Effects of Breviscapine on the Changes in Antioxidant Enzyme Activity Induced by Cerebral Ischemia-reperfusion in Rats

本文研究了灯盏花素对大鼠脑缺血再灌注引起抗氧化酶活性改变的影响，结果表明，灯盏花素明显提高脑缺血再灌注引起的脑组织超氧歧化酶（ＳＯＤ）、谷胱甘肽过氧化物酶（ＧＳＨｐｅｒｏｘｉｄａｓｅ）和过氧化氢酶（Ｃａｔａｌａｓｅ）的活性，减少脑组织丙二醛（ＭＤＡ）含量。这些作用有利于减轻脑缺血再灌注损伤。

Effects of L-Tetrahydropalmatine on the Expressions of bcl-2 and bax in Rat after Acute Global Cerebral Ischemia and Reperfusion

To investigate the effects of L-Tetrahydropalmatine (L-THP) on the expressions of bcl-2, bax and neuronal apoptosis after cerebral ischemia and reperfusion, 60 Wistars rats were randomly divided into 3 groups: sham-operation group (group S, n = 20), ischemic-reperfusion group treated with saline (group I, n=20) and ischemia-reperfusion group treated with L-THP (group T, n=20) .The rat model of global cerebral ischemia and reperfusion was induced by Pulsinelli's four-vessel occlusion method. The expression of bcl-2 and bax mRNA was detected by in situ hybridization and reverse transcriptional polymerase chain reaction (RT-PCR). The number of apoptotic neurons was examined by terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) method. Compared with group S, the expression of bcl-2 and bax mRNA in group I was increased significantly (P<0.01), and the number of apoptotic neurons increased either (P< 0.01). After L-THP treatment, the expression of bcl-2 mRNA was up-regulated (P<0.01) and that of bax mRNA was down-regulated (P<0.01); the number of apoptotic neurons was decreased (P<0.01). Our results indicated that bcl-2 may suppress apoptosis and bax promote apoptosis after cerebral ischemia and reperfusion. L-THP could ameliorate cerebral ischemia and reperfusion damage by reducing the apoptosis through regulating bcl-2 and bax.

A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

Vascular endothelial growth factor and its mimic peptide KLTWQELYQLKYKGI(QK)are widely used as the most potent angiogenic factors for the treatment of multiple ischemic diseases.However,conventional topical drug delivery often results in a burst release of the drug,leading to transient retention(inefficacy)and undesirable diffusion(toxicity)in vivo.Therefore,a drug delivery system that responds to changes in the microenvironment of tissue regeneration and controls vascular endothelial growth factor release is crucial to improve the treatment of ischemic stroke.Matrix metalloproteinase-2(MMP-2)is gradually upregulated after cerebral ischemia.Herein,vascular endothelial growth factor mimic peptide QK was self-assembled with MMP-2-cleaved peptide PLGLAG(TIMP)and customizable peptide amphiphilic(PA)molecules to construct nanofiber hydrogel PA-TIMP-QK.PA-TIMP-QK was found to control the delivery of QK by MMP-2 upregulation after cerebral ischemia/reperfusion and had a similar biological activity with vascular endothelial growth factor in vitro.The results indicated that PA-TIMP-QK promoted neuronal survival,restored local blood circulation,reduced blood-brain barrier permeability,and restored motor function.These findings suggest that the self-assembling nanofiber hydrogel PA-TIMP-QK may provide an intelligent drug delivery system that responds to the microenvironment and promotes regeneration and repair after cerebral ischemia/reperfusion injury.

Endoplasmic reticulum stress and autophagy in cerebral ischemia/reperfusion injury:PERK as a potential target for intervention

