PRDX1 Interfering Peptide Disrupts Amino Acids 70-90 of PRDX1 to Inhibit the TLR4/NF-κB Signaling Pathway and Attenuate Neuroinflammation and Ischemic Brain Injury.

Ischemic stroke ranks among the leading causes of death and disability in humans and is accompanied by motor and cognitive impairment. However, the precise mechanisms underlying injury after stroke and effective treatment strategies require further investigation. Peroxiredoxin-1 (PRDX1) triggers an extensive inflammatory cascade that plays a pivotal role in the pathology of ischemic stroke, resulting in severe brain damage from activated microglia. In the present study, we used molecular dynamics simulation and nuclear magnetic resonance to detect the interaction between PRDX1 and a specific interfering peptide. We used behavioral, morphological, and molecular experimental methods to demonstrate the effect of PRDX1-peptide on cerebral ischemia-reperfusion (I/R) in mice and to investigate the related mechanism. We found that PRDX1-peptide bound specifically to PRDX1 and improved motor and cognitive functions in I/R mice. In addition, pretreatment with PRDX1-peptide reduced the infarct area and decreased the number of apoptotic cells in the penumbra. Furthermore, PRDX1-peptide inhibited microglial activation and downregulated proinflammatory cytokines including IL-1ß, IL-6, and TNF-α through inhibition of the TLR4/NF-κB signaling pathway, thereby attenuating ischemic brain injury. Our findings clarify the precise mechanism underlying PRDX1-induced inflammation after ischemic stroke and suggest that the PRDX1-peptide can significantly alleviate the postischemic inflammatory response by interfering with PRDX1 amino acids 70-90 and thereby inhibiting the TLR4/NF-κB signaling pathway. Our study provides a theoretical basis for a new therapeutic strategy to treat ischemic stroke.

TAT-W61 peptide attenuates neuronal injury through blocking the binding of S100b to the V-domain of Rage during ischemic stroke.

Ischemic stroke is a devastative nervous system disease associated with high mortality and morbidity rates. Unfortunately, no clinically effective neuroprotective drugs are available now. In ischemic stroke, S100 calcium-binding protein b (S100b) binds to receptor for advanced glycation end products (Rage), leading to the neurological injury. Therefore, disruption of the interaction between S100B and Rage can rescue neuronal cells. Here, we designed a peptide, termed TAT-W61, derived from the V domain of Rage which can recognize S100b. Intriguingly, TAT-W61 can reduce the inflammatory caused by ischemic stroke through the direct binding to S100b. The further investigation demonstrated that TAT-W61 can improve pathological infarct volume and reduce the apoptotic rate. Particularly, TAT-W61 significantly improved the learning ability, memory, and motor dysfunction of the mouse in the ischemic stroke model. Our study provides a mechanistic insight into the abnormal expression of S100b and Rage in ischemic stroke and yields an invaluable candidate for the development of drugs in tackling ischemic stroke. KEY MESSAGES: S100b expression is higher in ischemic stroke, in association with a high expression of many genes, especially of Rage. S100b is directly bound to the V-domain of Rage. Blocking the binding of S100b to Rage improves the injury after ischemic stroke.

Receptor for advanced glycation end products aggravates cognitive deficits in type 2 diabetes through binding of C-terminal AAs 2-5 to mitogen-activated protein kinase kinase 3 (MKK3) and facilitation of MEKK3-MKK3-p38 module assembly.

In this study, we explored the precise mechanisms underlying the receptor for advanced glycation end products (RAGE)-mediated neuronal loss and behavioral dysfunction induced by hyperglycemia. We used immunoprecipitation (IP) and GST pull-down assays to assess the interaction between RAGE and mitogen-activated protein kinase kinase 3 (MKK3). Then, we investigated the effect of specific mutation of RAGE on plasticity at hippocampal synapses and behavioral deficits in db/db mice through electrophysiological recordings, morphological assays, and behavioral tests. We discovered that RAGE binds MKK3 and that this binding is required for assembly of the MEKK3-MKK3-p38 signaling module. Mechanistically, we found that activation of p38 mitogen-activated protein kinase (MAPK)/NF-κB signaling depends on mediation of the RAGE-MKK3 interaction by C-terminal RAGE (ctRAGE) amino acids (AAs) 2-5. We found that ctRAGE R2A-K3A-R4A-Q5A mutation suppressed neuronal damage, improved synaptic plasticity, and alleviated behavioral deficits in diabetic mice by disrupting the RAGE-MKK3 conjugation. High glucose induces direct binding of RAGE and MKK3 via ctRAGE AAs 2-5, which leads to assembly of the MEKK3-MKK3-p38 signaling module and subsequent activation of the p38MAPK/NF-κB pathway, and ultimately results in diabetic encephalopathy (DE).

