Activation of ERK1/2-CREB pathway during potentiating synaptic transmission of (-)clausenamide in rat dentate gyrus.

To investigate the signal mechanism of (-)clausenamide ((-)-3-hydroxy-5-(hydroxy-phenyl-methyl)-1-methyl-4-phenyl-pyrrolidin-2-one, 1) and for understanding its effect on synaptic transmission, electrophysiological recording was done for basal synaptic transmission determination. Western blot analysis was employed to examine the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and cAMP responsive element-binding protein (CREB). Immunohistochemistry and tissue in situ hybridization were applied to detect the expression of Zif268. The results showed that (-)clausenamide (1) increased the population spike of hippocampal dentate gyrus. The phosphorylation of ERK1/2 in hippocampus and cortex was increased and reached the maximum at 5 min and 30 min, respectively. (-)Clausenamide (1) promoted the phosphorylation of CREB, the downstream protein of ERK1/2. The expression of Zif268 protein and mRNA increased in both hippocampal dentate gyrus and cortex. Therefore, (-)clausenamide (1) activated the ERK1/2-CREB pathway, which may provide an explanation for its effect on potentiating synaptic transmission and improving learning and memory.

[Regulation of microglia on neurogenesis in adult mammals].

Adult neurogenesis had been confirmed in rodents and other mammals for decades. The mechanisms underlying neurogenesis have been investigated extensively in recent years. Microglial cells, an immuno-cell in the central nervous system, have been found playing important roles in modulating the process of adult neurogenesis. The inflammatory microenvironment contributed by microglia are harmful to the survival of new born neurons. On the other hand, by interacting with T cells, microglia could act as a trophic factor for neurogenesis, most possibly by releasing growth factors. We reviewed the progress on how microglia regulating adult neurogenesis in variant pathological conditions. To understand the relationship between microglia and neurogenesis will help us to approach for promising therapeutic strategy for neuronal injury.

Protective role of PI3-kinase/Akt/eNOS signaling in mechanical stress through inhibition of p38 mitogen-activated protein kinase in mouse lung.

AIM: To test the hypothesis that PI3K/Akt/eNOS signaling has a protective role in a murine model of ventilation associated lung injury (VALI) through down-regulation of p38 MAPK signaling. METHODS: Male C57BL/J6 (wild-type, WT) or eNOS knockout mice (eNOS(-/-)) were exposed to mechanical ventilation (MV) with low (LV(T), 7 mL/kg) and high tidal volume (HV(T), 20 mL/kg) for 0-4 h. A subset of WT mice was administered the specific inhibitors of PI3K (100 nmol/L Wortmannin [Wort], ip) or of p38 MAPK (SB203580, 2 mg/kg, ip) 1 h before MV. Cultured type II alveolar epithelial cells C10 were exposed to 18% cyclic stretch for 2 h with or without 20 nmol/L Wort pretreatment. At the end of the experiment, the capillary leakage in vivo was assessed by extravasation of Evans blue dye (EBD), wet/dry weight ratio and lung lavage protein concentration. The lung tissue and cell lysate were also collected for protein and histological review. RESULTS: MV decreased PI3K/Akt phosphorylation and eNOS expression but increased phospho-p38 MAPK expression along with a lung leakage of EBD. Inhibitions of phospho-Akt by Wort worsen the lung edema, whereas inhibition of p38 MAPK kinase restored activation of Akt together with alleviated capillary leakage. eNOS(-/-) mice showed an exacerbated lung edema and injury. The stretched C10 cells demonstrated that Wort diminished the activation of Akt, but potentiated phosphorylation of MAPK p38. CONCLUSION: Our results indicate that PI-3K/Akt/eNOS pathway has significant protective effects in VALI by preventing capillary leakage, and that there is a cross-talk between PI3K/Akt and p38 MAPK pathways in vascular barrier dysfunction resulting from VALI.

[Salvianolic acid B alleviate the disruption of blood-brain barrier in rats after cerebral ischemia-reperfusion by inhibiting MAPK pathway].

