Expression of Hypoxia Inducible Factor 1alpha Is Protein Kinase A-dependent in Primary Cortical Astrocytes Exposed to Severe Hypoxia.

The hypoxia inducible factor 1 (HIF-1) and the cyclic AMP-responsive element binding protein (CREB) are two transcription factors that have been studied in the context of neuronal survival and neurodegeneration. HIF-1 upregulation and CREB activation have been observed not only in neurons but also in astrocytes under conditions of hypoxia. We hypothesized that activation of CREB regulate HIF-1α expression in the nucleus of cortical astrocytes under in vitro ischemic condition. To test the hypothesis, we determined the effects of inhibiting the CREB activation pathway on the expression of HIF-1α protein in astrocytes exposed to CoCl2 and severe hypoxia (near anoxia, 0.1% O2). The results demonstrated that inhibition of CaMKII and CaMKIV had no effect on both HIF-1α and pCREB expression in cortical astrocytes exposed to CoCl2 and anoxia. In contrast, PKA inhibition lowered the expression of HIF-1α and pCREB expression. Furthermore, the inhibition of PKA but not CaMKII or CaMKIV increased cell death of astrocytes exposed to near anoxia. The results suggest that PKA plays an important role in the cell survival signaling pathways in astrocytes.

Iron Pathophysiology in Stroke.

Ischemic and hemorrhagic stroke are the common types of stroke that lead to brain injury neurological deficits and mortality. All forms of stroke remain a serious health issue, and there is little successful development of drugs for treating stroke. Incomplete understanding of stroke pathophysiology is considered the main barrier that limits this research progress. Besides mitochondria and free radical-producing enzymes, labile iron is an important contributor to oxidative stress. Although iron regulation and metabolism in cerebral stroke are not fully understood, much progress has been achieved in recent years. For example, hepcidin has recently been recognized as the principal regulator of systemic iron homeostasis and a bridge between inflammation and iron regulation. This review discusses recent research progress in iron pathophysiology following cerebral stroke, focusing molecular regulation of iron metabolism and potential treatment targets.

Factors controlling permeability of the blood-brain barrier.

As the primary protective barrier for neurons in the brain, the blood-brain barrier (BBB) exists between the blood microcirculation system and the brain parenchyma. The normal BBB integrity is essential in protecting the brain from systemic toxins and maintaining the necessary level of nutrients and ions for neuronal function. This integrity is mediated by structural BBB components, such as tight junction proteins, integrins, annexins, and agrin, of a multicellular system including endothelial cells, astrocytes, pericytes, etc. BBB dysfunction is a significant contributor to the pathogeneses of a variety of brain disorders. Many signaling factors have been identified to be able to control BBB permeability through regulating the structural components. Among those signaling factors are inflammatory mediators, free radicals, vascular endothelial growth factor, matrix metalloproteinases, microRNAs, etc. In this review, we provide a summary of recent progress regarding these structural components and signaling factors, relating to their roles in various brain disorders. Attention is also devoted to recent research regarding impact of pharmacological agents such as isoflurane on BBB permeability and how iron ion passes across BBB. Hopefully, a better understanding of the factors controlling BBB permeability helps develop novel pharmacological interventions of BBB hyperpermeability under pathological conditions.

Role of Hypoxia Inducible Factor 1 in Hyperglycemia-Exacerbated Blood-Brain Barrier Disruption in Ischemic Stroke.

Diabetes is a major stroke risk factor and is associated with poor functional recovery after stroke. Accumulating evidence indicates that the worsened outcomes may be due to hyperglycemia-induced cerebral vascular complications, especially disruption of the blood-brain barrier (BBB). The present study tested a hypothesis that the activation of hypoxia inducible factor-1 (HIF-1) was involved in hyperglycemia-aggravated BBB disruption in an ischemic stroke model. Non-diabetic control and Streptozotocin-induced type I diabetic mice were subjected to 90min transient middle cerebral artery occlusion (MCAO) followed by reperfusion. Our results demonstrated that hyperglycemia induced higher expression of HIF-1α and vascular endothelial growth factor (VEGF) in brain microvessels after MCAO/reperfusion. Diabetic mice showed exacerbated BBB damage and tight junction disruption, increased infarct volume as well as worsened neurological deficits. Furthermore, suppressing HIF-1 activity by specific knock-out endothelial HIF-1α ameliorated BBB leakage and brain infarction in diabetic animals. Moreover, glycemic control by insulin abolished HIF-1α up-regulation in diabetic animals and reduced BBB permeability and brain infarction. These findings strongly indicate that HIF-1 plays an important role in hyperglycemia-induced exacerbation of BBB disruption in ischemic stroke. Endothelial HIF-1 inhibition warrants further investigation as a therapeutic target for the treatment of stroke patients with diabetes.

