Tetramethylpyrazine inhibits neutrophil activation following permanent cerebral ischemia in rats.

Experimental studies have demonstrated the beneficial effects of tetramethylpyrazine (TMP) against ischemic stroke and highlighted its crucial role in anti-inflammatory activity. This study provides evidence of an alternative target for TMP and sheds light on the mechanism of its anti-inflammatory action against ischemic brain injury. We report a global inhibitory effect of TMP on inflammatory cell intracerebral activation and infiltration in a rat model of permanent cerebral ischemia. The results of immunohistochemistry, enzymatic assay, flow cytometric analysis, and cytological analysis revealed that intraperitoneal TMP administration reduced neuronal loss, macrophage/microglia activation, brain parenchyma infiltrative neutrophils, and circulating neutrophils after cerebral ischemia. Biochemical studies of cultured neutrophils further demonstrated that TMP attenuated neutrophil migration, endothelium adhesion, spontaneous nitric oxide (NO) production, and stimuli-activated NO production after cerebral ischemia. In parallel with these anti-neutrophil phenomena, TMP also attenuated the activities of ischemia-induced inflammation-associated signaling molecules, including plasma high-mobility group box-1 protein (HMGB1) and neutrophil toll-like receptor-4 (TLR4), Akt, extracellular signal-regulated kinase (ERK), and inducible nitric oxide synthase. Another finding in this study was that the anti-neutrophil effect of TMP was accompanied by a further elevated expression of NF-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in neutrophils after cerebral ischemia. Taken together, our results suggest that both the promotion of endogenous anti-inflammatory defense capacity and the attenuation of pro-inflammatory responses via targeting of circulating neutrophils by elevating Nrf2/HO-1 expression and inhibiting HMGB1/TLR4, Akt, and ERK signaling might actively contribute to TMP-mediated neuroprotection against cerebral ischemia.

Tetramethylpyrazine reduces cellular inflammatory response following permanent focal cerebral ischemia in rats.

Tetramethylpyrazine (TMP) has been used to treat ischemic stroke. However, scientific evidence related to its effectiveness or precise modes of neuroprotective action is largely unclear. This study provides evidence of an alternative target for TMP and sheds light on the mechanism of its physiological benefits. We report a global inhibitory effect of TMP on intracerebral cellular inflammatory response in a rat model of permanent cerebral ischemia. TMP exhibited a neuroprotective effect against ischemic deficits by reduction of behavioral disturbance, brain infarction, and edema. The results of immunohistochemistry, enzymatic assay, Western blot, real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and flow cytometric analysis revealed that TMP reduced the percentages of activated macrophages/microglia and infiltrative lymphocytes, neutrophils, and macrophages and pro-inflammatory cytokine expression after cerebral ischemia. In parallel with these immunosuppressive phenomena, TMP also attenuated the activities of ischemia-induced inflammation-associated signaling molecules and transcription factors. Another finding in this study was that the anti-inflammatory and neuroprotective effects of TMP were accompanied by a further elevated expression of NF-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in ipsilateral neurons and macrophages/microglia after cerebral ischemia. Taken together, our results suggest that both the promotion of endogenous defense capacity and the attenuation of the extent and composition percentage of the major cellular inflammatory responses via targeting of macrophages/microglia by elevating Nrf2/HO-1 expression might actively contribute to TMP-mediated neuroprotection against cerebral ischemia.

Signaling cascades mediate astrocyte death induced by zinc.

Zinc overload is known to cause the death of neural cells. Although the activation of extracellular signal-regulated kinase (ERK) and cytosolic phospholipase A(2) (cPLA(2)) have been implicated in zinc-induced astrocyte death, the detailed mechanisms of their activation and upstream regulatory cascades are incompletely understood. Here, we report that protein kinase C (PKC)- and Src-related Ras/Raf/ERK cascades and ERK-associated cPLA(2) participate in astrocyte death caused by ZnCl(2). Sustained exposure to ZnCl(2) caused damage to astrocytes in a time- and concentration-dependent manner. The cell death caused by ZnCl(2) was accompanied by increased reactive oxygen species (ROS) generation, PKC-α membrane association, Src phosphorylation, Ras membrane association, Raf phosphorylation, ERK phosphorylation, and cPLA(2) activation, and decreased protein phosphatase activity. Pharmacological studies revealed that these activations/inactivations all contributed to ZnCl(2)-induced astrocyte death. ROS, such as superoxide, appear to be a key trigger in response to ZnCl(2) treatment in astrocytes because of the attenuations in protein phosphatase inhibition, signaling activation, and cell death by antioxidant treatments. Mechanistic studies had suggested that ROS/PKC-α/Ras/Raf/ERK and ROS/Src/Ras/Raf/ERK were potential signals linking zinc and cPLA(2). These observations indicated that ROS/PKC-α/Ras/Raf/ERK and ROS/Src/Ras/Raf/ERK signaling and cPLA(2) were actively involved in zinc-induced astrocyte damage.