The aim of the study is to investigate the effect of salvianolic acid B (SalB) on blood-brain barrier (BBB) in rats after cerebral ischemia-reperfusion, and to illustrate its possible mechanisms. Cerebral ischemia-reperfusion was induced by middle cerebral artery occlusion in rats. The break-down of BBB was indicated by extravasations of immunoglobulin (IgG) monitored with immunohistochemistry. The expression of MMP-9 and NOS2 in the brain was determined by immunohistochemistry, and the expression of p-p38 and p-ERK1/2 was detected by Western blotting. It was shown that on day 2 after ischemia-reperfusion the IgG accumulated around the vascular boundary zone, suggesting the break-down of BBB, and the expression of MMP-9 and NOS2 up-regulated at the same time. The result of Western blotting suggested that the expression of p-p38 and p-ERK1/2 increased. On day 7 after ischemia-reperfusion the. expression of MMP-9 and NOS2 was about the same level as day 2, the expression of p-p38 was higher than that on day 2 and the expression of p-ERK1/2 was slightly lower than that on day 2. SalB (1 and 10 mg x kg(-1)) significantly alleviated the extravasations of immunoglobulin induced by cerebral ischemia-reperfusion (P < 0.05). On day 2 and day 7 SalB attenuated the expression of MMP-9 and NOS2 (P < 0.05). SalB (10 mg x kg(-1)) reduced the expression of p-p38 and p-ERK1/2 apparently on day 2 and 7 after ischemia-reperfusion (P < 0.05). SalB (1 mg x kg(-1)) inhibited the expression of p-p38 on day 7 after ischemia-reperfusion (P < 0.05). The results indicate that SalB protects blood-brain barrier in rats after cerebral ischemia-reperfusion by inhibiting the MAPK pathway.

Stereoselective plasma protein binding and target tissue distribution of clausenamide enantiomers in rats.

Stereoselective differences in pharmacokinetics between clausenamide (CLA) enantiomers have been found after intravenous and oral administration of each enantiomer to rats. The differences could be associated with protein binding of CLA enantiomers. By equilibrium dialysis methods, the binding of CLA enantiomers to rat plasma protein was investigated. The results showed that mean percentages of (-) and (+)CLA in the bound form were 28.5% and 38.0%, respectively, indicating that the unbound fraction of (-)CLA was higher than that of (+)CLA, which provided an explanation for stereoselective pharmacokinetics of CLA enantiomers in rats. The results also showed that there were species differences in plasma protein binding of (-)-isomer between rats (28.5%) and rabbits (47.2%). Furthermore, effects of plasma protein binding on the distribution of CLA enantiomers to their possible target tissues were observed. The amount of (-)CLA in brain was greater than that of (+)CLA 15 min after administration of each enantiomer to rats. But the results were reverse at 4 h postdose. Further studies in distributional kinetics showed that (-)CLA had a more rapid absorption and distribution to hippocampus, cortex, and cerebellum than (+) CLA. (+)CLA had greater values for T(max), t(1/2) (beta), and AUC(0) (-->infinity), and smaller ones for CL/F and V(d)/F than its antipode. The data indicated that the distribution of (-) and (+)CLA in their target tissues was stereoselective. The stereoselective distribution might be involved in the metabolism and transport of two enantiomers in the central nerve system.

Comparison of the pharmacological effects of Panax ginseng and Panax quinquefolium.