Several studies have shown that activation of unfolded protein response and endoplasmic reticulum(ER)stress plays a crucial role in severe cerebral ischemia/reperfusion injury.Autophagy occurs within hours after cerebral ischemia,but the relationship between ER stress and autophagy remains unclear.In this study,we established experimental models using oxygen-glucose deprivation/reoxygenation in PC12 cells and primary neurons to simulate cerebral ischemia/reperfusion injury.We found that prolongation of oxygen-glucose deprivation activated the ER stress pathway protein kinase-like endoplasmic reticulum kinase(PERK)/eukaryotic translation initiation factor 2 subunit alpha(e IF2α)-activating transcription factor 4(ATF4)-C/EBP homologous protein(CHOP),increased neuronal apoptosis,and induced autophagy.Furthermore,inhibition of ER stress using inhibitors or by si RNA knockdown of the PERK gene significantly attenuated excessive autophagy and neuronal apoptosis,indicating an interaction between autophagy and ER stress and suggesting PERK as an essential target for regulating autophagy.Blocking autophagy with chloroquine exacerbated ER stress-induced apoptosis,indicating that normal levels of autophagy play a protective role in neuronal injury following cerebral ischemia/reperfusion injury.Findings from this study indicate that cerebral ischemia/reperfusion injury can trigger neuronal ER stress and promote autophagy,and suggest that PERK is a possible target for inhibiting excessive autophagy in cerebral ischemia/reperfusion injury.

Upregulation of CDGSH iron sulfur domain 2 attenuates cerebral ischemia/reperfusion injury

CDGSH iron sulfur domain 2 can inhibit ferroptosis,which has been associated with cerebral ischemia/reperfusion,in individuals with head and neck cancer.Therefore,CDGSH iron sulfur domain 2 may be implicated in cerebral ischemia/reperfusion injury.To validate this hypothesis in the present study,we established mouse models of occlusion of the middle cerebral artery and HT22 cell models of oxygen-glucose deprivation and reoxygenation to mimic cerebral ischemia/reperfusion injury in vivo and in vitro,respectively.We found remarkably decreased CDGSH iron sulfur domain 2 expression in the mouse brain tissue and HT22 cells.When we used adeno-associated virus and plasmid to up-regulate CDGSH iron sulfur domain 2 expression in the brain tissue and HT22 cell models separately,mouse neurological dysfunction was greatly improved;the cerebral infarct volume was reduced;the survival rate of HT22 cells was increased;HT22 cell injury was alleviated;the expression of ferroptosis-related glutathione peroxidase 4,cystine-glutamate antiporter,and glutathione was increased;the levels of malondialdehyde,iron ions,and the expression of transferrin receptor 1 were decreased;and the expression of nuclear-factor E2-related factor 2/heme oxygenase 1 was increased.Inhibition of CDGSH iron sulfur domain 2 upregulation via the nuclear-factor E2-related factor 2 inhibitor ML385 in oxygen-glucose deprived and reoxygenated HT22 cells blocked the neuroprotective effects of CDGSH iron sulfur domain 2 up-regulation and the activation of the nuclear-factor E2-related factor 2/heme oxygenase 1 pathway.Our data indicate that the up-regulation of CDGSH iron sulfur domain 2 can attenuate cerebral ischemia/reperfusion injury,thus providing theoretical support from the perspectives of cytology and experimental zoology for the use of this protein as a therapeutic target in patients with cerebral ischemia/reperfusion injury.

Effects of anisodamine on altered [Ca^(2+)]i and cerebral cortex ultrastructure following acute cerebral ischemia/reperfusion injury in rabbits