H2S attenuates injury after ischemic stroke by diminishing the assembly of CaMKII with ASK1-MKK3-p38 signaling module.

Cerebral ischemia/reperfusion (I/R) injury is a leading cause of learning and memory dysfunction. Hydrogen sulfide (H2S) has been shown to confer neuroprotection in various neurodegenerative diseases, including cerebral I/R-induced hippocampal CA1 injury. However, the underlying mechanisms have not been completely understood. In the present study, rats were pretreated with SAM/NaHS (SAM, an H2S agonist, and NaHS, an H2S donor) only or SAM/NaHS combined with CaM (an activator of CaMKII) prior to cerebral ischemia. The Morris water maze test demonstrated that SAM/NaHS could alleviate learning and memory impairment induced by cerebral I/R injury. Cresyl violet staining was used to show the survival of hippocampal CA1 pyramidal neurons. SAM/NaHS significantly increased the number of surviving cells, whereas CaM weakened the protection induced by SAM/NaHS. The immunohistochemistry results indicated that the number of Iba1-positive microglia significantly increased after cerebral I/R. Compared with the I/R group, the number of Iba1-positive microglia in the SAM/NaHS groups significantly decreased. Co-Immunoprecipitation and immunoblotting were conducted to demonstrate that SAM/NaHS suppressed the assembly of CaMKII with the ASK1-MKK3-p38 signal module after cerebral I/R, which decreased the phosphorylation of p38. In contrast, CaM significantly inhibited the effects of SAM/NaHS. Taken together, the results suggested that SAM/NaHS could suppress cerebral I/R injury by downregulating p38 phosphorylation via decreasing the assembly of CaMKII with the ASK1-MKK3-p38 signal module.

Design, Preparation, and Characterization of Novel Calix[4]arene Bioactive Carrier for Antitumor Drug Delivery.

An amphiphilic and bioactive calix[4]arene derivative 8 (CA) is designed and successfully synthesized from tert-butyl calix[4] arene 1 by sequential inverse F-C alkylation, nitration, O-alkylation, esterification, aminolysis, reduction, and acylation reaction. The blank micelles of FA-CA and doxorubicin (DOX) loaded micelles FA-CA-DOX are prepared subsequently undergoing self-assembly and dialysis of CA and DSPE-PEG2000-FA. The drug release kinetics curve of the encapsulated-DOX micelle demonstrates a rapid release under mild conditions, indicating the good pH-responsive ability. Furthermore, the cytotoxicity of DOX-loaded micelle respect to the blank micelle against seven different human carcinoma (A549, HeLa, HepG2, HCT116, MCF-7, MDA-MB231, and SW480) cells has been also investigated. The results confirm the more significant inhibitory effect of DOX-loaded micelle than those of DOX and the blank micelles. The CDI calculations show a synergistic effect between blank micelles and DOX in inducing tumor cell death. In conclusion, FA-CA micelles reported in this work was a promising drug delivery vehicle for tumor targeting therapy.

The potential role of HO-1 in regulating the MLK3-MKK7-JNK3 module scaffolded by JIP1 during cerebral ischemia/reperfusion in rats.

Heme oxygenase (HO-1), which may be induced by Cobaltic protoporphyrin IX chloride (CoPPIX) or Rosiglitazone (Ros), is a neuroprotective agent that effectively reduces ischemic stroke. Previous studies have shown that the neuroprotective mechanisms of HO-1 are related to JNK signaling. The expression of HO-1 protects cells from death through the JNK signaling pathway. This study aimed to ascertain whether the neuroprotective effect of HO-1 depends on the assembly of the MLK3-MKK7-JNK3 signaling module scaffolded by JIP1 and further influences the JNK signal transmission through HO-1. Prior to the ischemia-reperfusion experiment, CoPPIX was injected through the lateral ventricle for 5 consecutive days or Ros was administered via intraperitoneal administration in the week prior to transient ischemia. Our results demonstrated that HO-1 could inhibit the assembly of the MLK3-MKK7-JNK3 signaling module scaffolded by JIP1 and could ultimately diminish the phosphorylation of JNK3. Furthermore, the inhibition of JNK3 phosphorylation downregulated the level of p-c-Jun and elevated neuronal cell death in the CA1 of the hippocampus. Taken together, these findings suggested that HO-1 could ameliorate brain injury by regulating the MLK3-MKK7-JNK3 signaling module, which was scaffolded by JIP1 and JNK signaling during cerebral ischemia/reperfusion.

