Noscapine Modulates Neuronal Response to Oxygen-glucose Deprivation/Reperfusion Injury Via Activation of Sigma-1 Receptor in Primary Cortical Cultures.

In the present study, we investigated the effects of noscapine (0.5-2 µM), an alkaloid from the opium poppy (Papaver somniferum), on primary murine cortical neurons exposed to 60 min oxygen-glucose deprivation (OGD) in the presence of 5 µM BD-1047, a selective sigma-1 receptor antagonist. The experiments were performed on cortical neurons after 11-16 days of culture. To initiate oxygen-glucose deprivation, the culture medium was transferred to glucose-free DMEM, and placed in a humidified incubation chamber containing a mixture of 95% N2 and 5% CO2 at 37 °C for 60 min. In order to explore the effect on neurons under oxygen-glucose deprivation in this condition, some cultures were pretreated with noscapine and BD1047 together, 24 h prior to OGD followed by 24 h recovery. Cell viability, nitric oxide (NO) production and intracellular calcium concentration ([Ca2+]i) levels were evaluated by MTT assay, the modified Griess method, and Fura-2, respectively. Pretreatment of the cultures with noscapine in the presence of BD1047 significantly increased cell viability and decreased NO generation in a dose-dependent manner compared to BD1047 alone. Pretreatment with 2 µM noscapine and BD-1047 was shown to decrease the rise in [Ca2+]i induced by sodium azide (NaN3) and glucose deprivation. We concluded that noscapine in the presence of BD1047 could protect primary cortical neurons after oxygen-glucose deprivation-induced cell injury but this effect was not complete. Our results indicate that neuroprotective effects of noscapine could be mediated partially through activation of sigma-1 receptor and by decreasing NO production and [Ca2+]i levels.

Protective Effect of 25Mg-Porphyrin-Fullerene Nanoparticles on Oxygen-Glucose Deprivation/Reperfusion Injury in PC12 Cells.

We investigated the effects of 25Mg-Porphyrin-Fullerene nanoparticles, (25MgPMC16) smart ferroporphyrin nanoparticles, on PC12 cells exposed to oxygen-glucose deprivation/reperfusion. In order to explore its effect on cells under oxygen-glucose deprivation conditions, the cultures were pretreated with 25MgPMC16 24 hours prior to oxygen-glucose deprivation/reperfusion. To initiate the oxygen-glucose deprivation/reperfusion, the cell culture medium was replaced with a glucose-free medium and the cells were transferred to a humidified incubation chamber in a mixture of 95% N2 and 5% CO2 at 37° C for 30, 60 and 120 min. Cell viability was assessed by MTT assay. Exposure of PC12 cells to 30, 60 and 120 min oxygen-glucose deprivation significantly decreased the cell viability. Pretreatment of the cultures with 25MgPMC16 significantly increased cell viability in a concentration-dependent manner. Pretreatment, the cultures with MK-801 (10 µM), a non-competitive NMDA antagonist, has attenuated the cell death after 30 min oxygen-glucose deprivation. We concluded that 25MgPMC16 could protect PC12 cells against oxygen-glucose deprivation/reperfusion-induced cell injury in a concentration-dependent manner. That could be due to the effect of 25MgPMC16 on ATP synthesis and the antioxidant effects of its components.

The Effect of Noscapine on Oxygen-Glucose Deprivation on Primary Murine Cortical Neurons in High Glucose Condition.

In the present work we set out to investigate the neuroprotective effects of noscapine (0.5-2 µM) in presence of D-glucose on primary murine foetal cortical neurons after oxygen-glucose deprivation/24 h. recovery. Cell viability, nitric oxide production and intracellular calcium ((ca(2+))i) levels were evaluated by MTT assay, the modified Griess method and Fura-2 respectively. 25 and 100 mM D-glucose could, in a concentration dependent manner, improve cell viability and decrease NO production and (ca(2+))i level in neuronal cells after ischemic insult. Moreover, pre-incubation of cells with noscapine, noticeably enhanced protective effects of 25 and 100 mM D-glucose compared to similar conditions without noscapine pre-treatment. In fact, noscapine attenuated NO production in a dose-dependent fashion, after 30 minutes (min) OGD, during high-glucose (HG) condition in cortical neurons. Pretreatment with 2 µM noscapine and 25 or 100 mM D-glucose, was shown to decrease the rise in (ca(2+))i induced by Sodium azide/glucose deprivation (chemical OGD) model. These effects were more pronounced than that of 25 or 100 mM D-glucose alone. The present study demonstrated that the neuroprotective effects of HG before an ischemic insult were augmented by pre-treatment with noscapine. Our results also suggested that the neuroprotection offered by both HG and noscapine involve attenuation of NO production and (ca(2+))i levels stimulated by the experimental ischemia in cortical neurons.

