Naohiro Iwata

Chronic Treatment with a Water-Soluble Extract from the Culture Medium of Ganoderma lucidum Mycelia Prevents Apoptosis and Necroptosis in Hypoxia/Ischemia-Induced Injury of Type 2 Diabetic Mouse Brain

Type 2 diabetes mellitus has been known to increase systemic oxidative stress by chronic hyperglycemia and visceral obesity and aggravate cerebral ischemic injury. On the basis of our previous study regarding a water-soluble extract from the culture medium of Ganoderma lucidum mycelia (designed as MAK), which exerts antioxidative and neuroprotective effects, the present study was conducted to evaluate the preventive effects of MAK on apoptosis and necroptosis (a programmed necrosis) induced by hypoxia/ischemia (H/I) in type 2 diabetic KKAy mice. H/I was induced by a combination of unilateral common carotid artery ligation with hypoxia (8% O2 for 20 min) and subsequent reoxygenation. Pretreatment with MAK (1 g/kg, p.o.) for a week significantly reduced H/I-induced neurological deficits and brain infarction volume assessed at 24 h of reoxygenation. Histochemical analysis showed that MAK significantly suppressed superoxide production, neuronal cell death, and vacuolation in the ischemic penumbra, which was accompanied by a decrease in the numbers of TUNEL- or cleaved caspase-3-positive cells. Furthermore, MAK decreased the expression of receptor-interacting protein kinase 3 mRNA and protein, a key molecule for necroptosis. These results suggest that MAK confers resistance to apoptotic and necroptotic cell death and relieves H/I-induced cerebral ischemic injury in type 2 diabetic mice.

Orally Administrated Ascorbic Acid Suppresses Neuronal Damage and Modifies Expression of SVCT2 and GLUT1 in the Brain of Diabetic Rats with Cerebral Ischemia-Reperfusion

Diabetes mellitus is known to exacerbate cerebral ischemic injury. In the present study, we investigated antiapoptotic and anti-inflammatory effects of oral supplementation of ascorbic acid (AA) on cerebral injury caused by middle cerebral artery occlusion and reperfusion (MCAO/Re) in rats with streptozotocin-induced diabetes. We also evaluated the effects of AA on expression of sodium-dependent vitamin C transporter 2 (SVCT2) and glucose transporter 1 (GLUT1) after MCAO/Re in the brain. The diabetic state markedly aggravated MCAO/Re-induced cerebral damage, as assessed by infarct volume and edema. Pretreatment with AA (100 mg/kg, p.o.) for two weeks significantly suppressed the exacerbation of damage in the brain of diabetic rats. AA also suppressed the production of superoxide radical, activation of caspase-3, and expression of proinflammatory cytokines (tumor necrosis factor-α and interleukin-1β) in the ischemic penumbra. Immunohistochemical staining revealed that expression of SVCT2 was upregulated primarily in neurons and capillary endothelial cells after MCAO/Re in the nondiabetic cortex, accompanied by an increase in total AA (AA + dehydroascorbic acid) in the tissue, and that these responses were suppressed in the diabetic rats. AA supplementation to the diabetic rats restored these responses to the levels of the nondiabetic rats. Furthermore, AA markedly upregulated the basal expression of GLUT1 in endothelial cells of nondiabetic and diabetic cortex, which did not affect total AA levels in the cortex. These results suggest that daily intake of AA attenuates the exacerbation of cerebral ischemic injury in a diabetic state, which may be attributed to anti-apoptotic and anti-inflammatory effects via the improvement of augmented oxidative stress in the brain. AA supplementation may protect endothelial function against the exacerbated ischemic oxidative injury in the diabetic state and improve AA transport through SVCT2 in the cortex.

Antidepressant-like effects of a water-soluble extract from the culture medium of Ganoderma lucidum mycelia in rats