Inhibition of iNOS alleviates cognitive deficits and depression in diabetic mice through downregulating the NO/sGC/cGMP/PKG signal pathway.

Diabetes mellitus often results in a number of complications involving impaired brain function, including cognitive deficits and depression. However, the potential mechanisms for diabetes-related cognitive deficits and depression are not fully understood. Neurons in the hippocampal, cortical and amygdala functional regions are more susceptible to damage during hyperglycemia. Neuroprotection in the brain can rescue cognitive deficits and depression induced by hyperglycemia. This study investigated the potential mechanisms underlying diabetes-related congnitive deficits and depression, determined whether the inflammatory factor inducible nitric oxide synthase (iNOS) and the nitric oxide (NO)/soluble guanylyl cyclases (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) pathway, play key roles in cognitive deficits and depression associated. In the present study, diabetic animal models were induced by streptozotocin (STZ, 150mg/kg) in mice, and aminoguanidine (AG), a selective inhibitor of iNOS, was given by intraperitoneal injection for 10 weeks. Blood glucose, activities of NOS and the levels of NO in serum and brain regions were measured. The spatial memory was detected using the Morris water maze test, depressive behavior was evaluated by the tail suspension test (TST), forced swimming test (FST), closed field test (CFT) and open field test (OFT). We also detected neuronal survival and cleaved caspase-3 positive ratios in three brain regions and the levels of iNOS, sGC, cGMP and PKG in hippocampus and frontal cortex. Data indicated that diabetic mice exerted impairments in spatial memory, decreased locomotor activity and increased immobile time in diabetic mice. In addition, diabetic mice had significantly decreased surviving neuronal density and showed signs of obvious neuronal injury in the hippocampus, frontal cortex and amygdala. iNOS overexpression and its associated signaling pathway NO/sGC/cGMP/PKG in the hippocampus and frontal cortex were implicated during hyperglycemia. However, AG improved the behavior disorders, reduced the activity of iNOS, protected nerve cells and inhibited the level of iNOS, sGC, PKG and cleaved caspase-3 in the hippocampus and cortex. These results suggested that iNOS/NO/sGC/cGMP/PKG signal pathway is a key feature of cognitive deficits and depression associated with diabetes. AG ameliorated cognitive deficits and depression in diabetic mice by exerting anti-inflammatory and neuroprotective effects by suppressing iNOS-associated signaling pathways.

Depression can be prevented by astaxanthin through inhibition of hippocampal inflammation in diabetic mice.

The critical factor considered in a depression induced by diabetes is the inflammation eliciting hippocampal, amygdala and thalamic neuronal injury. Therefore, inhibiting inflammatory reactions in the brain and reducing neuronal injury can alleviate depression in rodents suffering from diabetes mellitus. The oral administration of astaxanthin has been employed in emotional disorders and diabetic complications due to its anti-depressant, anti-inflammatory and anti-apoptotic functions. However, it has not been reported whether astaxanthin can improve diabetes-related depression-like behavior, and its potential mechanisms have not been elucidated. The objective of the present study is to elucidate the effect of astaxanthin on depression in diabetic mice and to understand the underlying molecular mechanisms. In this study, experimental diabetic mice were given a single intraperitoneal injection of streptozotocin (STZ, 150mg/kg, dissolved in citrate buffer) after fasting for 12h. The diabetic model was assessed 72h after STZ injection, and mice with a fasting blood glucose level more than or equal to 16.7mmol/L were used in this study, and oral astaxanthin (25mg/kg) was provided uninterrupted for ten weeks. Depression-like behavior was evaluated by the tail suspension test (TST) and forced swimming test (FST). The glial fibrillary acidic protein (GFAP) and cleaved caspase-3-positive cells were measured by immunohistochemistry, and the western blotting was used to test the protein levels of interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and cyclooxygenase (COX-2). The results showed that astaxanthin had an anti-depressant effect on diabetic mice. Furthermore, we observed that astaxanthin significantly reduced the number of GFAP-positive cells in the hippocampus and hypothalamus, and also the expression of cleaved caspase-3 in the hippocampus, amygdala and hypothalamus was decreased as well. Moreover, astaxanthin could down-regulate the expression of IL-6, IL-1ß and COX-2 in the hippocampus. These findings suggest that the mechanism of astaxanthin in preventing depression in diabetic mice involves the inhibition of inflammation/inflammation inhibition, thereby protecting neurons in hippocampus, amygdala and hypothalamus against hyperglycemic damage.