Medical application of Panax ginseng was first found in "Shen-Nong Herbal Classic"around 200 AD Panax quinquefolium was first introduced in "Essential of Materia Medica" in 1694 in China. The most important bioactive components contained in P ginseng and P quinquefolium are ginseng saponins (GS). The contents of ginsenoside Rb1, Re, and Rd in P quinquefolium are higher than they are in P ginseng. In P ginseng, the contents of Rg1,Rb2, and Rc are higher than they are in P quinquefolium. P ginseng had a higher ratio of Rg1: Rb1, and which was lower in P quinquefolium. After steaming for several hours, the total GS will decrease. However, some ginsenosides (Rg2, 20R-Rg2, Rg3, Rh1 and Rh2) increase, while others (Rb1, Rb2, Rb3, Rc, Rd, Re, and Rg1) decrease. However, variation, especially in P quinquefolium, is high. P ginseng and P quinquefolium are general tonics and adaptogens. Rg1 and Rb1 enhance central nervous system (CNS) activities, but the effect of the latter is weaker. Thus, for the higher contents of Rg1, P ginseng is a stimulant, whereas the Rb1 contents of P quinquefolium are mainly calming to the CNS. Re, Rg1, panaxan A and B from P ginseng are good for diabetes. Re and Rg1 enhance angiogenesis, whereas Rb1, Rg3 and Rh2 inhibit it. Rh2, an antitumor agent, can be obtained from Rb1 by steaming. The content of Re in P quinquefolium are higher than in P ginseng by 3-4 times. The vasorelax, antioxidant, antihyperlipidemic, and angiogenic effects of Re are reported. Thus, for the CNS "hot," wound healing and hypoglycemic effects, P ginseng is better than P quinquefolium. For anticancer effects, P quinquefolium is better.

Ginsenoside Rg1 activated CaMKIIalpha mediated extracellular signal-regulated kinase/mitogen activated protein kinase signaling pathway.

AIM: We carried out this study to investigate the effect of ginsenoside Rg1 on the extracellular signal-regulated kinase/mitogen activated protein kinase (ERK/ MAPK) pathway for understanding its effect on synaptic platicity. METHODS: Western blotting and immunostaining were used to examine the phosphorylation of ERK1/2, CaMKIIalpha and cAMP response element binding protein (CREB) in PC12 cells and synaptosomes. The confocal microscopy and fluorescent indicator Fluo-3 was applied to observe the intracellular calcium ion flux. RESULTS: The phosphorylation of ERK1/2 in PC12 cells and synaptosomes incubated with Rg1 was increased and reached maximum at 4 min. Rg1 also promoted the transient enhancement of upstream calcium ion and activated CaMKIIalpha, which reached maximum at 2 min. CREB, the downstream protein, was phosphorylated within 8 min in PC12 cells after being incubated with Rg1. Moreover, KN93 partially inhibited the activation of ERK1/2, and PD98059 also partially blocked the phosphorylation of CREB. CONCLUSIONS: Rg1 activated ERK/MAPK pathway by CaMKIIalpha, and the activation of CREB was not only dependent on ERK induced by Rg1, which may provide an explanation for the effect of Rg1 on long-term potentiation.

[Protective effect of (-) clausenamide against neurotoxicity induced by okadaic acid and beta-amyloid peptide25-35].

This study is to investigate the protective effect of (-) clausenamide against the neurotoxicity of okadaic acid in SH-SY5Y cell line, and injection beta-amyloid peptide25-35 (Abeta25-35) to the cerebral ventricle in ovariectomy (OVX) rats. MTT assay, LDH assay, and Hoechst 33258 staining were used to detect the effect of (-) clausenamide on the toxicity of okadaic acid in SH-SY5Y cell line. The animal model was induced by ovariectomized and injection of Abeta25-35 in the cerebroventricle of rats. The effect of (-) clausenamide on learning and memory deficiency was observed by step-through test. Electron microscope, Nissl body staining, and HE staining were used to examine the morphological changes in hippocampus and cerebral cortex neurons. Pretreatment of (-) clausenamide and LiCl decreased the rate of cell death from MTT, LDH release, and apoptosis from Hoechst 33258 staining in SH-SY5Y cell line. The step-through tests showed (-) clausenamide could improve the ability of learning and memory. The Nissl body staining and HE staining experiments also showed the neuroprotective effects of (-) clausenamide on the neurons of hippocampus and cerebral cortex. (-) Clausenamide has the protective effects against the neurotoxicity induced by okadaic acid and Abeta25-35.

[Effect of salvianolic acid B on neural cells damage and neurogenesis after brain ischemia-reperfusion in rats].