Ischemic tolerance in an in vivo model of glutamate preconditioning.

Ischemia initiates a complicated biochemical cascade of events that triggers neuronal death. This study focuses on glutamate-mediated neuronal tolerance to ischemia-reperfusion. We employed an animal model of lifelong excess release of glutamate, the glutamate dehydrogenase 1 transgenic (Tg) mouse, as a model of in vivo glutamate preconditioning. Nine- and twenty-two-month-old Tg and wild-type (wt) mice were subjected to 90 min of middle cerebral artery occlusion, followed by 24 hr of reperfusion. The Tg mice suffered significantly reduced infarction and edema volume compared with their wt counterparts. We further analyzed proteasomal activity, level of ubiquitin immunostaining, and microtubule-associated protein-2A (MAP2A) expression to understand the mechanism of neuroprotection observed in the Tg mice. We found that, in the absence of ischemia, the Tg mice exhibited higher activity of the 20S and 26S proteasomes, whereas there was no significant difference in the level of hippocampal ubiquitin immunostaining between wt and Tg mice. A surprising, significant increase was observed in MAP2A expression in neurons of the Tg hippocampus following ischemia-reperfusion compared with that in wt hippocampus. The results suggest that increased proteasome activity and MAP2A synthesis and transport might account for the effectiveness of glutamate preconditioning against ischemia-reperfusion.

HIF-1 is involved in high glucose-induced paracellular permeability of brain endothelial cells.

Experimental evidence from human patients and animal models of diabetes has demonstrated that hyperglycemia increases blood-brain barrier (BBB) permeability, which is associated with increased risk of neurological dysfunction. However, the mechanism underlying high glucose-induced BBB disruption is not understood. Here we investigated the role of hypoxia-inducible factor-1 (HIF-1) in high glucose-induced endothelial permeability in vitro using mouse brain microvascular endothelial cells (b.End3). Our results demonstrated that high glucose (30 mM) upregulated the protein level of HIF-1α, the regulatable subunit of HIF-1, and increased the transcriptional activity of HIF-1 in the endothelial cells. At the same time, high glucose increased the paracellular permeability associated with diminished expression and disrupted continuity of tight junction proteins occludin and zona occludens protein-1 (ZO-1) of the endothelial cells. Upregulating HIF-1 activity by cobalt chloride increased the paracellular permeability of the endothelial cells exposed to normal glucose (5.5 mM). In contrast, downregulating HIF-1 activity by HIF-1α inhibitors and HIF-1α specific siRNA ameliorated the increased paracellular permeability and the alterations of distribution pattern of occludin and ZO-1 induced by high glucose. In addition, high glucose increased expression of vascular endothelial growth factor (VEGF), a downstream gene of HIF-1. Inhibiting VEGF improved the expression pattern of occludin and ZO-1, and attenuated the endothelial leakage. Furthermore, key results were confirmed in human brain microvascular endothelial cells. These results strongly indicate that HIF-1 plays an important role in high glucose-induced BBB dysfunction. The results will help us understand the molecular mechanisms involved in hyperglycemia-induced BBB dysfunction and neurological outcomes.

Evaluating the effectiveness of lateral postural management for breech presentation: study protocol for a randomized controlled trial (BRLT study).