Methylene blue improves streptozotocin-induced memory deficit by restoring mitochondrial function in rats.

The pathogenesis of Alzheimer's disease (AD) is well documented to involve mitochondrial dysfunction which causes subsequent oxidative stress and energy metabolic failure in hippocampus. Methylene blue (MB) has been implicated to be neuroprotective in a variety of neurodegenerative diseases by restoring mitochondrial function. The present work was to examine if MB was able to improve streptozotocin (STZ)-induced Alzheimer's type dementia in a rat model by attenuating mitochondrial dysfunction-derived oxidative stress and ATP synthesis decline. MB was administrated at a dose of 0.5mg/kg/day for consecutive 7days after bilateral STZ intracerebroventricular (ICV) injection (2.5mg/kg). We first demonstrated that MB treatment significantly ameliorated STZ-induced hippocampus-dependent memory loss in passive avoidance test. We also found that MB has the properties to preserve neuron survival and attenuate neuronal degeneration in hippocampus CA1 region after STZ injection. In addition, oxidative stress was subsequently evaluated by measuring the content of lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). Importantly, results from our study showed a remarkable suppression of MB treatment on both MDA production and 4-HNE immunoactivity. Finally, energy metabolism in CA1 region was examined by detecting mitochondrial cytochrome c oxidase (CCO) activity and the resultant ATP production. Of significant interest, our result displayed a robust facilitation of MB on CCO activity and the consequent ATP synthesis. The current study indicates that MB may be a promising therapeutic agent targeting oxidative damage and ATP synthesis failure during AD progression.

Butylphthalide Suppresses Neuronal Cells Apoptosis and Inhibits JNK-Caspase3 Signaling Pathway After Brain Ischemia /Reperfusion in Rats.

Although Butylphthalide (BP) has protective effects that reduce ischemia-induced brain damage and neuronal cell death, little is known about the precise mechanisms occurring during cerebral ischemia/reperfusion (I/R). Therefore, the aim of this study was to investigate the neuroprotective mechanisms of BP against ischemic brain injury induced by cerebral I/R through inhibition of the c-Jun N-terminal kinase (JNK)-Caspase3 signaling pathway. BP in distilled non-genetically modified Soybean oil was administered intragastrically three times a day at a dosage of 15 mg/(kg day) beginning at 20 min after I/R in Sprague-Dawley rats. Immunohistochemical staining and Western blotting were performed to examine the expression of related proteins, and TUNEL-staining was used to detect the percentage of neuronal apoptosis in the hippocampal CA1 region. The results showed that BP could significantly protect neurons against cerebral I/R-induced damage. Furthermore, the expression of p-JNK, p-Bcl2, p-c-Jun, FasL, and cleaved-caspase3 was also decreased in the rats treated with BP. In summary, our results imply that BP could remarkably improve the survival of CA1 pyramidal neurons in I/R-induced brain injury and inhibit the JNK-Caspase3 signaling pathway.

Ischemic preconditioning protects the brain against injury via inhibiting CaMKII-nNOS signaling pathway.

