Hean Zhuang

Potential mechanism by which resveratrol, a red wine constituent, protects neurons

Abstract: Polyphenolic compounds, such as resveratrol, are naturally present at high concentration in grape skin, seeds, and red wine. Resveratrol is present in cis and trans isoforms and the major trans isomer is the biologically active one. Epidemiologic studies have revealed a reduced incidence of cardiovascular risk associated with consumers of red wine; this has been popularized as the French paradox. Resveratrol has been shown to have significant antioxidant properties in a variety of in vitro and in vivo models. It can reduce ischemic damage in heart ischemia reperfusion injury and also in brain ischemia/reperfusion in rodent models. Due to the high rate of oxygen consumption in the brain, and especially low levels of antioxidant defense enzymes, this organ is particularly susceptible of free radical damage. Most of the protective biological actions associated with resveratrol have been associated with its intrinsic radical scavenger properties. We have investigated the possibility of other indirect pathways by which resveratrol can exert its neuroprotective abilities. We have specifically tested whether heme oxygenase neuroprotective enzyme could be stimulated after resveratrol treatment. Using primary neuronal cultures, resveratrol was able to significantly induce heme oxygenase 1, whereas vehicle control showed no effect. No detectable toxicity was quantified. It is well established that after stroke significant levels of intracellular heme levels increase. The source of free heme comes mainly from several heme‐containing enzymes. Heme (iron‐protoporphyrin IX) is a pro‐oxidant and its rapid degradation by heme oxygenase is believed to be protective. Moreover, the generation of heme metabolites can also have their own intrinsic cellular properties. All together, increased heme oxygenase activity by resveratrol is a unique pathway by which this compound can exert its neuroprotective actions.

Resveratrol protects against experimental stroke: Putative neuroprotective role of heme oxygenase 1

Epidemiological and experimental reports have linked mild-to-moderate wine and/or grape consumption to a lowered incidence of cardiovascular, cerebrovascular, and peripheral vascular risk. This study revealed that resveratrol, an enriched bioactive polyphenol in red wine, selectively induces heme oxygenase 1 (HO1) in a dose- and time-dependent manner in cultured mouse cortical neuronal cells and provides neuroprotection from free-radical or excitotoxicity damage. This protection was lost when cells were treated with a protein synthesis or heme oxygenase inhibitor, suggesting that HO1 induction is at least partially required for resveratrols prophylactic properties. Furthermore, resveratrol pretreatment dose-dependently protected mice subjected to an optimized ischemic-reperfusion stroke model. Mice in which HO1 was selectively deleted lost most, if not all, of the beneficial effects. Together, the data suggest a potential intracellular pathway by which resveratrol can provide cell/organ resistance against neuropathological conditions.

Ginkgo Biloba Extract Neuroprotective Action Is Dependent on Heme Oxygenase 1 in Ischemic Reperfusion Brain Injury

Background and Purpose— Ginkgo biloba extracts are now prescribed in several countries for their reported health benefits, particularly for medicinal properties in the brain. The standardized Ginkgo extract, EGb761, has been reported to protect neurons against oxidative stress, but the underlying mechanisms are not fully understood. Methods— To characterize the oral consumption of EGb761 in transient ischemia, we performed the middle cerebral artery occlusion (MCAO) filament model in wild-type and heme oxygenase 1 (HO-1) knockouts. Mice were pretreated for 7 days before the transient occlusion or posttreated acutely during reperfusion; then neurobehavioral scores and infarct volumes were assessed. Furthermore, primary cortical neuronal cultures were used to investigate the contribution of the antioxidant enzyme HO-1 in the EGb761-associated cytoprotection. Results— Mice that were pretreated with EGb761 had 50.9±5.6% less neurological dysfunction and 48.2±5.3% smaller infarct volumes than vehicle-treated mice; this effect was abolished in HO-1 knockouts. In addition to the prophylactic properties of EGb761, acute posttreatment 5 minutes and 4.5 hours after reperfusion also led to significant reduction in infarct size (P<0.01). After our previous demonstration that EGb761 significantly induced HO-1 levels in a dose- and time-dependent manner in neuronal cultures, here we revealed that this de novo HO-1 induction was required for neuroprotection against free radical damage and excitotoxicity as it was significantly attenuated by the enzyme inhibitor. Conclusion— These results demonstrate that EGb761 could be used as a preventive or therapeutic agent in cerebral ischemia and suggest that HO-1 contributes, at least in part, to EGb761 neuroprotection.