This study is to observe the effect of salvianolic acid B (Sal B) on neural cells damage and neurogenesis in sub-granular zone (SGZ) and sub-ventricular zone (SVZ) after brain ischemia-reperfusion (I/R) in rats. A modified middle cerebral artery occlusion (MCAO) model of focal cerebral ischemia-reperfusion was used. The rats were divided into four groups: sham control group, ischemia-reperfusion group, Sal B 1 and 10 mg x kg(-1) groups. Sal B was consecutively administrated once a day by ip injection after MCAO. The neurogenesis in SGZ and SVZ was investigated by BrdU method 7 days after MCAO. The Nissl staining for neurons in the hippocampal CA1 and cerebral cortex was performed 14 days after MCAO. A beam-walking test was used to monitor the motor function recovery. We found that brain ischemia resulted in an increase of BrdU positive cells both in ipsilateral SGZ and SVZ at 7th day after MCAO. Sal B (10 mg x kg(-1)) significantly increased further the number of BrdU positive cells both in SGZ and SVZ (P < 0.01). Ipsilateral hippocampal neuron damage occurred and CA1 almost lost 14 days after MCAO. Sal B (10 mg x kg(-1)) obviously attenuated the neuron damage and increased the number of neuron both in ipsilateral CA1 and cerebral cortex (P < 0.01). We also observed an obvious improvement of motor function recovery when Sal B (10 mg x kg(-1)) administrated. From the results above we concluded that Sal B stimulated neurogenesis process both in SGZ and SVZ after brain ischemia, and also alleviated neural cells loss and improved motor function recovery after brain ischemia in rats.

Antidepressive effects of ginsenoside Rg1 via regulation of HPA and HPG axis.

BACKGROUND: Hypothalamic-pituitary-adrenal (HPA) axis hyperactivity is a well-established pathological feature of major depression, accompanied by the persistent increase of glucocorticoid level and the dysfunction of hypothalamic-pituitary-gonadal (HPG) axis. Ginsenoside Rg1 (Rg1) is one of the most active ingredients of Panax ginseng, which has various biological activity. OBJECTIVE: This study aimed to investigate the antidepressive effects of Rg1 and elucidate its impact on neuroendocrine system. METHODS: The antidepressive effects of Rg1 were first analysed in mice, and was further identified in the chronic-unpredictable-mild-stress (CUMS) model and the gonadectomized (GDX) model. The effects of Rg1 on depression-like behaviour were analysed by the forced swimming test (FST), tail suspension test (TST), sucrose preference test, and measurement of pentobarbital-induced sleep. The serum corticosterone and testosterone levels were detected by ELISA. The protein levels of glucocorticoid receptor (GR) and androgen receptor (AR) were analysed by western blot and immunohistochemistry analysis. RESULTS: Rg1 significantly decreased the immobility time of mice in FST and TST. Furthermore, Rg1 alleviated anhedonia and hopelessness, decreased serum corticosterone level, and increased serum testosterone level, and the GR protein level in the PFC and hippocampus of the CUMS-treated rats. Moreover, Rg1 improved sleep disruption, down-regulated the serum corticosterone level, and increased AR protein level in the PFC of the GDX-treated mice. CONCLUSION: Together, these studies suggest that Rg1 displayed antidepressant activity through the modulation of the HPA and the HPG axis. These findings provide new mechanism involved in the antidepressive effects of Rg1 and propose theoretical clues for clinical therapies.

Early melatonin supplementation alleviates oxidative stress in a transgenic mouse model of Alzheimer's disease.