BACKGROUND: Breech presentation is observed in 3-4% at term of pregnancy and is one of the leading causes of cesarean section. There is no established treatment for breech presentation before 36 weeks. A retrospective cohort study was conducted to demonstrate that the lateral position is effective for breech presentation. However, there are no randomized controlled trials evaluating lateral position management for breech presentation. Here, we described the methodology of a randomized controlled trial of a cephalic version for breech presentation in the third trimester by lateral postural management (BRLT study). METHODS: The BRLT study is an open-label, randomized controlled trial with two parallel groups allocated in a 1:1 ratio to examine the lateral position management for breech presentation, as compared with expectant management care. An academic hospital in Japan will enroll 200 patients diagnosed with a breech presentation by ultrasonography between 28 + 0 weeks and 30 + 0 weeks. Participants in the intervention group will be instructed to lie on their right sides for 15 min three times per day if the fetal back was on the left side or lie on their left sides if the fetal back was on the right side. The instruction will be given every 2 weeks after confirmation of fetal position, and the lateral position will be instructed until the cephalic version, and after the cephalic version, the reverse lateral position will be instructed until delivery. The primary outcome is cephalic presentation at term. The secondary outcomes are cesarean delivery, cephalic presentation 2, 4, and 6 weeks after the instruction, and at delivery, recurrent breech presentation after cephalic version, and adverse effects. DISCUSSION: This trial will answer whether the lateral positioning technique is effective in treating breech presentation and, depending on the results, may provide a very simple, less painful, and safe option for treating breech presentation before 36 weeks, and it may impact breech presentation treatment. TRIAL REGISTRATION: UMIN Clinical Trials Registry UMIN000043613. Registered on 15 March 2021 https://center6.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000049800 .

Abundance of TRPC6 protein in glomerular mesangial cells is decreased by ROS and PKC in diabetes.

The present study was performed to investigate the underlying mechanism, particularly the roles of reactive oxygen species (ROS) and protein kinase C (PKC), in the diabetes-induced canonical transient receptor potential 6 (TRPC6) downregulation. We found that high glucose (HG) significantly reduced TRPC6 protein expression in cultured mesangial cells (MCs). TRPC6 protein was also significantly reduced in the glomeruli but not in the heart or aorta isolated from streptozotocin-induced diabetic rats. In the cultured MCs, H(2)O(2) suppressed TRPC6 protein expression in a dose- and time-dependent manner, which emulated the HG effect. Catalase as well as superoxide dismutase were able to prevent the inhibitory effect of HG on TRPC6. The antioxidant effect observed in cultured cells was also observed in diabetic rats treated with tempol for 2 wk, which exhibited a preservation of TRPC6 in the glomeruli. Specific knockdown of Nox4, a component of NADPH oxidase, increased TRPC6 protein expression. Furthermore, the PKC activator phorbol 12-myristate 13-acetate (PMA), but not its analog 4α-phorbol 12, 13-didecanoate (4α-PDD), suppressed TRPC6 expression, and this PMA effect was not affected by catalase. Moreover, Gö6976, but not LY333531, attenuated the negative effect of HG on TRPC6 expression. Gö6976 also inhibited H(2)O(2) effect on TRPC6. Furthermore, either knockdown of TRPC6 or HG treatment significantly decreased ANG II-stimulated MC contraction, and the HG-impaired MC contraction was rescued by overexpression of TRPC6. These results suggest that hyperglycemia in diabetes downregulated TRPC6 protein expression in MCs through a NADPH oxidase Nox4-ROS-PKC pathway, proving a mechanism for impaired MC contraction in diabetes.

Recent advances in iron homeostasis and regulation - a focus on epigenetic regulation and stroke.

