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Angiotensin II receptor blockers (ARBs) have been shown to exert neuroprotective effects by suppressing inflammatory and apoptotic responses. In the present study, the effects of the ARB telmisartan on the NLRP3 inflammasome induced by oxygen-glucose deprivation (OGD) in neural stem cells (NSCs) were investigated, as well as their possible association with the activation of the PI3K pathway. Cultured NSCs were treated with different concentrations of telmisartan and subjected to various durations of OGD. Cell counting, lactate dehydrogenase, bromodeoxyuridine, and colony-forming unit assays were performed to measure cell viability and proliferation. In addition, the activity of intracellular signaling pathways associated with the PI3K pathway and NLRP3 inflammasome was evaluated. Telmisartan alone did not affect NSCs up to a concentration of 10 μM under normal conditions but showed toxicity at a concentration of 100 μM. Moreover, OGD reduced the viability of NSCs in a time-dependent manner. Nevertheless, treatment with telmisartan increased the viability and proliferation of OGD-injured NSCs. Furthermore, telmisartan promoted the expression of survival-related proteins and mRNA while inhibiting the expression of death-related proteins induced by OGD. In particular, telmisartan attenuated OGD-dependent expression of the NLRP3 inflammasome and its related signaling proteins. These beneficial effects of telmisartan were blocked by a PI3K inhibitor. Together, these results indicate that telmisartan attenuated the activation of the NLRP3 inflammasome by triggering the PI3K pathway, thereby contributing to neuroprotection.

Amlodipine, a L-type calcium channel blocker, has been reported to have a neuroprotective effect in brain ischemia. Mitochondrial calcium overload leads to apoptosis of cells in neurologic diseases. We evaluated the neuroprotective effects of amlodipine camsylate (AC) on neural stem cells (NSCs) injured by oxygen glucose deprivation (OGD) with a focus on mitochondrial structure and function. NSCs were isolated from rodent embryonic brains. Effects of AC on cell viability, proliferation, level of free radicals, and expression of intracellular signaling proteins were assessed in OGD-injured NSCs. We also investigated the effect of AC on mitochondrial structure in NSCs under OGD by transmission electron microscopy. AC increased the viability and proliferation of NSCs. This beneficial effect of AC was achieved by strong protection of mitochondria. AC markedly enhanced the expression of mitochondrial biogenesis-related proteins and mitochondrial anti-apoptosis proteins. Together, our results indicate that AC protects OGD-injured NSCs by protecting mitochondrial structure and function. The results of the present study provide insight into the mechanisms underlying the protective effects of AC on NSCs.

Cerebral infarction is one of the major causes of severe morbidity and mortality, and thus, research has focused on developing treatment options for this condition. Zinc (Zn) is an essential element in the central nervous system and has several neuroprotective effects in the brain. In this study, we examined the neuroprotective effects of Zn on neural stem cells (NSCs) exposed to hypoxia. After treatment with several concentrations of Zn, the viability of NSCs under hypoxic conditions was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Trypan blue staining, and a lactate dehydrogenase assay. To evaluate the effect of Zn on the proliferation of NSCs, bromodeoxyuridine/5-bromo-2'-deoxyuridine (BrdU) labeling and colony formation assays were performed. Apoptosis was also examined in NSCs exposed to hypoxia with and without Zn treatment. In addition, a western blot analysis was performed to evaluate the effect of Zn on intracellular signaling proteins. NSC viability and proliferation were decreased under hypoxic conditions, but treatment with sublethal doses of Zn restored viability and proliferation. Sublethal doses of Zn reduced apoptosis caused by hypoxia, increased the expression levels of proteins related to the phosphatidylinositol-3 kinase (PI3K) pathway, and decreased the expression levels of proteins associated with neuronal cell death. These findings confirm that in vivo, sublethal doses of Zn protect NSCs against hypoxia through the activation of the PI3K pathway. Thus, Zn could be employed as a therapeutic option to protect NSCs in ischemic stroke.