Luteolin inhibits cytokine expression in endotoxin/cytokine-stimulated microglia.

Microglial activation plays a pivotal role in the pathogenesis of neurodegenerative disease by producing excessive proinflammatory cytokines and nitric oxide (NO). Luteolin, a naturally occurring polyphenolic flavonoid antioxidant, has potent anti-inflammatory and neuroprotective properties both in vitro and in vivo. However, the molecular mechanism of luteolin-mediated immune modulation in microglia is not fully understood. In the present study, we report the inhibitory effect of luteolin on lipopolysaccharide (LPS)/interferon γ (IFN-γ)-induced NO and proinflammatory cytokine production in rat primary microglia and BV-2 microglial cells. Luteolin concentration-dependently abolished LPS/IFN-γ-induced NO, tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß) production as well as inducible nitric oxide synthase (iNOS) protein and mRNA expression. Luteolin exerted an inhibitory effect on transcription factor activity including nuclear factor κB (NF-κB), signal transducer and activator of transcription 1 (STAT1) and interferon regulatory factor 1 (IRF-1) in LPS/IFN-γ-activated BV-2 microglial cells. Biochemical and pharmacological studies revealed that the anti-inflammatory effect of luteolin was accompanied by down-regulation of extracellular signal-regulated kinase (ERK), p38, c-Jun N-terminal kinase (JNK), Akt and Src. Further studies have demonstrated that the inhibitory effect of luteolin on intracellular signaling execution and proinflammatory cytokine expression is associated with resolution of oxidative stress and promotion of protein phosphatase activity. Together, these results suggest that luteolin suppresses NF-κB, STAT1 and IRF-1 signaling, thus attenuating inflammatory response of brain microglial cells.

Glucose exacerbates zinc-induced astrocyte death.

Zinc and cytosolic phospholipase A(2) (cPLA(2)) have been implicated in the death of neural cells and the pathogenesis of ischemia, and hyperglycemia is a potential augmenting factor. However, their potential crosstalk and/or interaction in mediating cell damage have not yet been fully elucidated. Here, we report that a potential link between cPLA(2) activation and zinc-induced astrocyte damage involving reactive oxygen species (ROS)/protein kinase C-α (PKC-α)/extracellular signal-regulated kinase (ERK) signaling and glucose is able to increase zinc uptake and potentiate zinc-induced alterations and astrocyte damage. The cell death caused by ZnCl(2) was accompanied by increased ROS generation, PKC-α membrane translocation, ERK phosphorylation, and cPLA(2) phosphorylation and activity. Pharmacological studies revealed that these activations contributed to ZnCl(2)-induced astrocyte death. Mechanistic studies had suggested that ROS/PKC-α/ERK was a potential signal linking zinc and cPLA(2). Glucose increased zinc uptake and potentiated ZnCl(2)-induced alterations and astrocyte death. These observations indicated that ROS/PKC-α/ERK signaling and cPLA(2) were actively involved in zinc-induced astrocyte damage, and suggested zinc was a potential downstream effector in hyperglycemia-aggravated astrocyte injury.

Effect of acupressure on thirst in hemodialysis patients.

BACKGROUND: Thirst and dry mouth are common among hemodialysis (HD) patients. This paper reports a study to evaluate the impact of an acupressure program on HD patients' thirst and salivary flow rates. METHODS: The acupressure program included placebo, followed by true acupressure each applied for 4 weeks. Twenty-eight patients (mean age 57.6, SD = 16.13 years) first received a sticker as placebo acupressure at two acupoints CV23 and TE17 three times a week for 4 weeks, and then received true acupressure in the same area for the next 4 weeks. Salivary flow rate and thirst intensity were measured at baseline, during and after treatment completion for both the placebo and true acupressure program. RESULTS: The true acupressure program was associated with significantly increased salivary flow rate (0.09 +/- 0.08 ml/min at baseline to 0.12 +/- 0.08 ml/min after treatments completion, p = 0.04). The mean thirst intensity also improved from 4.21 +/- 2.66 at baseline to 2.43 +/- 2.32 (p = 0.008) after treatment completion in HD patients. There was no statistically significant difference in pre-post program salivary flow rate; however, significant improvement in thirst intensity scores was observed (p = 0.009) in the placebo acupressure program. CONCLUSION: This study provides preliminary evidence that acupressure may be effective in improving salivary flow rates and thirst intensity.

Graptopetalum paraguayense E. Walther leaf extracts protect against brain injury in ischemic rats.