Iron is an element with redox properties. It is active sites of many enzymes and plays an important role in various cellular and biological functions including ATP production and DNA synthesis. However, as a redox element, iron promotes free radical generation and lipid peroxidation, causing oxidative damage and cell death. Iron-mediated oxidation is a central player in ferroptosis, a type of cell death process that is different from apoptosis and necrosis. Thus, iron metabolism and homeostasis are sophisticatedly regulated. There has been exciting progress in understanding iron metabolism and regulation since hepcidin was recognized as the central regulator of iron homeostasis. Hepcidin mainly regulates the iron export function of the ferrous iron permease, ferroportin, which is the only known iron exporter expressed by mammalian cells. Particularly, epigenetic regulation has been a recent focus on iron homeostasis. Epigenetic phenomena have been demonstrated to modulate key proteins including hepcidin in iron metabolism. Here, we review the rapid progress in recent years in understanding molecular mechanisms of iron homeostasis with a focus on epigenetic regulation of hepcidin, ferritin, and ferroptosis. Interactions between methionine oxidation and iron is also discussed. Furthermore, many studies have suggested that the severity of neuronal damage after stroke is proportional to the magnitude of brain iron accumulation. Recent discoveries regarding iron metabolism in stroke is briefly discussed. Understanding the underlying mechanism in iron regulation could provide insight into the treatment of various intractable diseases including stroke.

Mitochondrial ferritin attenuates cerebral ischaemia/reperfusion injury by inhibiting ferroptosis.

Ischaemic stroke is becoming the most common cerebral disease in aging populations, but the underlying molecular mechanism of the disease has not yet been fully elucidated. Increasing evidence has indicated that an excess of iron contributes to brain damage in cerebral ischaemia/reperfusion (I/R) injury. Although mitochondrial ferritin (FtMt) plays a critical role in iron homeostasis, the molecular function of FtMt in I/R remains unknown. We herein report that FtMt levels are upregulated in the ischaemic brains of mice. Mice lacking FtMt experience more severe brain damage and neurological deficits, accompanied by typical molecular features of ferroptosis, including increased lipid peroxidation and disturbed glutathione (GSH) after cerebral I/R. Conversely, FtMt overexpression reverses these changes. Further investigation shows that Ftmt ablation promotes I/R-induced inflammation and hepcidin-mediated decreases in ferroportin1, thus markedly increasing total and chelatable iron. The elevated iron consequently facilitates ferroptosis in the brain of I/R. In brief, our results provide evidence that FtMt plays a critical role in protecting against cerebral I/R-induced ferroptosis and subsequent brain damage, thus providing a new potential target for the treatment/prevention of ischaemic stroke.

Arsenite induces endothelial cell permeability increase through a reactive oxygen species-vascular endothelial growth factor pathway.

As a potent environmental oxidative stressor, arsenic exposure has been reported to exacerbate cardiovascular diseases and increase vascular endothelial cell monolayer permeability. However, the underlying mechanism of this effect is not well understood. In this paper, we test our hypothesis that reactive oxygen species (ROS)-induced vascular endothelial growth factor (VEGF) expression may play an important role in an arsenic-caused increase of endothelial cell monolayer permeability. The mouse brain vascular endothelial cell bEnd3 monolayer was exposed to arsenite for 1, 3, and 6 days. The monolayer permeability, VEGF protein release, and ROS generation were determined. In addition, VE-cadherin and zonula occludens-1 (ZO-1), two membrane structure proteins, were immunostained to elucidate the effects of arsenite on the cell-cell junction. The roles of ROS and VEGF in arsenite-induced permeability was determined by inhibiting ROS with antioxidants and immuno-depleting VEGF with a VEGF antibody. We observed that arsenite increased bEnd3 monolayer permeability, elevated the production of cellular ROS, and increased VEGF release. VE-cadherin and ZO-1 disruptions were also found in cells treated with arsenite. Furthermore, both antioxidant (N-acetyl cysteine and tempol) and the VEGF antibody treatments significantly lowered the arsenite-induced permeability of the bEnd3 monolayer as well as VEGF expression. VE-cadherin and ZO-1 disruptions were also diminished by N-acetyl cysteine and the VEGF antibody. Our data suggest that the increase in VEGF expression caused by ROS may play an important role in the arsenite-induced increase in endothelial cell permeability.

The Antioxidant Enzyme Methionine Sulfoxide Reductase A (MsrA) Interacts with Jab1/CSN5 and Regulates Its Function.