BACKGROUND： Calcium ion （Ca^2＋） overload plays an important role in cerebral ischemia/reperfusion injury. Anisodamine, a type of alkaloid, can protect the myocardium from ischemia and reperfusion injury by inhibiting intracellular calcium [Ca^2＋]i overload. OBJECTIVE： To investigate effects of anisodamine on [Ca^2＋]i concentration and cortex ultrastructure following acute cerebral ischemia/reperfusion in rabbits. DESIGN, TIME AND SETTING： Randomized and controlled trial was performed at the Department of Emergency, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology from September to December 2006. MATERIALS： Forty healthy rabbits were used to establish models of acute cerebral ischemia/reperfusion. Anisodamine was provided by Lianyungang Dongfeng Pharmaceutical Factory; Fura-2 was purchased from Nanjing Jiancheng Bioengineering Institute; dual-wave length fluorescent spectrophotometry system and DM-300 software were provided by Bio-Rad, USA; OPTON-EM10C transmission electron microscope was product of Siemens, Germany. METHODS： Forty rabbits were randomly divided into the following groups： sham operation, ischemia, ischemia/reperfusion, and anisodamine, with ten rabbits in each group. Models of complete cerebral ischemia injury were established. In addition, blood was collected from the femoral artery of rats in the ischemia/reperfusion and anisodamine groups to induce hypotension and establish repeffusion injury models. The bilateral common carotid artery clamp was removed from the anisodamine group 20 minutes after ischemia, and anisodamine （10 mg/kg body mass） was injected via the femoral vein. Rabbits in the sham operation group underwent only venous cannulation. MAIN OUTCOME MEASURES： [Ca^2＋]i concentration was determined using a dual-wave length fluorescent spectrophotometry system, and cortical ultrastructure was observed following uranyl-lead citrate staining. RESULTS： The levels of [Ca^2＋]i in the ischemia and ischemia/reperfusion groups were significantly increased, compared with the sham operation group （P 〈 0.01）, and the levels of [Ca^2＋]i in the anisodamine group were remarkably less than the ischemia and ischemia/reperfusion groups （P 〈 0.01）. Ultrastructural damage to the cortex was greatly aggravated with increasing levels of [Ca^2＋]i. In the ischemia group, cortical neuronal membranes were fragmentally damaged, including the mitochoudria and endoplasmic reticulum, as well as neufite swelling, and slight chromatin margination. In the ischemia/reperfusion group, the cellular membrane was ruptured with aggravated mitochondrial swelling, increased chromatin margination, obscure neufite structure, and the disappearance of endoplasmic reticulum. However, in the anisodamine group, cellular damage was obviously alleviated. The appearance and structure of cortical neurons was relatively normal, with intact cells. There was slight swelling of the mitochondria and endoplasmic reticulum, as well as mild chromatin margination. CONCLUSION： Cerebral tissue injury was related to increased [Ca^2＋]i levels following ischemia/ reperfusion. Anisodamine exhibited a protective role on acute cerebral ischemia/reperfusion injury by inhibiting the increase in [Ca^2＋]i levels.

Effect of ephrin-B2 on the expressions of angiopoietin-1 and-2 after focal cerebral ischemia/reperfusion

Ephrin-B2 has been shown to participate in angiogenesis, but the underlying mechanisms involved remain unclear. In this study, a rat model of local cerebral ischemia was prepared by focal middle cerebral artery occlusion, followed by 24-hour reperfusion. Then, ephrin-B2 protein was administered intracerebroventricularly for 3 consecutive days via a micro-osmotic pump. Western blot assay and quantitative real-time reverse transcription PCR demonstrated the expression levels of angiopoietin-1 （Ang-1） mRNA and protein in the penumbra cortex of the ephrin-B2 treated group were decreased at day 4 after reperfusion, and increased at day 28, while the expression levels of angiopoietin-2 （Ang-2） were highly up-regulated at all time points tested. Double immunofluorescent staining indicated that Ang-1 and Ang-2 were both expressed in vascular endothelial cells positive for CD31. These findings indicate that ephrin-B2 influences the expressions of Ang-1 and Ang-2 during angiogenesis following transient focal cerebral ischemia.

Inhibition of cerebral ischemia/reperfusion injuryinduced apoptosis:nicotiflorin and JAK2/STAT3 pathway

Nicotiflorin is a flavonoid extracted from Carthamus tinctorius.Previous studies have shown its cerebral protective effect,but the mechanism is undefined.In this study,we aimed to determine whether nicotiflorin protects against cerebral ischemia/reperfusion injury-induced apoptosis through the JAK2/STAT3 pathway.The cerebral ischemia/reperfusion injury model was established by middle cerebral artery occlusion/reperfusion.Nicotiflorin（10 mg/kg） was administered by tail vein injection.Cell apoptosis in the ischemic cerebral cortex was examined by hematoxylin-eosin staining and terminal deoxynucleotidyl transferase d UTP nick end labeling assay.Bcl-2 and Bax expression levels in ischemic cerebral cortex were examined by immunohistochemial staining.Additionally,p-JAK2,p-STAT3,Bcl-2,Bax,and caspase-3 levels in ischemic cerebral cortex were examined by western blot assay.Nicotiflorin altered the shape and structure of injured neurons,decreased the number of apoptotic cells,down-regulates expression of p-JAK2,p-STAT3,caspase-3,and Bax,decreased Bax immunoredactivity,and increased Bcl-2 protein expression and immunoreactivity.These results suggest that nicotiflorin protects against cerebral ischemia/reperfusion injury-induced apoptosis via the JAK2/STAT3 pathway.