Multiple lines of evidence demonstrated that increased brain oxidative stress is a key feature of Alzheimer's disease (AD). Melatonin is a potent endogenous antioxidant and free radical scavenger. A transgenic mouse model for AD mimics the accumulation of senile plaques, neuronal loss, and memory impairment. Four-month-old transgenic mice were administrated melatonin at 10 mg/kg for 4 months. We investigated the long-term influence of melatonin on these mice before amyloid plaques were deposited. We found an increase in the levels of brain thiobarbituric acid-reactive substances (TBARS) and a decrease in glutathione (GSH) content, as well as accelerated upregulation of the apoptotic-related factors, such as Bax, caspase-3, and prostate apoptosis response-4 (Par-4) in transgenic mice, but not in wild-type (WT) littermates. Significantly, the increase in TBARS levels, reduction in superoxide dismutase activity, and GSH content were reinstated by melatonin. In addition, transgenic mice administered melatonin (10 mg/kg) showed a significant reduction in upregulated expression of Bax, caspase-3 and Par-4, indicating inhibited triggering of neuronal apoptosis. These results supported the hypothesis that oxidative stress was an early event in AD pathogenesis and that antioxidant therapy may be beneficial only if given at this stage of the disease process. In sharp contrast to conventional antioxidants, melatonin crosses the blood-brain barrier, is relatively devoid of toxicity, and constitutes a potential therapeutic candidate in AD treatment.

Ginsenoside Rb1 promotes neurotransmitter release by modulating phosphorylation of synapsins through a cAMP-dependent protein kinase pathway.

Ginseng, the root of Panax ginseng C.A. Meyer (Araliaceae), has been extensively used in traditional oriental medicine for the prevention and treatment of aging-related disorders for over 2000 years. Accumulating evidence suggests that ginsenosides such as Rg1 and Rb1, which are the pharmacologically active ingredients of ginseng, modulate neurotransmission. Synapsins are abundant phosphoproteins essential for regulating neurotransmitter release. All synapsins contain a short amino-terminal domain A that is highly conserved and phosphorylated by cAMP-dependent protein kinase (PKA), which plays a key role in regulating neurotransmitter release. In the present study, we demonstrated that both Rg1 and Rb1 increased neurotransmitter release in undifferentiated and differentiated PC12 cells. However, in the presence of the PKA inhibitor H89, Rg1, but not Rb1, still induced neurotransmitter release. Moreover, Rb1, but not Rg1, enhanced the phosphorylation of synapsins via PKA pathway. In summary, Rb1 promotes neurotransmitter release by increasing the phosphorylation of synapsins through the PKA pathway, whereas the similar effects observed with Rg1 are independent of the phosphorylation of synapsins.

[Comparison of the inhibitory activities of salvianolic acid B and Ginkgo biloba extract EGb 761 on neurotoxicity of beta-amyloid peptide].

AIM: To compare the effects of salvianolic acid B (Sal B) and Ginkgo biloba extract EGb 761 on beta-amyloid peptide (beta-AP) fibril formation and cytotoxicity to PC12 cells. METHODS: The inhibitory effects of Sal B and EGb 761 on beta-AP1-40 fibril formation were determined by using fluorescence analysis with Thioflavin T (ThT) and electron microscopic image. beta-AP25-35 was aged by incubating at 37 degrees C for 7 d, then the protein was incubated with PC12 cells. The protective effects of Sal B and EGb 761 against cytotoxicity induced by aged beta-AP25-35 in PC12 cells were evaluated by MTT reduction assay and flow cytometric analysis. beta-AP25-35-induced accumulation of intracellular reactive oxygen species (ROS) was determined by fluorescence analysis. RESULTS: Both Sal B and EGb 761 inhibited the formation of amyloid fibrils, protected PC12 cells from beta-AP25-35-induced cytotoxicity, and decreased ROS accumulation caused by beta-AP25-35. The effective doses of Sal B were far lower than those of EGb 761. CONCLUSION: Sal B was much more efficient than EGb 761 in inhibiting beta-AP aggregation and in protecting PC12 cells from beta-AP-induced cytotoxicity.

[The mechanism of ginsenosides Rg1 and Rb1 promoting glutamic acid release from PC12 cells].