Methionine sulfoxide (MetO) is an oxidative posttranslational modification that primarily occurs under oxidative stress conditions, leading to alteration of protein structure and function. This modification is regulated by MetO reduction through the evolutionarily conserved methionine sulfoxide reductase (Msr) system. The Msr type A enzyme (MsrA) plays an important role as a cellular antioxidant and promotes cell survival. The ubiquitin- (Ub) like neddylation pathway, which is controlled by the c-Jun activation domain-binding protein-1 (Jab1), also affects cell survival. Jab1 negatively regulates expression of the cell cycle inhibitor cyclin-dependent kinase inhibitor 1B (P27) through binding and targeting P27 for ubiquitination and degradation. Here we report the finding that MsrA interacts with Jab1 and enhances Jab1's deneddylase activity (removal of Nedd8). In turn, an increase is observed in the level of deneddylated Cullin-1 (Cul-1, a component of E3 Ub ligase complexes). Furthermore, the action of MsrA increases the binding affinity of Jab1 to P27, while MsrA ablation causes a dramatic increase in P27 expression. Thus, an interaction between MsrA and Jab1 is proposed to have a positive effect on the function of Jab1 and to serve as a means to regulate cellular resistance to oxidative stress and to enhance cell survival.

Specific inhibition of hypoxia inducible factor 1 exaggerates cell injury induced by in vitro ischemia through deteriorating cellular redox environment.

Hypoxia inducible factor 1 (HIF-1) has been suggested to play a critical role in the fate of cells exposed to hypoxic stress. However, the mechanism of HIF-1-regulated cell survival is still not fully understood in ischemic conditions. Redox status is critical for decisions of cell survival, death and differentiation. We investigated the effects of inhibiting HIF-1 on cellular redox status in SH-SY5Y cells exposed to hypoxia or oxygen and glucose deprivation (OGD), coupled with cell death analyses. Our results demonstrated that inhibiting HIF-1alpha expression by HIF-1alpha specific small interfering RNA (siRNA) transfection increased reactive oxygen species generation, and transformed the cells to more oxidizing environments (low GSH/GSSG ratio, low NADPH level) under either hypoxic or OGD exposure. Cell death increased dramatically in the siRNA transfected cells, compared to non-transfected cells after hypoxic/OGD exposures. In contrast, increasing HIF-1alpha expression by desferrioxamine, a metal chelator and hydroxylase inhibitor, induced a more reducing environment (high GSH/GSSG ratio, high NADPH level) and reduced cell death. Further studies showed that HIF-1 regulated not only glucose transporter-1 expression, but also the key enzymes of the pentose phosphate pathway such as glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. These enzymes are important in maintaining cellular redox homeostasis by generating NADPH, the primary reducing agent in cells. Moreover, catalase significantly decreased cell death in the siRNA-transfected cells induced by hypoxia and OGD. These results suggest that maintenance of cellular redox status by HIF-1 protects cells from hypoxia and ischemia mediated injuries.

Dual Effects of Chinese Herbal Medicines on Angiogenesis in Cancer and Ischemic Stroke Treatments: Role of HIF-1 Network.

Hypoxia-inducible factor-1 (HIF-1)-induced angiogenesis has been involved in numerous pathological conditions, and it may be harmful or beneficial depending on the types of diseases. Exploration on angiogenesis has sparked hopes in providing novel therapeutic approaches on multiple diseases with high mortality rates, such as cancer and ischemic stroke. The HIF-1 pathway is considered to be a major regulator of angiogenesis. HIF-1 seems to be involved in the vascular formation process by synergistic correlations with other proangiogenic factors in cancer and cerebrovascular disease. The regulation of HIF-1-dependent angiogenesis is related to the modulation of HIF-1 bioactivity by regulating HIF-1α transcription or protein translation, HIF-1α DNA binding, HIF-1α and HIF-1α dimerization, and HIF-1 degradation. Traditional Chinese herbal medicines have a long history of clinical use in both cancer and stroke treatments in Asia. Growing evidence has demonstrated potential proangiogenic benefits of Chinese herbal medicines in ischemic stroke, whereas tumor angiogenesis could be inhibited by the active components in Chinese herbal medicines. The objective of this review is to provide comprehensive insight on the effects of Chinese herbal medicines on angiogenesis by regulating HIF-1 pathways in both cancer and ischemic stroke.

A network pharmacology-based study on the anti-hepatoma effect of Radix Salviae Miltiorrhizae.