DNA hypomethylation promotes learning and memory recovery in a rat model of cerebral ischemia/reperfusion injury

Cerebral ischemia/reperfusion injury impairs learning and memory in patients.Studies have shown that synaptic function is involved in the formation and development of memory,and that DNA methylation plays a key role in the regulation of learning and memory.To investigate the role of DNA hypomethylation in cerebral ischemia/reperfusion injury,in this study,we established a rat model of cerebral ischemia/reperfusion injury by occlusion of the middle cerebral artery and then treated the rats with intraperitoneal 5-aza-2′-deoxycytidine,an inhibitor of DNA methylation.Our results showed that 5-aza-2′-deoxycytidine markedly improved the neurological function,and cognitive,social and spatial memory abilities,and dose-dependently increased the synaptic density and the expression of SYP and SHANK2 proteins in the hippocampus in a dose-dependent manner in rats with cerebral ischemia/reperfusion injury.The effects of 5-aza-2′-deoxycytidine were closely related to its reduction of genomic DNA methylation and DNA methylation at specific sites of the Syp and Shank2 genes in rats with cerebral ischemia/reperfusion injury.These findings suggest that inhibition of DNA methylation by 5-aza-2′-deoxycytidine promotes the recovery of learning and memory impairment in a rat model of cerebral ischemia/reperfusion injury.These results provide theoretical evidence for stroke treatment using epigenetic methods.

Time-dependent changes of glial fibriliary acidic protein and cytosolic phospholipase A2 in hippocampal area of focal cerebral ischemia/reperfusion in rats

BACKGROUND： Interaction between astrocyte and neuron may two-dimensionally influence on ischemic injury; however, glial fibriliary acidic protein （GFAP） and cytosolic phospholipase A2 （cPLA2） are both important markers to reflect changes of astrocyte and neuron after cerebral ischemia, respectively. OBJECTIVE： To observe the changes of GFAP and positive cPLA2 cells in hippocampal area of model rats with focal cerebral ischemia in various phases of cerebral ischemia/reperfusion. DESIGN ： Randomized contrast observation SETTING： Department of Basic Medical Science of Human Anatomy and Histology ＆ Embryology, Medical College of Wuhan Polytechnic University; Faculty Medical College of Wuhan University. MATERIALS： The experiment was carried out in the Department of Basic Medical Science, Medical College of Wuhan Industry College from May to June 2004. A total of 28 healthy SD rats of either gender and weighing 200-250 g were provided by Animal Department of Medical College of Jianghan University. METHODS： All 28 rats were randomly divided into 7 groups, including sham operation group, 2-, 6-, 12-, 24- and 48-reperfusion groups, and triphenyltetrazolium chloride （TTC） group, with 4 in each group. Two hours after ischemia, ischemia/reperfusion models were established in left middle cerebral artery （MCA）; common carotid artery was ligated and line cork was inserted into it with the depth of （1.8±0.5） cm. Rats in sham operation group were inserted with the depth of 1.0 cm, and other operations were as the same as those in 2-hour ischemia/reperfusion groups. Models in TTC group were established as the same as those in 2-hour ischemia/24-hour reperfusion group, and they were used to evaluate the therapeutic effect. Changes of GFAP and cPLA2 in hippocampal area in various phases were detected with immunohisto- chemical method. MAIN OUTCOME MEASURES ： Changes of GFAP and positive cPLA2 cells in hippocampal area of rats with focal cerebral ischemia in various phases of ischemia/reperfusion. RESULTS： All 28 rats were involved in the final analysis without any loss. （1） Animal models successfully showed the effect of focal cerebral ischemia. （2） Changes of GFAP and cPLA2 in hippocampal area in various phases： Two hours after ischemia/reperfusion, changes of GFAP and cPLA2 were increased gradually, reached at peak at 24 hours, and decreased gradually. CONCLUSION ： Courses of GFAP and cPLA2 are changed at the onset of focal cerebral ischemia, and this suggests that both of them participate in injury or protection of brain tissue of focal cerebral ischemia.