BackgroundGanoderma lucidum is a popular medicinal mushroom used for promoting health and longevity in Asian countries. Previously, we reported that a water-soluble extract from a culture medium of Ganoderma lucidum mycelia (MAK) exerts antioxidative and cerebroprotective effects against ischemia–reperfusion injury in vivo. Here, we evaluated the antidepressant and anxiolytic activities of MAK in rats.MethodsMAK (0.3 or 1 g/kg, p.o.) was administered in the experimental animals 60 min before the forced swimming, open-field, elevated plus-maze, contextual fear-conditioning, and head twitch tests. Additionally, the mechanisms involved in the antidepressant-like action of MAK were investigated by the serotonin precursor 5-hydroxy-L-tryptophan (5-HTP)- or 5-HT2A agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)-induced head twitch responses.ResultsTreatment with MAK (1 g/kg) exhibited antidepressant-like effects in the forced swimming test, attenuated freezing behavior in the contextual fear-conditioning test, and decreased the number of head twitches induced by DOI, but not with 5-HTP. No significant response was observed in locomotion or anxiety-like behavior, when the animals were evaluated in the open-field or elevated plus-maze test, respectively.ConclusionsThese data suggest that MAK has antidepressant-like potential, which is most likely due to the antagonism of 5-HT2A receptors, and possesses anxiolytic-like effects toward memory-dependent and/or stress-induced anxiety in rats.

Effects of Etanercept against Transient Cerebral Ischemia in Diabetic Rats

Diabetes mellitus is known to exacerbate acute cerebral ischemic injury. Previous studies have demonstrated that infarction volumes caused by transient cerebral ischemia were greater in diabetic rats than in nondiabetic rats. Tumor necrosis factor-α (TNF-α) is a proinflammatory protein produced in the brain in response to cerebral ischemia that promotes apoptosis. Etanercept (ETN), a recombinant TNF receptor (p75)-Fc fusion protein, competitively inhibits TNF-α. Therefore, we evaluated the neuroprotective effects of chronic or acute treatment with ETN on cerebral injury caused by middle cerebral artery occlusion/reperfusion (MCAO/Re) in rats with streptozotocin-induced diabetes. Furthermore, we evaluated the effects of ETN against the apoptosis and myeloperoxidase activity. Single administration of ETN before MCAO significantly suppressed exacerbation of cerebral damage in nondiabetic rats, as assessed by infarct volume. In contrast, the diabetic state markedly aggravated MCAO/Re-induced cerebral damage despite ETN treatment within 24 h before MCAO. However, the damage was improved by repeated administration of ETN at 900 μg/kg/daily in rats in an induced diabetic state. These results suggested that repeated administration of ETN can prevent exacerbation of cerebral ischemic injury in the diabetic state and is mainly attributed to anti-inflammatory effects.

Diabetes-Mediated Exacerbation of Neuronal Damage and Inflammation After Cerebral Ischemia in Rat: Protective Effects of Water-Soluble Extract from Culture Medium of Ganoderma lucidum Mycelia

Diabetes mellitus is a metabolic disorder, suffered by hundreds of millions of people throughout the world, which is characterized by hyperglycemia resulting from insufficiency of insulin secretion and/or action (Wild et al., 2004). Complications associated with diabetes affecting vessels, eyes, kidney, and peripheral nerves reduce the QOL of diabetic patients. Also, diabetes is widely recognized as a major risk factor for atherosclerotic disease such as acute brain ischemia. Indeed, diabetic patients have a higher risk of stroke compared with non-diabetic patients (Baynes 1991; Stephens et al., 2009). Additionally, they are more likely to have a poor prognosis and increased mortality after stroke (Biller et al., 1993; Vinik et al., 2002). Previous studies have demonstrated that the diabetic state increases oxidative stress in the brain and aggravates cerebral ischemic injury in both type I (Li et al., 2004; Saito et al., 2005; Rizk et al., 2005) and type II diabetic animal models (Anabela et al., 2006; Tureyen et al., 2011). In addition to neuronal damage attributed to hypoxia and ATP depletion caused by vascular obstruction in ischemic core region, cerebral injury caused by subsequent reperfusion is also involved in the pathophysiology of transient ischemia (Doyle et al., 2008; Nakka et al., 2008; Wang et al., 2010). During reperfusion, the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is enhanced by abrupt re-oxygenation (Cuzzocrea et al., 2001; Saito et al., 2005). Besides direct injurious effects to the cell membrane, proteins and DNA by

Protective Effects of Ferulic Acid against Chronic Cerebral Hypoperfusion-Induced Swallowing Dysfunction in Rats