Neuroprotective effect of noscapine on cerebral oxygen-glucose deprivation injury.

BACKGROUND: The present study aims to investigate the effect of noscapine (0.5-2.5 µM), an alkaloid from the opium poppy, on primary murine fetal cortical neurons exposed to oxygen-glucose deprivation (OGD), an in vitro model of ischemia. METHODS: Cells were transferred to glucose-free DMEM and were exposed to hypoxia in a small anaerobic chamber. Cell viability and nitric oxide production were evaluated by MTT assay and the Griess method, respectively. RESULTS: The neurotoxicities produced by all three hypoxia durations tested were significantly inhibited by 0.5 µM noscapine. Increasing noscapine concentration up to 2.5 µM produced a concentration-dependent inhibition of neurotoxicity. Pretreatment of cells with MK-801 (10 µM), a non-competitive NMDA antagonist, and nimodipine (10nM), an L-type Ca(2+) channel blockers, increased cell viability after 30 min OGD, while the application of NBQX (30 µM), a selective AMPA-kainate receptor antagonist partially attenuated cell injury. Subsequently, cells treated with noscapine in the presence of thapsigargin (1 µM), an inhibitor of endoplasmic reticulum Ca(2+) ATPases. After 60 min OGD, noscapine could inhibit the cell damage induced by thapsigargin. However, noscapine could not reduce cell damage induced by 240 min OGD in the presence of thapsigargin. Noscapine attenuated nitric oxide (NO) production in cortical neurons after 30 min OGD. CONCLUSIONS: We concluded that noscapine had a neuroprotective effect, which could be due to its interference with multiple targets in the excitotoxicity process. These effects could be mediated partially by a decrease in NO production and the modulation of intracellular calcium levels.

Protective Effects of Berberine on Oxygen-Glucose Deprivation/Reperfusion on Oligodendrocyte Cell Line (OLN-93).

BACKGROUND: Oligodendrocytes, the myelinating glial cells of central nervous system, are highly vulnerable to ischemic-induced excitotoxic insult, a phenomenon in which calcium overload triggers cell death. Berberine is an alkaloid extracted from medicinal herbs as Coptidis Rhizoma with several pharmacological effects like inhibition of neuronal apoptosis in cerebral ischemia. METHODS: We examined the effects of berberine (0.5-4 µM) and glutamate receptors antagonists (MK-801 [10 µM] and NBQX [30 µM]) on OLN-93 cell line (a permanent immature rat oligodendrocyte) during (30, 60, 240 min) oxygen-glucose deprivation (OGD)/24 h reperfusion. The cells were cultured in 12-well plates. The cells were exposed to glucose-free medium and hypoxia in a small anaerobic chamber. Cell viability was evaluated by MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay. The intracellular calcium levels also were evaluated by Ca(2+)-sensitive indicator Fura-2/AM in presence or absence of berberine (2 µM) during 30 min chemical OGD by NaN3 (20 mM). Student's t-test and ANOVA were used for statistical analysis. RESULTS: Berberine, MK-801and NBQX significantly increased oligodendrocyte viability in all 3 time-scheduled oxygen-glucose deprivation/reperfusion. Berberine at 2 µM produced peak of protection, and increased cell viability to 83%, 77%, and 79% during 30, 60, 240 min ischemic experiments, respectively (P < 0.001). Berberine significantly attenuated intracellular Ca(2+) rise induced by chemical ischemia, and this effect of berberine was significantly stronger than MK-801 and NBQX (P < 0.001). CONCLUSIONS: We concluded that berberine protected OLN-93 oligodendrocyte against ischemic induced excitotoxic injury. Attenuation of intracellular Ca(2+) overload by berberine may be the key mechanism that saved OLN-93 from excitotoxicity damage.

The effects of low and high concentrations of luteolin on cultured human endothelial cells under normal and glucotoxic conditions: involvement of integrin-linked kinase and cyclooxygenase-2.