Protective effects of imperatorin against cerebral ischemia/reperfusion-induced oxidative stress through Nrf2 signaling pathway in rats

OBJECTIVE To investigates the effects of imperatorin on the oxidative stress in the cerebral cortex and hippocampus after focal cerebral ischemia/reperfusion injury.METHODS Transient focal cerebral ischemia/reperfusion model in male Sprague-Dawley rats was induced by 2 h middle cerebral artery occlusion followed by 24 h reperfusion.Imperatorin(1.25 and 2.5 mg·kg-1)or vehicle were administered intraperitoneally at 1,5 and 9 h after the onset of ischemia.At 24 h after reperfusion,the biomarkers of oxidative stress such as the levels of reactive oxygen species(ROS),lipid peroxidation products malondialdehyde(MDA),nitric oxide(NO)and total antioxidant capacity(T-AOC),the activities of inducible nitric oxide synthase(iN OS),superoxide dismutase(SOD)and catalase(CAT)in the cerebral cortex and hippocampus were observed.We also assessed the nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase-1(HO-1),and the NAD(P)H-quinone oxidoreductase 1(NQO-1)protein expression by Western blot.RESULTS As compared to vehicle-treated animals,imperatorin treatment significantly reduced the ROS,MDA,NO levels and i NOS activity,increased T-AOC and the activities of SOD and CAT.Furthermore,imperatorin treatment also significantly induced the nuclear translocation of Nrf2,enhanced the protein expression of HO-1 and NQO-1 in the cerebral cortex and hippocampus.CONCLUSION Our findings indicate that imperatorin can protect the brain against the excessive oxidative stress induced by cerebral ischemia/reperfusion through activation of Nrf2 signaling pathway.

Angiopoietin-1 mRNA and Bcl-2 expression following estradiol treatment in ovariectomized rats with focal cerebral ischemia/reperfusion injury

BACKGROUND： Estrogen has been clinically demonstrated to attenuate ischemic brain injury. However, the precise mechanisms remain controversial. OBJECTIVE： To investigate the effects of estradiol on angiopoietin-1 mRNA and Bcl-2 expression, as well as apoptosis and cerebral blood flow, in ovadectomized rats with focal cerebral ischemia following reperfusion. DESIGN, TIME AND SETTING： Randomized, controlled, animal experiment. The study was performed at the Central Laboratory, Chongqing Medical University from September to December 2005. MATERIALS： Estradiol benzoate was purchased from Shanghai Ninth Pharmaceutical Factory; corn oil was purchased from Walmart Supercenter; TUNEL kit, rabbit anti-rat Bcl-2 polyclonal antibody, and biotin-labeled goat anti-rabbit antibody were purchased from Wuhan Boster, China. METHODS： Healthy, female, 6-month-old Wistar rats-wild-type and estrogen alpha receptor gene knockout （ERKO）-were randomly divided into estradiol and control groups with 25 animals in each group. The rats were intramuscularly injected with estradiol benzoate （100 μg/kg per day） at 30 days following bilateral ovariectomy or corn oil （1 mL/kg per day） for seven consecutive days. Following administration, cerebral ischemia/reperfusion models were established using the right middle cerebral artery occlusion （MCAO） method. After 30 minutes of MCAO, estradiol and control groups were separately injected with estradiol benzoate and corn oil with the above-mentioned doses. MAIN OUTCOME MEASURES： Cell apoptosis was determined by TUNEL; angiopoietin-1 mRNA and Bcl-2 gene expression was determined, respectively, by immunohistochemical staining and RT-PCR. In addition, changes in cerebral blood flow were measured by laser Doppler flowmetry. RESULTS： Changes in angiopoietin-1 mRNA and cerebral blood flow in estradiol-treated, wild-type, MCAO rats following ischemia/reperfusion were greater than in control rats （P 〈 0.01 or 0.05）. However, no significant difference was observed between estradiol-treated ERKO MCAO rats and control rats. In addition, estradiol-treated wild-type and ERKO MCAO rats exhibited significantly increased Bcl-2 expression （P 〈 0.05） and decreased number of apoptotic cells in brain tissues compared with control groups （P 〈 0.05）. CONCLUSION： Estradiol upregulated angiopoietin-1 mRNA and Bcl-2 expression, suggesting that estradiol might be involved in protective mechanisms of cerebral ischemia/reperfusion injury.