Phosphatidylinositol 3-kinases (PI3Ks) have recently been implicated in apoptosis and ischemic cell death. We tested the efficacy of early intervention with a peptide PI3K activator in focal cerebral ischemia. After determining the most effective dose (24 μg/kg) and time window (2 h after MCAO) of treatment, a total of 48 rats were subjected to middle cerebral artery occlusion (MCAO). Diffusion weighted MRI (DWI) was performed 1 h after MCAO and rats with lesion sizes within a predetermined range were randomized to either PI3K activator or vehicle treatment arms. Fluid attenuated inversion recovery (FLAIR) MRI, neurological function, western blots, and immunohistochemistry were blindly assessed. Initial DWI lesion volumes were nearly identical between two groups prior to treatment. However, FLAIR showed significantly smaller infarct volumes in the PI3K activator group compared with vehicle (146 ± 81 mm 3 and 211 ± 96 mm 3 , p = 0.045) at 48 h. The PI3K activator group also had better neurological function for up to 2 weeks. In addition, PI3K activator decreased the number of TUNEL-positive cells in the peri-infarct region compared with the control group. Western blot and immunohistochemistry showed increased expression of phosphorylated Akt (Ser473) and GSK-3β (Ser9) and decreased expression of cleaved caspase-9 and caspase-3. Our results suggest a neuroprotective role of early activation of PI3K in ischemic stroke. The use of DWI in the randomization of experimental groups may reduce bias.

Although statins, or hydroxymethylglutaryl coenzyme A (HMG-Co A) reductase inhibitors, are generally used to decrease levels of circulating cholesterol, they have also been reported to have neuroprotective effects through various mechanisms. However, recent results have indicated that they may be harmful in patients with amyotrophic lateral sclerosis (ALS). In this study, we investigate whether atorvastatin protects motor neuron-like cells (NSC-34D) from oxidative stress. To evaluate the effects of atorvastatin or hydrogen peroxide or both on NSC-34D cells, the cells were treated with various combinations of these agents. To evaluate the viability of the cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and trypan blue staining were performed. Levels of free radicals and intracellular signaling proteins were evaluated using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and Western blotting, respectively. Atorvastatin protected NSC-34D cells against oxidative stress in a concentration-dependent manner. This neuroprotective effect of atorvastatin was blocked by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor and by FR180204, a selective extracellular signal-related kinase (ERK) inhibitor. Atorvastatin treatment increased the expression levels of p85αPI3K, phosphorylated Akt, phosphorylated glycogen synthase kinase-3β, phosphorylated ERK, and Bcl-2, which are proteins related to survival. Furthermore, atorvastatin decreased the levels of cytosolic cytochrome C, Bax, cleaved caspase-9, and cleaved caspase-3, which are associated with death in oxidative stress-injured NSC-34D cells. We conclude that atorvastatin has a protective effect against oxidative stress in motor neurons by activating the PI3K and ERK pathways as well as by scavenging free radicals. These findings indicate that statins could help protect motor neurons from oxidative stress.

Cerebral infarction causes permanent neuronal loss inducing severe morbidity and mortality. Because hypertension is the main risk factor for cerebral infarction and most patients with hypertension take antihypertensive drugs daily, the neuroprotective effects and mechanisms of anti-hypertensive drugs need to be investigated. Cilnidipine, a long-acting, new generation 1,4-dihydropyridine inhibitor of both L- and N-type calcium channels, was reported to reduce oxidative stress. In this study, we investigated whether cilnidipine has therapeutic effects in an animal model of cerebral infarction. After determination of the most effective dose of cilnidipine, a total of 128 rats were subjected to middle cerebral artery occlusion. Neurobehavioral function test and brain MRI were performed, and rats with similar sized infarcts were randomized to either the cilnidipine group or the control group. Cilnidipine treatment was performed with reperfusion after 2-h occlusion. Western blots and immunohistochemistry were also performed after 24-h occlusion. Initial infarct volume on diffusion-weighted MRI was not different between the cilnidipine group and the control group; however, fluid-attenuated inversion recovery MRI at 24 h showed significantly reduced infarct volume in the cilnidipine group compared with the control group. Cilnidipine treatment significantly decreased the number of triphosphate nick end labeling-positive cells compared to the control group. Western blot and immunohistochemistry showed increased expression of phosphorylated Akt (Ser473), phosphorylated glycogen synthase kinase-3β, and Bcl-2 and decreased expression of Bax and cleaved caspase-3. These results suggest that cilnidipine, which is used for the treatment of hypertension, has neuroprotective effects in the ischemic brain through activation of the PI3K pathway. We investigated whether cilnidipine has neuroprotective effects on ischemic stroke in an animal model. We have demonstrated that the neuroprotective effect of cilnidipine is associated with the activation of the PI3K pathway. Considering the daily use of antihypertensive drugs for patients with hypertension, cilnidipine could be beneficial for patients with ischemic stroke., (© 2015 International Society for Neurochemistry.)