BACKGROUND: Radix Salviae Miltiorrhizae (RSM), a well-known traditional Chinese medicine, has been shown to inhibit tumorigenesis in various human cancers. However, the anticancer effects of RSM on human hepatocellular carcinoma (HCC) and the underlying mechanisms of action remain to be fully elucidated. METHODS: In this study, we aimed to elucidate the underlying molecular mechanisms of RSM in the treatment of HCC using a network pharmacology approach. In vivo and in vitro experiments were also performed to validate the therapeutic effects of RSM on HCC. RESULTS: In total, 62 active compounds from RSM and 72 HCC-related targets were identified through network pharmacological analysis. RSM was found to play a critical role in HCC via multiple targets and pathways, especially the EGFR and PI3K/AKT signaling pathways. In addition, RSM was found to suppress HCC cell proliferation, and impair cancer cell migration and invasion in vitro. Flow cytometry analysis revealed that RSM induced cell cycle G2/M arrest and apoptosis, and western blot analysis showed that RSM up-regulated the expression of BAX and down-regulated the expression of Bcl-2 in MHCC97-H and HepG2 cells. Furthermore, RSM administration down-regulated the expression of EGFR, PI3K, and p-AKT proteins, whereas the total AKT level was not altered. Finally, the results of our in vivo experiments confirmed the therapeutic effects of RSM on HCC in nude mice. CONCLUSIONS: We provide an integrative network pharmacology approach, in combination with in vitro and in vivo experiments, to illustrate the underlying therapeutic mechanisms of RSM action on HCC.

Glucose up-regulates HIF-1 alpha expression in primary cortical neurons in response to hypoxia through maintaining cellular redox status.

It has been suggested that hypoxia-inducible factor 1 (HIF-1), a key regulator in cell's adaptation to hypoxia, plays an important role in the fate of neurons during ischemia. However, the mechanism of HIF-1 regulation is still not fully understood in neurons subjected to ischemia. In this study, we demonstrated that glucose up-regulated the expression of HIF-1alpha, the oxygen-dependent subunit of HIF-1, in rat primary cortical neurons exposed to hypoxia. To understand the mechanism of glucose-regulated HIF-1alpha expression, we investigated the relationships between HIF-1alpha expression, reactive oxygen species (ROS), and redox status. Low levels of HIF-1alpha protein expression were observed in the neurons exposed to in vitro ischemic conditions that had high levels of ROS (oxidizing environments), and vice versa. The glutathione (GSH) precursor, N-acetyl cysteine, induced HIF-1alpha protein expression in hypoxic neurons while the GSH synthesis inhibitor, l-buthionine sulfoximine, inhibited the expression. Moreover, (-)-epicatechin gallate, a ROS scavenger, elevated HIF-1alpha expression in the neurons subjected to in vitro ischemia. Furthermore, results from a systemic hypoxia model showed that a reducing environment increased HIF-1alpha expression in rat brains. Taken together, these data presented the first evidence that glucose promoted HIF-1alpha stabilization through regulating redox status in primary neurons exposed to hypoxia. The results imply that hypoxia only may not be sufficient to stabilize HIF-1alpha and that a reducing environment is required to stabilize HIF-1alpha in neurons exposed to hypoxia.

A reducing environment stabilizes HIF-2alpha in SH-SY5Y cells under hypoxic conditions.

Accumulating evidence suggests that hypoxia-inducible factor-2 (HIF-2) is important for the cellular response to hypoxia. However, it is not clear how HIF-2 is regulated under hypoxic conditions. We investigated kinetic changes in redox status and HIF-2alpha accumulation in hypoxic SH-SY5Y cells. Our results demonstrated that hypoxia caused a reducing environment and increased HIF-2alpha protein levels. Experiments with redox modulations (N-acetylcysteine and l-buthionine sulfoximine) confirmed that a reducing environment induced HIF-2alpha accumulation while an oxidizing environment decreased it. In addition, experiments with SOD mimic, catalase, and exogenous H2O2 provided evidence that the presence of H2O2 down-regulated the amount of HIF-2alpha protein. This study offers novel evidence supporting redox status regulation of HIF-2alpha accumulation under hypoxic conditions.