Although studies have shown that cerebral ischemic preconditioning (IPC) can ameliorate ischemia/reperfusion (I/R) induced brain damage, but its precise mechanisms remain unknown. Therefore, the aim of this study was to investigate the neuroprotective mechanisms of IPC against ischemic brain damage induced by cerebral I/R and to explore whether the Calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated up-regulation of nNOS ser847-phosphorylation signaling pathway contributed to the protection provided by IPC. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The rats were pretreated with 3 min of IPC alone or KN62 (selective antagonist of CaMKII) treatment before IPC, after reperfusion for 3 days, 6 min ischemia was induced. Cresyl violet staining was used to examine the survival of hippocampal CA1 pyramidal neurons. Immunoblotting was performed to measure the phosphorylation of CaMKII, nNOS, c-Jun and the expression of FasL. Immunoprecipitation was used to examine the binding between PSD95 and nNOS. The results showed that IPC could significantly protect neurons against cerebral I/R injury, furthermore, the combination of PSD95 and nNOS was increased, coinstantaneously the phosphorylation of CaMKII and nNOS (ser847) were up-regulated, however the activation of c-Jun and FasL were reduced. Conversely, KN62 treatment before IPC reversed all these effects of IPC. Taken together, the results suggest that IPC could diminish ischemic brain injury through CaMKII-mediated up-regulation of nNOS ser847-phosphorylation signaling pathway.

The complete mitochondrial genome of the Fossorochromis rostratus.

The Fossorochromis rostratus is a species of cichlid endemic to Lake Malawi in East Africa. In this study, we first reported the complete mitochondrial genome of F. rostratus. The whole mitochondrial genome is 16 581 bp in length, which contains 13 protein-coding genes, 22 transfer RNA genes, and two ribosomal RNA genes. The GC content of this mitochondrial genome is 45.96% (27.47% A, 26.57%T, 30.12% C, and 15.84% G), similar to Astatotilapia calliptera (the GC content of 45.90%). We constructed a phylogenetic tree on the complete mitochondrial genomes of these two species and other 10 closely related species to show their phylogenic relationship. The complete mitochondrial genome of F. rostratus and its phylogenic relationship with other related species would facilitate our understanding of the evolution of Cichlidae mitochondrial genome.

Neuroprotection of Sevoflurane Against Ischemia/Reperfusion-Induced Brain Injury Through Inhibiting JNK3/Caspase-3 by Enhancing Akt Signaling Pathway.

In this study, we investigated the neuroprotective effect of sevoflurane against ischemic brain injury and its underlying molecular mechanisms. Transient global brain ischemia was induced by 4-vessel occlusion in adult male Sprague-Dawley rats. The rats were pretreated with sevoflurane alone or sevoflurane combined with LY294002/wortmannin (selective inhibitor of PI3K) before ischemia. Cresyl violet staining was used to examine the survival of hippocampal CA1 pyramidal neurons. Immunoblotting and immunoprecipitation were performed to measure the phosphorylation of Akt1, PRAS40, ASK1, and JNK3 and the expression of cleaved-caspase-3. The results demonstrated that a moderate dose of sevoflurane inhalation of 2% for 2 h had significant neuroprotective effects against ischemia/reperfusion induced hippocampal neuron death. Sevoflurane significantly increased Akt and PRAS40 phosphorylation and decreased the phosphorylation of ASK1 at 6 h after reperfusion and the phosphorylation of JNK3 at 3 days after reperfusion following 15 min of transient global brain ischemia. Conversely, LY294002 and wortmannin significantly inhibited the effects of sevoflurane. Taken together, the results suggest that sevoflurane could suppress ischemic brain injury by downregulating the activation of the ASK1/JNK3 cascade via increasing the phosphorylation of Akt1 during ischemia/reperfusion.

Anti-inflammatory Effect of Astaxanthin on the Sickness Behavior Induced by Diabetes Mellitus.