As practice in folk medicine, Graptopetalum paraguayense E. Walther possesses several biological/pharmacological activities including hepatoprotective, anti-oxidant, and anti-inflammatory. We investigated the neuroprotective potential of Graptopetalum paraguayense E. Walther leaf extracts on inflammation-mediated ischemic brain injury. Water (GWE), 50% alcohol (GE50) extracts of Graptopetalum paraguayense E. Walther, and extracts obtained from further extraction of GE50 with ethyl acetate (GEE) were used. Oral administration of GEE, but not GWE or GE50, for 2 weeks protected animals against cerebral ischemia/reperfusion brain injury. The neuroprotective effect of GEE was accompanied by reductions in brain infarction, neurological deficits, caspase-3 activity, malondialdehyde content, microglia activation, and inducible nitric oxide synthase (iNOS) expression. Since microglia-mediated inflammation plays critical roles in ischemic brain injury, anti-inflammatory potential of Graptopetalum paraguayense E. Walther leaf extracts was further investigated on lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma-activated BV-2 microglial cells. GEE decreased H(2)O(2)- and LPS/IFN-gamma-induced free radical generation and LPS/IFN-gamma-induced iNOS expression. Mechanistic study revealed that the neuroactive effects of GEE were markedly associated with anti-oxidative potential, activation of serine/threonine and tyrosine phosphatases, and down-regulation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38, Akt, Src, Janus kinase-1, Tyk2, signal transducer and activator of transcription-1, and NF-kappaB and might be attributed to the presence of polyphenolic compounds such as gallic acid, genistin, daidzin, and quercetin. Together, our findings point out its potential therapeutic strategies that target microglia activation, oxidative stress, and iNOS expression to reduce ischemic brain injury and suggest that Graptopetalum paraguayense E. Walther leaf extracts represent a valuable source for the development of neuroprotective agents.

Inhibition of nitric oxide production by quercetin in endotoxin/cytokine-stimulated microglia.

AIMS: Flavonoids possess several biological and pharmacological activities. Quercetin, a naturally occurring flavonoid, has been shown to down-regulate inflammatory responses and provide neuroprotection. However, the mechanisms underlying the anti-inflammatory properties of quercetin are poorly understood. In the present study, we investigated the modulatory effect of quercetin against neuroinflammation. MAIN METHODS: We herein describe a potential regulatory mechanism by which quercetin suppresses nitric oxide (NO) production by lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma)-stimulated BV-2 microglial cells. The underlying regulatory cascades were approached by biochemical and pharmacological strategies. KEY FINDINGS: Quercetin produced an inhibitory effect on inducible nitric oxide synthase (iNOS) expression and NO production. Biochemical studies revealed that the anti-inflammatory effect of quercetin was accompanied by the down-regulation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38, Akt, Src, Janus kinase-1, Tyk2, signal transducer and activator of transcription-1, and NF-kappaB. In addition, quercetin scavenged free radicals and produced inhibitory effects on serine/threonine and tyrosine phosphatase activities. Intriguingly, the accumulation of lipid rafts, which is the critical step for signaling, was disrupted by quercetin. SIGNIFICANCE: The data indicate that the anti-inflammatory action of quercetin may be attributable to its raft disrupting and anti-oxidant effects. These distinct mechanisms work in synergy to down-regulate iNOS expression and NO production.

Protective effect of docosahexaenoic acid against brain injury in ischemic rats.

Evidence suggests that inactivation of cell-damaging mechanisms and/or activation of cell-survival mechanisms may provide effective preventive or therapeutic interventions to reduce cerebral ischemia/reperfusion (I/R) injuries. Docosahexaenoic acid (DHA) is an essential polyunsaturated fatty acid in the central nervous system that has been shown to possess neuroprotective effects. We examined whether different preadministrative protocols of DHA have effects on brain injury after focal cerebral I/R and investigated the potential neuroactive mechanisms involved. Sprague-Dawley rats were intraperitoneally pretreated with DHA once 1 h or 3 days being subjected to focal cerebral I/R or daily for 6 weeks before being subjected to focal cerebral I/R. Reduction of brain infarction was found in all three DHA-pretreated groups. The beneficial effect of DHA on the treatment groups was accompanied by decreases in blood-brain barrier disruption, brain edema, malondialdehyde (MDA) production, inflammatory cell infiltration, interleukin-6 (IL-6) expression and caspase-3 activity. Elevation of antioxidative capacity, as evidenced by decreased MDA level and increased superoxide dismutase activity and glutathione level, was detected only in the chronic daily-administration group. The two single-administration groups showed increased phosphorylation of extracellular-signal-regulated kinase (ERK). Elevation of Bcl-2 expression was detected in the chronic daily-administration and 3-day-administration groups. In vitro study demonstrated that DHA attenuated IL-6 production from stimulated glial cells involving nuclear factor kappaB inactivation. Therefore, the data suggest that the neuroprotective mechanisms of DHA pretreatment are, in part, mediated by attenuating damaging mechanisms through reduction of cytotoxic factor production and by strengthening survival mechanisms through ERK-mediated and/or Bcl-2-mediated prosurvival cascade.