Heme–Hemopexin Complex Attenuates Neuronal Cell Death and Stroke Damage:

Hemoproteins undergo degradation during hypoxic/ischemic conditions, but the prooxidant free heme that is released cannot be recycled and must be degraded. The extracellular heme associates with its high-affinity binding protein, hemopexin (HPX). Hemopexin is shown here to be expressed by cortical neurons and it is present in mouse cerebellum, cortex, hippocampus, and striatum. Using the transient ischemia model (90-min middle cerebral artery occlusion followed by 96-h survival), we provide evidence that HPX is protective in the brain, as neurologic deficits and infarct volumes were significantly greater in HPX−/− than in wild-type mice. Addressing the potential protective HPX cellular pathway, we observed that exogenous free heme decreased cell survival in primary mouse cortical neuron cultures, whereas the heme bound to HPX was not toxic. Heme-HPX complexes induce HO1 and, consequently, protect primary neurons against the toxicity of both heme and prooxidant tert-butyl hydroperoxide; such protection was decreased in HO1−/− neuronal cultures. Taken together, these data show that HPX protects against heme-induced toxicity and oxidative stress and that HO1 is required. We propose that the heme-HPX system protects against stroke-related damage by maintaining a tight balance between free and bound heme. Thus, regulating extracellular free heme levels, such as with HPX, could be neuroprotective.

Heme oxygenase-1 protects brain from acute excitotoxicity.

Heme oxygenase is a rate-limiting enzyme that degrades heme, a pro-oxidant, into carbon monoxide, iron, and bilirubin. Heme oxygenase has two active isoforms: heme oxygenase-1 and heme oxygenase-2. Heme oxygenase-1 can be induced by various insults. Several investigators have postulated that it has cytoprotective activities, although its role in the nervous system is not fully understood, especially considering that normally heme oxygenase-2 accounts for the vast majority of heme oxygenase activity in the brain. Here, the basal effect of heme oxygenase-1 was investigated in acute glutamatergic excitotoxicity to test the hypothesis that N-methyl-D-aspartate-induced acute toxicity in brain is attenuated by heme oxygenase-1. N-methyl-D-aspartate was unilaterally injected into the striatum of wildtype and heme oxygenase-1 knockout mice. After 48 h, brains were harvested, sectioned, and stained with Cresyl Violet to measure the lesion size. Lesion volume was significantly (P<0.05) greater in brains of heme oxygenase-1 knockout mice (15.2+/-3.1 mm(3); n=10) than in those of wildtype mice (6.2+/-1.5 mm(3); n=11). In addition, Western blot analysis indicated no detectable differences between wildtype and heme oxygenase-1 knockout mouse brains in the levels of the glutamate or N-methyl-D-aspartate receptors studied. To test whether heme oxygenase-1 could specifically protect neurons, mouse primary neuronal cell cultures of wildtype and heme oxygenase-1 knockout mice were treated with or without N-methyl-D-aspartate. Cell viability of the heme oxygenase-1 knockout neurons was significantly less than that of wildtype neurons at each of the N-methyl-D-aspartate concentrations tested (12.8+/-1.3%, 16.0+/-1.4%, and 18.4+/-1.8% at 30, 100, and 300 microM N-methyl-D-aspartate, respectively). These results indicate that heme oxygenase-1 provides neuroprotection against acute excitotoxicity and suggest that potential intervention that can increase heme oxygenase-1 activity within the brain should be considered as a therapeutic target in acute and potentially chronic neurological disorders.

Stimulation of prostaglandin E2-EP3 receptors exacerbates stroke and excitotoxic injury.

The effect of PGE(2) EP3 receptors on injury size was investigated following cerebral ischemia and induced excitotoxicity in mice. Treatment with the selective EP3 agonist ONO-AE-248 significantly and dose-dependently increased infarct size in the middle cerebral artery occlusion model. In a separate experiment, pretreatment with ONO-AE-248 exacerbated the lesion caused by N-methyl-d-aspartic acid-induced acute excitotoxicity. Conversely, genetic deletion of EP3 provided protection against N-methyl-d-aspartic acid-induced toxicity. The results suggest that PGE(2), by stimulating EP3 receptors, can contribute to the toxicity associated with cyclooxygenase and that antagonizing this receptor could be used therapeutically to protect against stroke- and excitotoxicity-induced brain damage.

Elevated microsomal prostaglandin-E synthase–1 in Alzheimer’s disease

The proinflammatory prostaglandin E2 (PGE2) fluctuates over time in the cerebrospinal fluid of patients with Alzheimers disease (AD), but the cerebral distribution and expression patterns of microsomal prostaglandin‐E synthase (mPGES)–1 have not been compared with those of normal human brains.