Nano-liposomes of lycopene reduces ischemic brain damage in rodents by regulating iron metabolism.

In order to discover new drug delivery approaches and to understand the mechanism of iron overload in cerebral ischemia/reperfusion (I/R), we aimed to investigate the effects of lycopene (LYC) in the form of nano-liposomes (L-LYC) on iron-regulating proteins and ischemic brain injury. We found that L-LYC significantly increased the LYC content in serum and the brain. Adult male Sprague-Dawley rats treated with L-LYC for 14 days were subjected to 60 min of ischemia and 7 days of reperfusion. The effects of L-LYC were evaluated by infarction volume, neurological score, neuronal apoptosis, and markers for oxidative stress. Levels of iron-regulating protein such as hepcidin and ferroportin (FPN1) were examined. L-LYC reduced cerebral infarction and improved neurobehavior of the rats more efficiently than "naked" LYC. L-LYC reduced protein levels of oxidases (e.g. nitric oxide synthase and NOX2), increased the level of Bcl-2, lowered caspase-3, and suppressed apoptosis through inhibiting MAPK-JNK. Furthermore, L-LYC suppressed hepcidin-mediated decrease in FPN1, a sole iron exporter, and normalized the levels of iron. We further demonstrated that the effect of L-LYC on hepcidin expression might result from its ability to attenuate the release of the inflammatory factor interleukin 6. The results demonstrated that nano-liposomal encapsulation significantly improved LYC efficacy in providing neuronal protection against I/R injury. The data also revealed a novel mechanism of L-LYC's neuroprotection by regulating iron metabolism in an ischemic brain.

Cerebral tissue oxygenation and oxidative brain injury during ischemia and reperfusion.

The brain requires glucose and oxygen to maintain neuronal metabolism and function. Cerebral ischemia causes heterogeneous changes in tissue oxygenation and cellular metabolism, with a region of decreased blood flow, the penumbra, surrounding a severely damaged ischemic core. Because oxygenation is central in ischemic neuronal death, it is critical to understand exactly what actual changes occur in interstitial oxygen tension (pO2) in ischemic regions during stroke, particularly the penumbra and ischemic core. Cerebral ischemia induces a complex series of molecular pathways involving signaling mechanisms, gene transcription, and protein formation. Free radicals and oxidative stress have been suggested to be involved in each of the steps in the injury cascade. The goal of this review paper is to summarize the current literature concerning our understanding about cerebral tissue oxygenation changes after cerebral ischemia and reperfusion, the subsequent cellular and physiological changes in response to alteration in tissue oxygenation, and treatment strategies utilized to minimize the detrimental effects caused by stroke.

Heat shock protein 70 regulates cellular redox status by modulating glutathione-related enzyme activities.

Heat shock protein (Hsp) 70 has been reported to protect various cells and tissues from ischemic damage. However, the molecular mechanisms of the protection are incompletely understood. Ischemia induces significant alterations in cellular redox status that plays a critical role in cell survival/death pathways. We investigated the effects of Hsp70 overexpression on cellular redox status in Madin-Darby canine kidney (MDCK) cells under both hypoxic and ischemic conditions with 3 different approaches: reactive oxygen species (ROS) measurement by a fluorescence probe, redox environment evaluation by a hydroxylamine spin probe, and redox status assessment by the glutathione/glutathione disulfide (GSH/GSSG) ratio. Results from each of these approaches showed that the redox status in Hsp70 cells was more reducing than that in control cells under either hypoxic or oxygen and glucose deprivation (OGD) conditions. In order to determine the mechanisms that mediated the alterations in redox state in Hsp70 cells, we measured the activities of glutathione peroxidase (GPx) and glutathione reductase (GR), two GSH-related antioxidant enzymes. We found that OGD exposure increased GPx and GR activities 47% and 55% from their basal levels (no stress) in Hsp70 cells, compared to only 18% and 0% increase in control cells, respectively. These data, for the first time, indicate that Hsp70 modulates the activities of GPx and GR that regulate cellular redox status in response to ischemic stress, which may be important in Hsp70's cytoprotective effects.