Opioids modulate post-ischemic progression in a rat model of stroke.

Alterations in the opioidergic system have been found in cerebral ischemia. Neuroprotection studies have demonstrated the involvement of the opioidergic system in cerebral ischemia/reperfusion (I/R). However, the neuroprotective mechanisms remain largely unclear. This study was conducted to investigate whether intracerebroventricular administration of opioidergic agonists has a neuroprotective effect against cerebral ischemia in rats and, if this proved to be the case, to determine the potential neuroprotective mechanisms. Using a focal cerebral I/R rat model, we demonstrated that the opioidergic agents, BW373U86 (delta agonist) and Dynorphin A 1-13 (kappa agonist), but not TAPP (mu agonist), attenuated cerebral ischemic injury as manifested in the reduction of cerebral infarction and preservation of neurons. The antagonism assay showed that the neuroprotective effect of Dynorphin A was attenuated by nor-Binaltorphimine (kappa antagonist). Surprisingly, BW373U86-induced neuroprotection was not changed by Naltrindole (delta antagonist). These findings indicate that BW373U86 and Dynorphin A exerted distinct neuroprotection against ischemia via opioid-independent and -dependent mechanisms, respectively. The post-ischemic protection in beneficial treatments was accompanied by alleviations in brain edema, inflammatory cell infiltration, and pro-inflammatory cytokine interleukin 6 (IL-6) expression. In vitro cell study further demonstrated that the opioidergic agonists, delta and kappa, but not mu, attenuated IL-6 production from stimulated glial cells. Our findings indicate that opioidergic agents have a role in post-ischemic progression through both opioid-dependent and -independent mechanisms. In spite of the distinct-involved action mechanism, the potential neuroprotective effect of opioidergic compounds was associated with immune suppression. Taken together, these findings suggest a potential role for opioidergic agents in the therapeutic consideration of neuroinflammatory diseases. However, a better understanding of the mechanisms involved is necessary before this therapeutic potential can be realized.

Neuroprotection by tetramethylpyrazine against ischemic brain injury in rats.

In traditional Chinese medicine, Ligusticum wallichii Franchat (Chuan Xiong) and its active ingredient tetramethylpyrazine (TMP) have been used to treat cardiovascular diseases and to relieve various neurological symptoms such as ischemic deficits. However, scientific evidence related to their effectiveness or precise modes of neuroprotective action is largely unclear. In the current study, we elicited the neuroprotective mechanisms of TMP after focal cerebral ischemic/reperfusion (I/R) by common carotid arteries and middle cerebral artery occlusion model in rats. TMP was administrated 60 min before occlusion via intraperitoneal injection. TMP concentration-dependently exhibited significant neuroprotective effect against ischemic deficits by reduction of behavioral disturbance. Neuronal loss and brain infarction in the ischemic side of rats were markedly lowered by treatment with TMP. Cerebral I/R-induced internucleosomal DNA fragmentation, caspase-8, caspase-9, and caspase-3 activation, and cytochrome c release were reduced by TMP treatment. Western blot analysis revealed the down-regulation of Bcl-2 and Bcl-xL and the up-regulation of Bax and Bad by cerebral I/R insult. Among them, only the alteration in Bcl-xL expression was reversed by TMP treatment. Moreover, the activation of microglia and/or recruitment of inflammatory cells within the ischemic side and the consequent production of monocyte chemoattractant protein 1 (MCP-1) were suppressed by TMP pre-treatment. Our findings suggest that TMP might provide neuroprotection against ischemic brain injury, in part, through suppression of inflammatory reaction, reduction of neuronal apoptosis, and prevention of neuronal loss.

Tetramethylpyrazine reduces ischemic brain injury in rats.

Tetramethylpyrazine (TMP), which is widely used in the treatment of ischemic stroke by Chinese herbalists, is one of the most important active ingredients of the traditional Chinese herbal medicine, Ligusticum wallichii Franchat (Chung Xiong). However, the mechanism by which TMP protects the brain is still not clear. We examined neuroprotective effects of TMP after transient focal cerebral ischemia using common carotid artery and middle cerebral artery occlusion model in rats and evaluated the involvement of anti-inflammation. TMP administrated intraperitoneally significantly protected the brain against ischemic insult as evidenced by the reduction in infarction volume, preservation of neurons, and decrease in brain edema. TMP markedly reduced cerebral ischemia/reperfusion-induced inflammatory cell activation and proinflammatory mediator production. Moreover, TMP suppressed lipopolysaccharide/interferon-gamma-induced inflammation and prostaglandin E(2) production in cultured glial cells. Our findings suggest that one of neuroprotective effects of TMP against ischemic brain injury might involve its anti-inflammatory potential.