β-arrestin 2 negatively regulates NOD2 mediated inflammatory signaling through the association with TRAF6 in cerebral ischemia-reperfusion injury

We recently reported that nucleotide-binding oligomerization domain （NOD） 2, an important cytoplasmic pattern recognition receptor, is involved in cerebral ischemia-reperfusion （I/R） injury. β-arrestins, in addition to regulate desensitization of G protein-coupled receptors （GPCRs） , have emerged as potential mediators of innate im- mune activation. However, the role and mechanism of β-arrestin2 in NOD2-triggered signaling in the cerebral I/R remain to be established. Methods BV2 cells were transfected with either β-arrestin2-shRNA plasmid or β-arres- tin2 full-length plasmid and control vector. Middle cerebral artery occlusion （MCAO） was induced in male wild- type mice and in wild type （WT） and β-arrestin2 deficient mice. Results muramyl dipeptide （MDP）, an extrin- sic ligand of NOD2, significantly increased the expression of TRAF6 and COX-2 and enhanced the activation of NF- KB in the microglia time-dependently. MDP stimulation also promoted the expression and activation of MMP-9 time- dependently, but did not affect MMP-2 obviously. Additionally, β-arrestin 2 interacted with TRAF6 after MDP stim- ulation rapidly. Overexpression of β-arrestin2 inhibited NF-KB and MMP-9 activation and COX-2 upregulation in- duced by MDP, while silence of β-arrestin2 enhanced NOD2-triggered inflammatory signaling. Finally, Deletion of β-arrestin 2 markedly aggravated brain infarction, neurological deficit and inflammation induced by MDP in mice subjected to MCAO. Conclusion The results provide the first evidence that β-arrestin 2 is an essential negatively regulator of NOD2 triggered inflammatory signaling in the cerebral I/R injury.

Neuroprotective effect of Shenqi Fuzheng injection pretreatment in aged rats with cerebral ischemia/reperfusion injury

Shenqi Fuzheng injection is extracted from the Chinese herbs Radix Astragali and Radix Codonopsis. The aim of the present study was to investigate the neuroprotective effects of Shenqi Fuzheng injection in cerebral ischemia and reperfusion. Aged rats（20–22 months） were divided into three groups： sham, model, and treatment. Shenqi Fuzheng injection or saline（40 m L/kg） was injected into the tail vein daily for 1 week, after which a cerebral ischemia/reperfusion injury model was established. Compared with model rats that received saline, rats in the treatment group had smaller infarct volumes, lower brain water and malondialdehyde content, lower brain Ca2＋ levels, lower activities of serum lactate dehydrogenase and creatine kinase, and higher superoxide dismutase activity. In addition, the treatment group showed less damage to the brain tissue ultrastructure and better neurological function. Our findings indicate that Shenqi Fuzheng injection exerts neuroprotective effects in aged rats with cerebral ischemia/reperfusion injury, and that the underlying mechanism relies on oxygen free radical scavenging and inhibition of brain Ca2＋ accumulation.

Neuroprotective effects of atorvastatin against cerebral ischemia/reperfusion injury through the inhibition of endoplasmic reticulum stress

Cerebral ischemia triggers secondary ischemia/reperfusion injury and endoplasmic reticulum stress initiates cell apoptosis. However, the regulatory mechanism of the signaling pathway remains unclear. We hypothesize that the regulatory mechanisms are mediated by the protein kinase-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α in the endoplasmic reticulum stress signaling pathway. To verify this hypothesis, we occluded the middle cerebral artery in rats to establish focal cerebral ischemia/reperfusion model. Results showed that the expression levels of protein kinase-like endoplasmic reticulum kinase and caspase-3, as well as the phosphorylation of eukaryotic initiation factor 2α, were increased after ischemia/reperfusion. Administration of atorvastatin decreased the expression of protein kinase-like endoplasmic reticulum kinase, caspase-3 and phosphorylated eukaryotic initiation factor 2α, reduced the infarct volume and improved ultrastructure in the rat brain. After salubrinal, the specific inhibitor of phosphorylated eukaryotic initiation factor 2α was given into the rats intragastrically, the expression levels of caspase-3 and phosphorylated eukaryotic initiation factor 2α in the were decreased, a reduction of the infarct volume and less ultrastructural damage were observed than the untreated, ischemic brain. However, salubrinal had no impact on the expression of protein kinase-like endoplasmic reticulum kinase. Experimental findings indicate that atorvastatin inhibits endoplasmic reticulum stress and exerts neuroprotective effects. The underlying mechanisms of attenuating ischemia/reperfusion injury are associated with the protein kinase-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/caspase-3 pathway.