Luteolin protects against high glucose (HG)-induced endothelial dysfunction whereas its cytotoxicity has been reported against normal endothelial cells. This study was undertaken to determine luteolin cytoprotective and cytotoxic dose ranges and to elucidate their respective mechanisms. Luteolin prevented HG-induced human umbilical vein endothelial cell (HUVEC) death with an EC50 value of 2.0 ± 0.07 µM. The protective effect of luteolin was associated with decreased intracellular reactive oxygen species (ROS) and Ca(2+) (Cai(2+)) levels and enhanced nitric oxide (NO) production. At high concentrations, luteolin caused HUVEC death in normal glucose (NG) and HG states (LC50 40 ± 2.23 and 38 ± 1.12 µM, respectively), as represented by increased ROS and Cai(2+) and decreased NO. Western blots illustrated that exposure to HG increased cyclooxygenase-2 (COX-2) and integrin-linked kinase (ILK) expression. Luteolin at low concentrations suppressed HG-mediated up-regulation of COX-2 but maintained HG-induced over-expression of ILK while at high concentrations significantly increased COX-2 and decreased ILK expression in both HG and NG states. Our data indicated that cytoprotective action of luteolin was manifested with much lower concentrations, by a factor of approximately 20, compared with cytotoxic activity under both normal or glucotoxic conditions. It appears that luteolin exerts its action, in part, by modulating ILK expression which is associated with regulation of COX-2 expression and NO production in endothelial cells.

Effect of berberine on nitric oxide production during oxygen-glucose deprivation/reperfusion in OLN-93 oligodendrocytes.

In this study, the effect of berberine, an isoquinoline alkaloid isolated from Coptidis rhizoma, on Nitric Oxide (NO) production, as a possible involved factor, during excitotoxic injury in oligodendroglial cells were evaluated. The overactivation of ionotropic glutamate receptors which is known as the excitotoxicity, is an important phenomenon because of the contribution in acute injury to the central nervous system, chronic neurodegenerative disorders, oligodendrocyte loss and demyelinating diseases as Multiple Sclerosis (MS). Intracellular Ca2+ overload, have a key role during excitotoxic injury and such increase in cytoplasmic Ca2+ triggers a series of events such as production of NO that end to cell death. Previous report showed the protective effects of berberine on ischemic-induced excitotoxic insult in oligodendrocytes. Hereby, we intended to know if the NO production could be associated with oxygen-glucose deprivation/reperfusion-induced excitotoxic damage in oligodendrocyte; moreover, the alteration of NO production could be considered as an involved mechanism for protective effect of berberine in such condition. Therefore, the effect of berberine (2 µM) on NO production during oxygen-glucose deprivation/24 h reperfusion in oligodendrocytes were examined. The OLN-93 cell line (a permanent immature rat oligodendrocyte) was used as a model of oligodendrocyte. Thirty minutes-oxygen-glucose deprivation/24 h reperfusion was used to induce excitotoxicity. NO production was evaluated by Griess method. Our results demonstrated that berberine (2 µM) significantly decreased NO production during 30 min oxygen-glucose deprivation/reperfusion. It seems that blockade of NO production by berberine may also participate in oligodendroglial cell protection against oxygen-glucose deprivation/reperfusion-induced insult.

Delivery of epidermal neural crest stem cells (EPI-NCSC) to hippocamp in Alzheimer's disease rat model.

BACKGROUND: Alzheimer's disease (AD) is characterized by progressive neuronal loss in hippocamp. Epidermal neural crest stem cells (EPI-NCSC) can differentiate into neurons, astrocytes and oligodendrocytes. The purpose of this study was to evaluate the effects of transplanting EPI-NCSC into AD rat model. METHODS: Two weeks after induction of AD by injection of Amyloid-ß1-40 into CA1 area of rat hippocamp, Y-maze and single-trial passive avoidance tests were used to show deficit of learning and memory abilities. EPI-NCSC were obtained from the vibrissa hair follicle of rat, cultured and labeled with bromodeoxyuridine. When Alzheimer was proved by behavioral tests, EPI-NCSC was transplanted into CA3 area of hippocamp in AD rat model. The staining of EPI-NCSC markers (nestin and SOX10) was done in vitro. Double-labeling immunofluorescence was performed to study survival and differentiation of the grafted cells. RESULTS: We showed that transplanted EPI-NCSC survive and produce many neurons and a few glial cells, presenting glial fibrillary acidic protein. Total number of granule cells in hippocamp was estimated to be more in the AD rat model with transplanted cells as compared to AD control group. We observed that rats with hippocampal damage made more errors than control rats on the Y-maze, when reward locations were reversed. CONCLUSION: Transplanted cells were migrated to all areas of hippocamp and the total number of granule cell in treatment group was equal compared to control group. Transplantation of EPI-NCSC into hippocamp might differentiate into cholinergic neurons and could cure impairment of memory in AD rat model.