Neurogenesis in the adult brain is important for memory and learning, and the alterations in neural stem cells (NSCs) may be an important part of Alzheimer's disease pathogenesis. The phosphatidylinositol 3-kinase (PI3K) pathway has been suggested to play an important role in neuronal cell survival and is highly involved in adult neurogenesis. Recently, coenzyme Q10 (CoQ10) was found to affect the PI3K pathway. We investigated whether CoQ10 could restore amyloid β (Aβ)25-35 oligomer-inhibited proliferation of NSCs by focusing on the PI3K pathway. To evaluate the effects of CoQ10 on Aβ25-35 oligomer-inhibited proliferation of NSCs, NSCs were treated with several concentrations of CoQ10 and/or Aβ25-35 oligomers. BrdU labeling, Colony Formation Assays, and immunoreactivity of Ki-67, a marker of proliferative activity, showed that NSC proliferation decreased with Aβ25-35 oligomer treatment, but combined treatment with CoQ10 restored it. Western blotting showed that CoQ10 treatment increased the expression levels of p85α PI3K, phosphorylated Akt (Ser473), phosphorylated glycogen synthase kinase-3β (Ser9), and heat shock transcription factor, which are proteins related to the PI3K pathway in Aβ25-35 oligomers-treated NSCs. To confirm a direct role for the PI3K pathway in CoQ10-induced restoration of proliferation of NSCs inhibited by Aβ25-35 oligomers, NSCs were pretreated with a PI3K inhibitor, LY294002; the effects of CoQ10 on the proliferation of NSCs inhibited by Aβ25-35 oligomers were almost completely blocked. Together, these results suggest that CoQ10 restores Aβ25-35 oligomer-inhibited proliferation of NSCs by activating the PI3K pathway.