Chronic inflammation appears to play a critical role in sickness behavior caused by diabetes mellitus. Astaxanthin has been used in treating diabetes mellitus and diabetic complications because of its neuroprotective and anti-inflammatory actions. However, whether astaxanthin can improve sickness behavior induced by diabetes and its potential mechanisms are still unknown. The aim of this study was to investigate the effects of astaxanthin on diabetes-elicited abnormal behavior in mice and its corresponding mechanisms. An experimental diabetic model was induced by streptozotocin (150 mg/kg) and astaxanthin (25 mg/kg/day) was provided orally for 10 weeks. Body weight and water consumption were measured, and the sickness behavior was evaluated by the open field test (OFT) and closed field test (CFT). The expression of glial fibrillary acidic protein (GFAP) was measured, and the frontal cortical cleaved caspase-3 positive cells, interleukin-6 (IL-6), and interleukin-1ß (IL-1ß) expression levels were also investigated. Furthermore, cystathionine ß-synthase (CBS) in the frontal cortex was detected to determine whether the protective effect of astaxanthin on sickness behavior in diabetic mice is closely related to CBS. As expected, we observed that astaxanthin improved general symptoms and significantly increase horizontal distance and the number of crossings in the OFT and CFT. Furthermore, data showed that astaxanthin could decrease GFAP-positive cells in the brain and down-regulate the cleaved caspase-3, IL-6, and IL-1ß, and up-regulate CBS in the frontal cortex. These results suggest that astaxanthin provides neuroprotection against diabetes-induced sickness behavior through inhibiting inflammation, and the protective effects may involve CBS expression in the brain.

Mangiferin regulates interleukin-6 and cystathionine-b-synthase in lipopolysaccharide-induced brain injury.

Mangiferin has been extensively applied in different fields due to its anti-inflammatory properties. However, the precise mechanism used by mangiferin on lipopolysaccharide (LPS)-induced inflammation has not been elucidated. Here, we discuss the potential mechanism of mangiferin during a LPS-induced brain injury. Brain injury was induced in ICR mice via intraperitoneal LPS injection (5 mg/kg). Open- and closed-field tests were used to detect the behaviors of mice, while immunoblotting was performed to measure the expression of interleukin-6 (IL-6) and cystathionine-b-synthase (CBS) in the hippocampus after mangiferin was orally administered (p.o.). Mangiferin relieved LPS-induced sickness 6 and 24 h after LPS injection; in addition, this compound suppressed LPS-induced IL-6 production after 24 h of LPS induction as well as the downregulation of LPS-induced CBS expression after 6 and 24 h of LPS treatment in the hippocampus. Therefore, mangiferin attenuated sickness behavior by regulating the expression of IL-6 and CBS.

Heme oxygenase-1 regulates the JNK signaling pathway through the MLK3-MKK7-JNK3 signaling module in brain ischemia injury.

Although previous researches indicated that heme oxygenase-1 (HO-1) plays a conspicuous role in neuronal injury induced by reperfusion following the brain ischemia, reasonable mechanisms for the role of HO-1 are not clear. In this work, we investigated whether HO-1 was involved in the regulation of the c-Jun N-terminal kinase (JNK) signaling pathway and neuronal cell injury induced by the brain ischemia followed by reperfusion. Cobaltic protoporphyrin (CoPP), an activator of HO-1, was administrated to induce the overexpression of HO-1 by intracerebroventricular infusion 20 min before ischemia. The results showed that the combination of HO-1-mixed lineage kinase 3 (MLK3), MLK3-mitogen-activated kinase kinase 7 (MKK7) and MKK7-JNK3 increased to a peak at 6h of reperfusion following 15 min of ischemia induced by four-vessel occlusion in rats, and these effects were downregulated by CoPP. In addition, CoPP could inhibit the activation of JNK3, c-Jun and caspase-3. Furthermore, pretreatment with CoPP significantly increased the survival of neurons after 5 days of reperfusion. In contrast, all of the above effects of CoPP were reversed by zinc protoporphyrin (ZnPP), a selective inhibitor of HO-1. Our results suggested that HO-1 could protect neurons against brain ischemic injury by downregulating the JNK signaling pathway through the MLK3-MKK7-JNK3 signaling module.

Inhibition of cerebral ischemia/reperfusion-induced injury by adenovirus expressed C-terminal amino acids of GluR6.

GluR6 kainate receptor subunit is largely expressed in hippocampus of brain regions and plays an important role in brain ischemia/reperfusion-mediated neuronal cell death. Our previous researches have shown that cerebral ischemia/reperfusion could facilitate the assembly of GluR6 and postsynaptic density protein 95(PSD95) as well as mixed lineage kinase 3(MLK3) and further induce the activation of c-Jun NH2-terminal kinase 3(JNK3), leading to neuronal death of hippocampal CA1. Here, we show that over-expression of C-terminal amino acids of GluR6 can interrupt the combination of GluR6 with PSD95, inhibit the assembly of GluR6.PSD-95.MLK3 signaling module, suppress the activation of JNK3 and the downstream signaling pathway. Thus, our results imply that over-expression of C-terminal amino acids of GluR6 induce neuroprotection against ischaemic brain injury in rat hippocampal CA1 region via suppressing proapoptosis signaling pathways, which can be an experimental foundation for gene therapy of stroke.