AIM: To study the mechanism of ginsenosides Rg1 and Rb1 promoting glutamic acid release from PC12 cells. METHODS: The amount of glutamic acid released from PC12 cells was measured by high performance liquid chromatography (HPLC). The effect of Rg1 and Rb1 on the phosphorylation of synapsins was detected with immunofluorescent staining and Western blotting. RESULTS: Both Rg1 (10 micromol x L(-1)) and Rb1 (10 micromol x L(-1)) increased glutamic acid release from PC12 cells. The release of glutamic acid was decreased by pre-incubating with the PKA inhibitor H89. H89 inhibited the release of glutamic acid induced by Rb1, but had no effect on the release of glutamic acid induced by Rg1. Moreover, Rb1 enhanced the phosphorylation of synapsins via PKA pathway, Rg1 was out of touch with this. CONCLUSION: Rb1 may promote release of neurotransmitters by increasing the phosphorylation of synapsins via PKA pathway, whereas the up-regulation of neurotransmitters release induced by Rg1 is independent of the phosphorylation of synapsins.

Long-term melatonin or 17beta-estradiol supplementation alleviates oxidative stress in ovariectomized adult rats.

Melatonin is an endogenously generated potent antioxidant. Our previous results indicated that melatonin improved learning and memory deficits in the transgenic mouse model of Alzheimer's disease (AD) and ovariectomized (OVX) rats by improving cholinergic nerve system dysfunction, preventing apoptosis. In this study we aim to investigate the antioxidative effects of melatonin or estradiol in the brains of ovariectomized rats. OVX Sprague-Dawley rats received daily injections of melatonin (5, 10, or 20 mg/kg), 17beta-estradiol (80 microg/kg), or sesame oil for 16 weeks. We found an increase in brain mitochondrial thiobarbituric acid-reactive substances (TBARS) levels, a decrease in mitochondrial glutathione (GSH) content as well as mitochondrial superoxide dismutase (SOD) activity and upregulation of the apoptotic-related factors, such as Bax, Caspase-3, and Prostate apoptosis response-4 (Par-4) in the frontal cortex of OVX rats. In addition to oxidative stress, OVX also caused decreased activities of mitochondrial respiration complex I and complex IV, which implicated mitochondrial dysfunction. Melatonin or 17beta-estradiol antagonized the detrimental effects induced by OVX. Furthermore, immunohistochemistry results revealed that the abnormal upregulation of the apoptotic related factor such as Bax, Caspase-3, and (Par-4) greatly reduced expression after melatonin or 17beta-estradiol supplement action. These findings demonstrate the important effects of melatonin or 17beta-estradiol on postmenopausal neuropathy and support the potential application of melatonin in the treatment of dementia in postmenopausal women. Early, long-term melatonin application is a promising strategy which could potentially be applied in a clinical setting.

Expression of nicotinic receptors on primary cultures of rat astrocytes and up-regulation of the alpha7, alpha4 and beta2 subunits in response to nanomolar concentrations of the beta-amyloid peptide(1-42).

Neuronal nicotinic acetylcholine receptors (nAChRs) are thought to be involved in the pathogenesis of Alzheimer's disease (AD). Interestingly, in the brains of patients with this disease, losses of several subtypes of nAChRs on neurons have been reported, while an increase in alpha7 nAChRs was recently detected in the astrocytes. However, little is presently known about the expressions of individual subunits of nAChR on rat astrocytes in primary culture or the possible influence of exposure to beta-amyloid peptide (Abeta), a neuropathological hallmark of AD, on this expression. Thus, in the present investigation the levels of individual nAChR subunits on primary rat astrocytes and the possible direct influence of Abetas on the receptors were examined by RT-PCR, Western blotting, monitoring intracellular free calcium and immunohistochemistry. The alpha4, alpha7, beta2 and beta3 subunits and related calcium channel responses were found in these cells, whereas neither alpha2 nor alpha3 could be detected. Elevation in the levels of alpha7, alpha4 and beta2 mRNAs and proteins were observed in astrocytes exposed to 0.1-100nM Abeta(1-42). In contrast, incubation with 1muM Abeta(1-42) or Abeta(35-25) did not affect these levels. We propose that the enhanced expression of alpha7, alpha4 and beta2 nAChRs by astrocytes stimulated directly by nanomolar concentrations of Abeta(1-42) might be related to ongoing defensive or compensative mechanisms.

Protective effect of melatonin on beta-amyloid-induced apoptosis in rat astroglioma C6 cells and its mechanism.

