cells may be mediated via an AT1 receptor/NF-kappaB pathway.

Protective effects of hydroxyethylpuerarin on cultured bovine cerebral microvascular endothelial cells damaged by hydrogen peroxide.

AIM: To observe the damages induced by hydrogen peroxide in cultured bovine cerebral microvascular endothelial cells (BCMEC) and evaluate the protective effects of hydroxyethylpuerarin on hydrogen peroxide-injured BCMEC. METHODS: BCMEC were cultured and transferred into modified Eagle medium (MEM). The viability of cells was detected by MTT assay. Cell injury was determined by lactate dehydrogenase (LDH) activity in the extracellular medium. Flow cytometry was employed to observe the occurrence of apoptosis. Morphologic changes of cells were visualized under phase contrast and electron microscopes. RESULTS: Hydrogen peroxide (200 micromol x L(-1) for 4 hours) inhibited the viability of cultured BCMEC and stimulated LDH release. Hydrogen peroxide (100 micromol x L(-1) for 4 hours) induced the occurrence of apoptosis. Hydroxyethylpuerarin was shown to increase the survival rate and decrease the activity of LDH of BCMEC damaged by hydrogen peroxide. Hydroxyethylpuerarin was also found to protect BCMEC against apoptosis induced by hydrogen peroxide. CONCLUSION: Hydrogen peroxide induces BCMEC injury either by apoptosis or through necrosis. Hydroxyethylpuerarin protects BCMEC against hydrogen peroxide-induced injury in a concentration-dependent manner. Its antioxidant effects might be involved as the mechanism protection.

Anti-inflammatory effect of hydroxyethylpuerarin on focal brain ischemia/reperfusion injury in rats.

The objective of this study is to investigate the anti-inflammatory effect of hydroxyethylpuerarin on focal brain ischemia injury in rats and to explore its mechanisms of action. After 24 h of reperfusion following 2 h of cerebral ischemia, the infiltration of neutrophils was observed by myeloperoxidase (MPO) activity determination, the expression of intercellular adhesion molecule-1 (ICAM-1) was observed by western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, and the nuclear translocation and DNA binding activity of nuclear factor-kappaB (NF-kappaB) were observed by western blot and electrophoretic mobility shift assay (EMSA). The results showed that hydroxyethylpuerarin could obviously inhibit the MPO activity and ICAM-1 expression following 2 hours of ischemia with 24 hours of reperfusion. The nuclear translocation and DNA binding activity were also decreased by hydroxyethylpuerarin treatment. These results suggested that hydroxyethylpuerarin could inhibit neutrophil-mediated inflammatory response after brain ischemia reperfusion in rats. This effect may be mediated by down-regulation of ICAM-1 and NF-kappaB activity.