Antiproliferative activity of flavonoids: influence of the sequential methoxylation state of the flavonoid structure.

Dracocephalum kotschyi Boiss. has been used as part of an ethnobotanical remedy against many forms of human cancer in Iran. It has been demonstrated that a flavonoid named xanthomicrol from D. kotschyi contributes to its preferential antiproliferative activity against malignant cells. In the present study, the antiproliferative activity of its flavonoid fraction was further characterized. Using liquid-liquid extraction and a semi-preparative reversed-phase HPLC method, eight flavonoid aglycones were isolated from the aerial parts of the plant and their identities were confirmed through MS and NMR analyses as luteolin, naringenin, apigenin, isokaempferide, cirsimaritin, penduletin, xanthomicrol and calycopterin. The in vitro antiproliferative activity of each compound was evaluated against a panel of established normal and malignant cell lines using the MTT assay and some structure-activity relationships were observed. The hydroxyflavones (luteolin, apigenin and isokaempferide) exerted comparable antiproliferative activities against malignant and normal cells, while the methoxylated hydroxyflavones (cirsimaritin, penduletin, xanthomicrol and calycopterin) showed preferential activities against tumor cells. This activity may be of value in treating tumors as it would exert few side effects in normal tissues. Xanthomicrol selectively inhibited the growth of human gastric adenocarcinoma, while calycopterin selectively prevented human acute promyelocytic leukemia and human colon carcinoma cells proliferation.

Anti-inflammatory effects of anti-hypertensive agents: influence on interleukin-1ß secretion by peripheral blood polymorphonuclear leukocytes from patients with essential hypertension.

The effects of clinically relevant concentrations of anti-hypertensive agents on lipopolysaccharide (LPS)-induced interleukin-1beta (IL-1ß) secretion by polymorphonuclear leukocytes (PMNs) were investigated in vitro. Lipopolysaccharide-induced secretion of IL-1ß by PMNs from 15 hypertensive and 15 normotensive subjects after incubation with losartan, captopril, amlodipine, atenolol, and hydrochlorothiazide were assessed. IL-1ß secretion by PMNs markedly increased in hypertensive patients versus normotensive subjects. Losartan, captopril, and amlodipine caused a concentration-dependent attenuation of IL-1ß levels in both groups. Losartan, captopril, and amlodipine demonstrated marked in vitro anti-inflammatory effects at clinically relevant serum concentrations but atenolol and hydrochlorothiazide did not.

Neuroprotective effects of mebudipine and dibudipine on cerebral oxygen-glucose deprivation/reperfusion injury.

In the present study, we investigated the effects of mebudipine and dibudipine, two new Ca(2+) channel blockers, on primary murine cortical neurons exposed to oxygen-glucose deprivation/reperfusion. The experiments were performed on cells after 11-16 days of culture. To initiate oxygen-glucose deprivation /reperfusion, the culture medium was replaced by glucose-free medium, and the cells were transferred to a humidified incubation chamber in a mixture of 95% N(2) and 5% CO(2) at 37 degrees C for 30 min. The cultures were pretreated with mebudipine and dibudipine 3 h prior to oxygen-glucose deprivation/reperfusion, in order to explore their effects on neurons under oxygen-glucose deprivation conditions. Cell viability and nitric oxide (NO) production were assessed by MTT assay and the modified Griess method, respectively. Exposure of murine cortical neuronal cells to 30 min oxygen-glucose deprivation significantly decreased cell viability and increased NO production. Pretreatment of the cultures with mebudipine and dibudipine significantly increased cell viability and decreased NO generation in a dose-dependent manner. However, the drugs had no protective effect in cells subjected to oxygen-glucose deprivation for 60 min. Pretreatment of cultures with MK-801 (10 microM), a non-competitive NMDA antagonist, decreased neuronal death after 30-min oxygen-glucose deprivation, while application of NBQX (30 microM), a selective AMPA-kainate receptor antagonist, partially attenuated the cell injury. oxygen-glucose deprivation -induced cytotoxicity and NO production were also inhibited by N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor and MK-801. We conclude that mebudipine and dibudipine could protect cortical neurons against oxygen-glucose deprivation /reperfusion-induced cell injury in a dose-dependent manner, and that this could be mediated partially by decreased NO production.