S-nitrosylation of PTEN Invovled in ischemic brain injury in rat hippocampal CA1 region.

The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) is not only a protein, but also a lipid phosphatase that can negatively regulate the serine/threonine kinase Akt. It has been reported that PTEN can be regulated by means of phosphorylation. However, whether PTEN can be regulated by another post-translational protein modification (S-nitrosylation) was not fully elucidated. In this study, we investigated the S-nitrosylation of PTEN during transient cerebral ischemia/reperfusion in rat hippocampus. Transient brain ischemia was induced by the four-vessel occlusion in Sprague-Dawley rats. Our data show that S-nitrosylation of PTEN was increased significantly after 12 h of reperfusion compared with sham control. Pretreatment with the inhibitor of nNOS (7-NI) and the inhibitor of iNOS could inhibit PTEN's activity and decrease S-nitrosylation of PTEN. Taken together, these results indicate that nitric oxide could regulate PTEN's activity via S-nitrosylation during transient global ischemia in rat hippocampus.

Exogenous nitric oxide negatively regulates c-Jun N-terminal kinase activation via inhibiting endogenous NO-induced S-nitrosylation during cerebral ischemia and reperfusion in rat hippocampus.

Nitric oxide (NO), synthesized from l-arginine by NO synthases, is a small endogenous free radical with multiple functions. The c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in mediating apoptosis in cerebral ischemia and reperfusion. In this study, we found that the NO donor sodium nitroprusside (SNP) can decrease the damage of hippocampal neurons induced by cerebral ischemia and reperfusion. Our current study demonstrates that SNP can suppress the phosphorylation of JNK3 by suppressing the increased S-nitrosylation of JNK3 induced by cerebral ischemia and reperfusion. In contrast, dithiothreitol reversed the effect of SNP on S-nitrosylation of JNK3. Furthermore, the inhibitor of nNOS (7-NI) and the inhibitor of iNOS (AMT) can decrease JNK3 phosphorylation through decreasing S-nitrosylation of JNK3. Our data suggest that endogenous NO synthesized by NO synthases can increase JNK3 phosphorylation by means of S-nitrosylation during global ischemia/reperfusion in rat hippocampus. However, the exogenous NO (SNP) can reverse the effect of endogenous NO by inhibiting S-nitrosylation of JNK3. Together, these results suggest that the exogenous NO may provide a new clue for stroke therapy.

Neuroprotection of hypothermia against neuronal death in rat hippocampus through inhibiting the increased assembly of GluR6-PSD95-MLK3 signaling module induced by cerebral ischemia/reperfusion.

Kainate receptor containing GluR6 subunit (KAR) is involved in the neuronal cell death induced by cerebral ischemia/reperfusion (I/R). Hypothermia is an effective neuroprotectant in brain ischemia, whereas the neuroprotective mechanisms have not been clearly established. The present study was set out to examine whether hypothermia would cause the alternation of the assembly of the GluR6-PSD95-MLK3 signaling module and the activation of c-Jun N-terminal kinase (JNK) pathway through KAR. Hypothermia (32 degrees C) was induced 10 min before ischemia and was maintained for 3 h after ischemia. Our results indicated that hypothermia could inhibit the assembly of GluR6-PSD95-MLK3 signaling module and suppressed the activation of MLK3, MKK4/7, and JNK3. The inhibition of JNK3 activation by hypothermia diminished the phosphorylation of the transcription factor c-Jun and downregulated FasL expression in hippocampal CA1. Meanwhile, the inhibition of JNK3 activation by hypothermia attenuated bax translocation, the release of cytochrome c, and the activation of caspase-3 in CA1 subfields. Both GluR6 antagonist NS102 and GluR6 antisense oligodeoxynucleotides partly blocked the aforementioned effects of hypothermia, which was further confirmed by histology. Taken together, our results strongly suggest that hypothermia decreased the increased assembly of the GluR6-PSD95-MLK3 signaling module and the activation of JNK pathway induced by I/R through KAR, which gave a new insight into the ischemic therapy.