Cilnidipine, a calcium channel blocker, has been reported to have neuroprotective effects. We investigated whether cilnidipine could protect neurons from hypoxia and explored the role of the phosphatidylinositol 3-kinase (PI3K) and extracellular signal-related kinase (ERK) pathways in the neuroprotective effect of cilnidipine. The viability of a primary culture of cortical neurons injured by hypoxia, measured by trypan blue staining and lactate dehydrogenase (LDH) assay, was dramatically restored by cilnidipine treatment. TUNEL and DAPI staining showed that cilnidipine significantly reduced apoptotic cell death induced by hypoxia. Free radical stress and calcium influx induced by hypoxia were markedly decreased by treatment with cilnidipine. Survival signaling proteins associated with the PI3K and ERK pathways were significantly increased while death signaling proteins were markedly decreased in the primary culture of cortical neurons simultaneously exposed to cilnidipine and hypoxia when compared with the neurons exposed only to hypoxia. These neuroprotective effects of cilnidipine were blocked by treatment with a PI3K inhibitor or an ERK inhibitor. These results show that cilnidipine protects primary cultured cortical neurons from hypoxia by reducing free radical stress, calcium influx, and death-related signaling proteins and by increasing survival-related proteins associated with the PI3K and ERK pathways, and that activation of those pathways plays an important role in the neuroprotective effects of cilnidipine against hypoxia. These findings suggest that cilnidipine has neuroprotective effects against hypoxia through various mechanisms, as well as a blood pressure-lowering effect, which might help to prevent ischemic stroke and reduce neuronal injury caused by ischemic stroke., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Oxidative stress plays critical roles in the pathogenic mechanisms of several neurodegenerative disorders including Alzheimer's disease (AD), thus much research effort has focused on antioxidants as potential treatment agents for AD. Coenzyme Q10 (CoQ10) is known to have powerful antioxidant effects. We investigated the neuroprotective effects of CoQ10 against Amyloid beta(25-35) (Aβ(25-35))-induced neurotoxicity in rat cortical neurons. To evaluate the neuroprotective effects of CoQ10 on Aβ(25-35)-injured neurons, primary cultured cortical neurons were treated with several concentrations of CoQ10 and/or Aβ(25-35) for 48h. CoQ10 protected neuronal cells against Aβ(25-35)-induced neurotoxicity in a concentration-dependent manner. These neuroprotective effects of CoQ10 were blocked by LY294002 (10μM), a phosphatidylinositol 3-kinase (PI3K) inhibitor. Aβ(25-35) concentration-dependent increased free radical levels in rat cortical neurons, while combined treatment with CoQ10 reduced these free radical levels in a dose-dependent manner. Meanwhile, CoQ10 treatment of Aβ(25-35)-injured primary cultured cortical neurons increased the expression levels of p85aPI3K, phosphorylated Akt, phosphorylated glycogen synthase kinase-3β, and heat shock transcription factor, which are proteins related to neuronal cell survival, and decreased the levels of cytosolic cytochrome c and cleaved caspase-3, which are associated with neuronal cell death. Together, these results suggest that the neuroprotective effects of CoQ10 on Aβ(25-35) neurotoxicity are mediated by inhibition of oxidative stress together with activation of the PI3-K/Akt pathway., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Neurotoxic effects have been suggested for l-3,4-dihydroxyphenylalanine (L-DOPA), while neuroprotective effects have been proposed for statins. We investigated whether certain statins (simvastatin or pitavastatin) could inhibit L-DOPA neurotoxicity. Neuronally-differentiated PC12 (nPC12) cells were treated with L-DOPA and/or statins for 24h, and their viabilities were measured using a cell counting kit, trypan blue staining, and 4',6-diamidino-2-phenylindole (DAPI) staining. Free radical and specific intracellular signaling protein levels were measured with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and Western blotting, respectively. High concentrations of l-DOPA reduced nPC12 cell viability, but combined treatment with statins restored viability. Treatment with 200 μM L-DOPA increased free radical and hydroxyl radical levels, but combined treatment with 5 μM statins decreased these levels. Survival-related signaling proteins were decreased in nPC12 cells treated with 200 μM L-DOPA, but combined treatment with 5μM statins significantly increased the levels of these proteins. Treatment with 200 μM L-DOPA significantly increased death-related signaling proteins, while combined treatment with 5 μM statins reduced the levels of these proteins. Pretreatment with LY294002, a phosphatidylinositol 3 kinase (PI3K) inhibitor, before combined treatment with statins and L-DOPA almost completely blocked the protective effects of statins. These results indicate that statins reduce L-DOPA neurotoxicity by lowering oxidative stress and by enhancing survival signals and inhibiting death signals via activation of the PI3K pathway., (Copyright © 2010 Elsevier B.V. All rights reserved.)