PGD2 DP1 receptor protects brain from ischemia-reperfusion injury

Prostaglandin D2 is the most abundant prostaglandin in the brain. It has long been described as a modulator of the neuroinflammatory process, but little is known regarding the role of its Gαs‐coupled receptor, DP1. Therefore, in this study, the effect of the DP1 receptor on the outcome of cerebral ischemia in wildtype (WT) and DP1 knockout (DP1–/–) C57Bl/6 mice was investigated. Ischemia‐reperfusion injury was produced by a 90‐min occlusion of the right middle cerebral artery followed by a 4‐day reperfusion. Infarct size was 49.0 ± 11.0% larger in DP1–/– mice (n = 11; P < 0.01) than in WT mice (n = 9 per group). However, no differences were detected in the relative cerebral blood flow (CBF) or any of the physiological parameters measured (n = 5 per group) or in the large blood vessel anatomy (n = 3 per group). To further address whether the DP1 protective role in the brain could be extended to neurons, mouse primary corticostriatal neuronal cultures were exposed to the DP1‐selective agonist, BW245C, which provided dose‐dependent protection against excitotoxicity induced by glutamate. Protection was significant at a dose as low as 0.05 µm. The results indicate that the DP1 receptor is neuroprotective in both in vivo and in vitro paradigms. Development of drugs to stimulate the DP1 receptor in brain could provide a new therapeutic strategy against cerebral ischemia and potentially other neurological conditions.

1-HydroxyPGE1 reduces infarction volume in mouse transient cerebral ischemia

Differential neurological outcomes due to prostaglandin E2 activating G‐protein‐coupled prostaglandin E (EP) receptors have been observed. Here, we investigated the action of the EP4/EP3 agonist 1‐hydroxyPGE1 (1‐OHPGE1) in modulating transient ischemic brain damage. C57BL/6 mice were pretreated 50 min before transient occlusion of the middle cerebral artery with an intraventricular injection of 1‐OHPGE1 (0.1, 0.2, 2.0 nmol/0.2 µL). Brain damage 4 days after reperfusion, as estimated by infarct volume, was significantly reduced by more than 19% with 1‐OHPGE1 in the two higher‐dose groups (P < 0.05). To further address whether protection also was extended to neurons, primary mouse cultured neuronal cells were exposed to N‐methyl‐d‐aspartate. Co‐treatment with 1‐OHPGE1 resulted in significant neuroprotection (P < 0.05). To better understand potential mechanisms of action and to test whether changes in cyclic adenosine monophosphate (cAMP) levels and downstream signaling would be neuroprotective, we measured cAMP levels in primary neuronal cells. Brief exposure to 1‐OHPGE1 increased cAMP levels more than twofold and increased the phosphorylation of extracellular‐regulated kinases at positions Thr‐202/Tyr‐204. In a separate cohort of animals, 1‐OHPGE1 at all doses tested produced no significant effect on the physiological parameters of core body temperature, mean arterial pressure and relative cerebral blood flow observed following drug treatment. Together, these results suggest that modulation of PGE2 receptors that increase cAMP levels and activate extracellular‐regulated kinases 1/2 caused by treatment with 1‐OHPGE1 can be protective against neuronal injury induced by focal ischemia.

Prostaglandins of J Series Control Heme Oxygenase Expression

Abstract: Cyclopentenone prostaglandins (cyPGs) are a subfamily of prostaglandins that are characterized by the cyclopentenone ring in their structure. They exert their effect after active transportation into the cell, probably by interacting with cellular target proteins or DNA sequences. The cyPGs have anti‐inflammatory activities, especially important during the resolution of inflammation, anticancer, and cytoprotective properties. Here, we show that the cyPGs, especially the 15‐deoxy‐Δ12,14 PGJ2, can specifically induce heme oxygenase 1 in mouse primary neuronal cells. Heme oxygenase is the enzyme responsible for the degradation of heme into biliverdin, ferrous iron, and carbon monoxide. This enzyme conveys protection to oxidative cellular injury by degrading the pro‐inflammatory heme; producing biliverdin and bilirubin, potent antioxidants; producing carbon monoxide, a neurotransmitter that also has anti‐inflammatory and vasodilatory properties; and assisting in keeping iron cellular homeostasis. CyPGs appear to possess a promising future in designing therapeutics for many neurologic diseases, such as Alzheimers disease, vascular‐related dementia, multiple sclerosis, ischemic conditions, and many others in which inflammation is a part of the pathophysiology.