Mebudipine and dibudipine protect PC12 cells against oxygen-glucose deprivation and glutamate-induced cell death.

The protective effect of two new L-type calcium-channel blockers, mebudipine and dibudipine on neurotoxic effects induced by glutamate and oxygen-glucose deprivation (OGD) in PC12 cells was investigated. PC12 cells were intoxicated with two different methods. First, the cells were incubated with glutamate (10muM/L), glutamate and mebudipine (10muM/L), dibudipine (10muM/L) or nimodipine (10muM/L), on three different treatment schedules (concurrently, pre-3h and pre-24h). In the second method PC12 cells were exposed to in vitro oxygen-glucose deprivation for 30min and 60min alone or with the drugs in the same time schedules described above. Cellular viability was assessed by MTT assay. Glutamate-induced cell death and OGD-induced cell injury were attenuated significantly by mebudipine, dibudipine in comparison with nimodipine in all three different treatment schedules. Application of MK801 (10muM/L), an antagonist of NMDA glutamate receptors inhibited PC12 cell death in both methods. Our study suggests that mebudipine and dibudipine, like nimodipine, may have protective effects against glutamate and oxygen-glucose deprivation-induced neurotoxicity.

Apoptotic pathway induced by noscapine in human myelogenous leukemic cells.

It has been shown that noscapine, an opium-derived phthalideisoquinoline alkaloid that is currently being used as an oral antitussive drug, induces apoptosis in myeloid leukemia cells. The molecular mechanism responsible for the anticancer effects of noscapine is poorly understood. In the current study, the apoptotic effects of noscapine on two myeloid cell lines, apoptosis-proficient HL60 cells and apoptosis-resistant K562 cells, were analyzed. An increase in the activity of caspase-2, -3, -6, -8 and -9, poly(ADP ribose) polymerase cleavage, detection of phosphatidylserine on the outer layer of the cell membrane, nucleation of chromatin, and DNA fragmentation suggested the induction of apoptosis. Noscapine increased the Bax/Bcl-2 ratio with a significant decrease of Bcl-2 expression accompanied with Bcl-2 phosphorylation. Using an inhibitory approach, the activation of the caspase cascade involved in the noscapine-induced apoptosis was analyzed. We observed no inhibitory effect of the caspase-8 inhibitor on caspase-9 activity. In view of these results and taking into consideration that K562 cells are Fas-null, we suggested that caspase-8 is activated in a Fas-independent manner downstream of caspase-9. In conclusion, noscapine can induce apoptosis in both apoptosis-proficient and apoptosis-resistant leukemic cells, and it can be a novel candidate in the treatment of hematological malignancies.

Xanthomicrol is the main cytotoxic component of Dracocephalum kotschyii and a potential anti-cancer agent.

Spinal-Z, a methanolic mixture of dried powdered seeds of Peganum harmala Linn. and leaf of Dracocephalum kotschyii Boiss. is an Iranian ethno-medical remedy. It has been used for the treatment of various types of cancer for many years. To evaluate the use of Spinal-Z in treatment of cancer, we examined its effects against a panel of malignant cell lines and tumors induced in mice. The in vitro antiproliferative activities of Spinal-Z, the seed extract of P. harmala and the leaf extract of D. kotschyii were determined using the MTT assay. The concentration of the agent required to inhibit cell growth by 50% (IC50) was estimated. In addition, the anti-tumor activities of the remedy and its constituents were investigated. Viability of cells treated with Spinal-Z and its components decreased in a dose dependent manner. Spinal-Z and its components showed cytotoxic effects against all cell lines tested. The leaf extract of D. kotschyii showed a greater preferential cytotoxic effect than the seed extract of P. harmala and Spinal-Z, on all cell lines tested. Harmine showed cytotoxicity against HL60 and K562 cell lines. This could explain the cytotoxic effect of P. harmala on these cells. The leaf extract of D. kotschyii was able to inhibit tumor proliferation in mice. The active ingredient in the leaf extract of D. kotschyii appears to be a flavone identified as xanthomicrol. Xanthomicrol was able to inhibit proliferation of a number of malignant cells. The cytotoxic effects of xanthomicrol were more selective towards malignant cells than doxorubicin.