Ferulic acid (FA), a phenolic phytochemical, has been reported to exert antioxidative and neuroprotective effects. In this study, we investigated the protective effects of FA against the dysfunction of the swallowing reflex induced by ligation of bilateral common carotid arteries (2VO) in rats. In 2VO rats, topical administration of water or citric acid to the pharyngolaryngeal region evoked a diminished number of swallowing events with prolonged latency compared to sham-operated control rats. 2VO rats had an increased level of superoxide anion radical, and decreased dopamine and tyrosine hydroxylase enzyme levels in the striatum, suggesting that 2VO augmented cerebral oxidative stress and impaired the striatal dopaminergic system. Furthermore, substance P (SP) expression in the laryngopharyngeal mucosa, which is believed to be positively regulated by dopaminergic signaling in the basal ganglia, was decreased in 2VO rats. Oral treatment with FA (30 mg/kg) for 3 weeks (from one week before 2VO to two weeks after) improved the swallowing reflex and maintained levels of striatal dopamine and laryngopharyngeal SP expression in 2VO rats. These results suggest that FA maintains the swallowing reflex by protecting the dopamine-SP system against ischemia-induced oxidative damage in 2VO rats.

Early Release of HMGB1 may Aggravate Neuronal Damage after Transient Focal Ischemia in Diabetic Rat Brain

Objective: High Mobility Group Box 1 (HMGB1) released extracellularly from necrotic cells evokes delayed inflammatory processes via interaction with the Receptor for Advanced Glycation End Products (RAGE) or Toll-like Receptors (TLR) in postischemic brain. The diabetic state (DM) aggravated cerebral ischemic injury following the stroke in rats. Therefore, we examined the behavior of HMGB1 and the expression of RAGE in non-DM and DM rat brain after middle cerebral artery occlusion followed by reperfusion (MCAO/Re). Methods: Diabetes was induced by a single injection of streptozotocin in male Sprague Dawley rats (DM group). MCAO/ Re was performed in non-DM and DM rats (ischemic groups) using a standard intraluminal procedure, and postischemic neurological deficits. Both brain infarction and edema were evaluated at various times after reperfusion. Control non-DM and DM rats underwent sham operation using the same manipulation, but without insertion of the occlusion filament. The behavior of HMGB1 and the expression of its receptors in the rat brain were examined using immunohistochemical and western blot analyses. Results: In sham-operated DM rat brain, immunoreactivity of HMGB1, which was localized in the neuronal nuclei of the cortex, was markedly increased compared with that in non-DM shamoperated rat brain. In the ischemic groups, the DM state aggravated MCAO/Re-induced neurological deficits and cerebral injury assessed by the infarction volume. Enhancement of translocation of HMGB1 from the nucleus to the cytoplasm induced by MCAO/ Re was markedly accelerated in the penumbral region of DM rat cortex. Immunoblot analysis revealed that the ischemia-induced increase in the release of HMGB1 into the cerebrospinal fluid and plasma was also enhanced in DM rats. Moreover, the expression of RAGE was upregulated in the brains of DM ischemic and control rats. Conclusions: The early release of HMGB1 and the expression of its receptors may be involved in the aggravation of neuronal damage caused by transient cerebral ischemia in DM rats. Therefore, it is important to inhibit the HMGB1 released in response to ischemia during the treatment of postischemic injury in diabetic patients.

Matrin 3 Augments the Transcriptional Activity of an SV40 Promoter-MediatedLuciferase Gene with a Highly Repetitive DNA Component

We examined the transcriptional augmentation of matrin 3, a nuclear matrix protein, of the SV40 promotermediated luciferase gene (pGL3) following transient transfection of recombinant plasmids into cells. It has been reported that the interaction of the Xmn I fragment, a highly repetitive DNA component as one of a typical matrix- or scaffold-attachment regions (MAR/SAR) tethered upstream from the SV40 promoter (pGL3-Xmn I) with matrin 3 appeared to be required for augmentation of luciferase gene transcription. In this study, we investigated the levels of induction in cells overexpressing the wild type and several deletion mutants of matrin 3. It appeared that pGL3-Xmn I augmented luciferase production to 4-times the control level in Ac2F cells, but 23-fold in cells overexpressing matrin 3. Electrophoretic mobility shift assay showed that the Xmn I fragment augmented luciferase gene transcription through interaction with matrin 3. Furthermore, our findings suggest that all of the functional domains tested in matrin 3 were necessary for transcriptional augmentation. We aim not only to describe the transcriptional augmentation of matrin 3 with MAR/SAR, but also to strengthen interest in their use to mediate the expression of therapeutic transgenes.