L-3,4-Dihydroxyphenylalanine (L-DOPA) is one of the most important drugs for the treatment of Parkinson's disease (PD). Although neurotoxicity of L-DOPA remains controversial, there are many reports suggesting that L-DOPA causes neuronal death. We investigated whether the neurotoxic effect of L-DOPA could be inhibited by the activation of the phosphatidylinositol 3-kinase (PI3K) pathway. Cell counting kit-8, trypan blue staining, and DAPI staining all showed that L-DOPA decreased nPC12 cell viability at high concentrations. However, combined treatment with the PI3K activator and L-DOPA significantly increased the viability of nPC12 cells when compared with treatment with L-DOPA only. Phosphorylated Akt (Ser473), phosphorylated glycogen synthase kinase-3beta (GSK-3beta) (Ser9), and heat shock transcription factor-1, which are survival-related signaling proteins, were decreased in nPC12 cells treated with 200 microM L-DOPA, but were significantly increased with combined treatment with the PI3K activator in a concentration-dependent manner. However, treatment of L-DOPA significantly increased expressions of cytosolic cytochrome c and cleaved caspase-3, which are death-related signaling proteins, in nPC12 cells, but combined treatment with the PI3K activator reduced those expressions. To confirm whether the effect of the PI3K activator is associated with direct activation of PI3K, LY294002, a PI3K inhibitor, was used to pretreat the nPC12 cells prior to combined treatment with the PI3K activator and L-DOPA. The protective effect of the PI3K activator was almost completely blocked. Together, these results suggest that L-DOPA neurotoxicity can be prevented by PI3K activation.

We investigated the neuroprotective effect and mechanisms of action of cilnidipine, a long-acting, second-generation 1,4-dihydropyridine inhibitor of L- and N-type calcium channels, in PC12 cells that were neuronally differentiated by treatment with nerve growth factor (nPC12 cells). To evaluate the effect of cilnidipine on viability, nPC12 cells were treated with several concentrations of this drug before performing viability assays. Free radical levels and intracellular signaling proteins were measured with the fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate and western blotting, respectively. Cell viability was not affected by low concentrations of cilnidipine up to 150 microM, but it was slightly decreased at 200 microM cilnidipine. Following H(2)O(2) exposure, the viability of nPC12 cells decreased significantly; however, treatment with cilnidipine increased the viability of H(2)O(2)-injured nPC12 cells in a concentration-dependent manner. Treatment with H(2)O(2) resulted in a concentration-dependent increase in free radical levels in nPC12 cells, and cilnidipine treatment reduced free radical levels in H(2)O(2)-injured nPC12 cells in a dose-dependent manner. Cilnidipine treatment increased the expression of p85aPI3K (phosphatidylinositol 3-kinase) phosphorylated Akt, phosphorylated glycogen synthase kinase-3 (pGSK-3beta), and heat shock transcription factor (HSTF-1) which are proteins related to neuronal cell survival, and decreased levels of cytosolic cytochrome c, activated caspase 3, and cleaved poly (ADP-ribose) polymerase (PARP), which are associated with neuronal cell death, in H(2)O(2)-injured nPC12 cells. These results indicate that cilnidipine mediates its neuroprotective effects by reducing oxidative stress, enhancing survival signals (e.g., PI3K, phosphorylated Akt, pGSK-3beta, and HSTF-1), and inhibiting death signals from cytochrome c release, caspase 3 activation, and PARP cleavage.

The phosphatidylinositol-3-kinase (PI3-K) pathway has been suggested to play a pivotal role in neuronal survival. Although PI3-K has been recently identified as a neuroprotectant, there are no reports regarding the effect of a direct PI3-K activator on Abeta-induced neurotoxicity. We investigated whether direct PI3-K activation prevents Abeta-induced neurotoxicity. To evaluate the effect of Abeta on neuronal cells, we treated primary cultured cortical neurons with several doses of Abeta for 72 h. To investigate the protective effect that PI3-K activation has on Abeta-induced neurotoxicity, cells were simultaneously treated with several doses of a PI3-K activator for 72 h. An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, trypan blue staining, and DAPI staining showed that Abeta decreased neuronal cell viability in a concentration-dependent manner and also that PI3-K activation effectively prevented Abeta-induced neuronal cell death. Abeta significantly decreased survival signals, including phosphorylated Akt, glycogen synthase kinase-3beta, and heat shock transcription factor-1. Abeta also increased death signals, such as phosphorylated tau (pThr231) and activated caspase-3. Treatment with a PI3-K activator restored the survival signals and inhibited the death signals. These results suggest that the neurotoxic effect of Abeta can be partially prevented by PI3-K activation.