Astrocytosis is a common feature of amyloid plaques. The Abeta-astrocyte interaction produces a detrimental effect on neurons, which may contribute to neurodegeneration in Alzheimer disease (AD). The regulation of astrocyte apoptosis is essential to physiological and pathological processes in the CNS. Melatonin is a potent antioxidant and free radical scavenger. Previously, we showed that melatonin alleviated the learning and memory deficits in the APP 695 transgenic mouse model of AD. In this study, the importance of melatonin in the management of Abeta-induced apoptosis in an astrocyte-like cell is discussed. We found that rat astroglioma C6 cells treated with Abeta25-35 or Abeta1-42 undergo apoptosis and that melatonin pretreatment at 10(-5), 10(-6), and 10(-7) M significantly attenuates Abeta25-35- or Abeta1-42-induced apoptosis. The antiapoptotic effects of melatonin were extremely reproducible and corroborated by multiple quantitative methods, including an MTT cell viability assay, Hoechst 33342 nuclei staining, DNA fragmentation analysis, and flow cytometric analysis. In addition, melatonin effectively suppressed Abeta1-42-induced nitric oxide formation, remarkably prevented Abeta1-40-induced intracellular calcium overload, and significantly alleviated Abeta1-40-induced membrane rigidity. Our results demonstrate that, in addition to the beneficial effects of providing direct antioxidant protection to neurons, melatonin may enhance neuroprotection against Abeta-induced neurotoxicity by promoting the survival of glial cells.

Mechanism of (-)clausenamide induced calcium transient in primary culture of rat cortical neurons.

(-)clausenamide is a compound isolated from Clausena lansium (lour) Skeel with nootropic effects. At the present study, we investigated the clausenamide induced Ca2+ signaling in primary cultures of rat cortical neurons by using laser confocal microscopy. The mean amplitude of (-)clausenamide (1 microM) induced Ca2+ transient was similar in extracellular solution with or without calcium; and (-)clausenamide failed to trigger calcium transient after treatment with endoplasmic reticulum Ca2+ pumps inhibitor BHQ to exhaust intracellular Ca2+ stores. This result suggested that the primary source of (-)clausenamide induced Ca2+ transient was from internal stores. Application of IP3 receptor inhibitor MgCl2 and PLC-gamma inhibitor U73122 suppressed (-)clausenamide induced Ca2+ transient, suggesting that the major source of (-)clausenamide induced Ca2+ transient was from IP3 receptor pathway. We also found that mitochondria were involved in (-)clausenamide triggered Ca2+ transient. The distinctive spatial and temporal characteristic of (-)clausenamide induced Ca2+ transient may play an important role in its action.

(-)Clausenamide improves long-term potentiation impairment and attenuates apoptosis after transient middle cerebral artery occlusion in rats.

The effects of (-)clausenamide (clau) on long-term potentiation (LTP) and neuronal DNA damage were investigated in a rat model of middle cerebral artery (MCA) occlusion. Four days after reperfusion, electrophysiology records revealed reduced LTP in the ipsilateral dentate gyrus of ischemic rats, while treatment with clau (10 mg kg-1 p.o. once daily) improved LTP impairment. The fractional increase of population spike amplitude 20-50 min after tetanus was significantly larger in ischemic rats treated with clau than vehicle treated animals. Terminal deoxynucleotidyltransferase mediated dUTP end labeling (TUNEL) assay revealed occurrence of apoptosis in the ipsilateral striatum. The numbers of TUNEL-positive particles were significantly reduced after treatment with clau compared with vehicle group (78.8 +/- 17.9 versus 105.8 +/- 27.2). Mitochondrial rhodamine 123 accumulation showed that clau treatment (55.0 +/- 8.5) elevated numbers of rhodamine 123-positive particles in the ipsilateral striatum compared with vehicle group (40.4 +/- 7.5) These results demonstrate that clau can improve LTP impairment in the ipsilateral dentate gyrus and enhance cell survival in the striatum compared to the vehicle-treated rats four days following ischemic damage and its protective effect on mitochondria may partially underlie its action.