Protective Effect of GRK2 and Effect of Sanguis Draconis Flavones on Focal Cerebral Ischemia-Reperfusion Injury in Rats

[Objectives] To explore the protective effect of Sanguis Draconis flavones (SDF) on rat focal cerebral ischemia-reperfusion injury (CIRI) models established by middle cerebral artery occlusion (MCAO).[Methods] A total of 60 healthy adult male Sprague-Dawley rats were selected. They were evenly and randomly divided into sham group, model group, edaravone group (12 mg/kg) and SDF group (360 mg/kg), and administered intragastrically and intraperitoneally. The middle cerebral artery of each rat was blocked by suture-occluded method to establish a CIRI model. After ischemia for 2 h and reperfusion for 48 h, the pathological injury on the ischemic side was observed by HE staining;the neuron and myelin sheath structure was observed by transmission electron microscopy;the expression of G protein-coupled receptor kinase 2 (GRK2) was preserved by immunohistochemistry;and the transfer of GRK2 was detected by western-blot.[Results] After 48 h of CIRI, the nuclei of the penumbral cortical neurons shrank, the chromatin was unevenly distributed, the nuclear membrane was dissolved and the mitochondria in the cytoplasm were swollen and vacuolated. The myelin layer was disordered. With this change, the distribution of GRK2 subcellular cells in the penumbra of the injured lateral cortex transferred from the cytoplasm to the membrane. SDF can effectively restore neuronal and myelin sheath structural damage and reduce the functional (membrane coupling) expression of GRK2.[Conclusions] GRK2 may be an effective target for SDF to protect the impaired blood-brain barrier (BBB) in CIRI.

Puerarin partly counteracts the inflammatory response after cerebral ischemia/reperfusion via activating the cholinergic anti-inflammatory pathway

Puerarin, a major isoflavonoid derived from the Chinese medical herb radix puerariae （Gegen）, has been reported to inhibit neuronal apoptosis and play an anti-inflammatory role in focal cerebral ischemia model rats. Recent findings regarding stroke pathophysiology have recognized that anti-inflammation is an important target for the treatment of ischemic stroke. The cholinergic anti-inflammatory pathway is a highly robust neural-immune mechanism for inflammation control. This study was to investigate whether activating the cholinergic anti-inflammatory pathway can be involved in the mechanism of inhibiting the inflammatory response during puerarin-induced cerebral ischemia/reperfusion in rats. Results showed that puerarin pretreatment （intravenous injection） re- duced the ischemic infarct volume, improved neurological deficit after cerebral ischemia/reperfusion and decreased the levels of interleukin-1β, interleukin-6 and tumor necrosis factor-a in brain tissue. Pretreatment with puerarin （intravenous injection） attenuated the inflammatory response in rats, which was accompanied by janus-activated kinase 2 （JAK2） and signal transducers and activators of transcription 3 （STAT3） activation and nuclear factor kappa B （NF-KB） inhibition. These observa- tions were inhibited by the alpha7 nicotinic acetylcholine receptor （a7nAchR） antagonist a-bungarotoxin （a-BGT）. In addition, puerarin pretreatment increased the expression of a7nAchR mRNA in ischemic cerebral tissue. These data demonstrate that puerarin pretreatment strongly protects the brain against cerebral ischemia/reperfusion injury and inhibits the inflammatory re- sponse. Our results also indicated that the anti-inflammatory effect of puerarin may partly be medi- ated through the activation of the cholinergic anti-inflammatory pathway.

Comparison of the anti-apoptotic effects of 15-and 35-minute suspended moxibustion after focal cerebral ischemia/reperfusion injury

Heat-sensitive suspended moxibustion has a neuroprotective effect against focal cerebral ischemia/reperfusion injury, but the underly- ing mechanisms remain unclear. The duration of heat-sensitive suspended moxibustion （usually from 30 minutes to 1 hour） is longer than traditional suspended moxibustion （usually 15 minutes）. However, the effects of 15- and 35-minute suspended moxibustion in rats with cerebra/ischemia/reperfusion injury are poorly understood. In this study, we performed 15- or 35-minute suspended moxibustion at acupoint Dazhui （GV14） in an adult rat model of focal cerebral ischemia/reperfusion injury. Infarct volume was evaluated with the 2,3,5-triphenyltetrazolium chloride assay. Histopathological changes and neuronal apoptosis at the injury site were assessed by hematoxy- lin-eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Caspase-9 and caspase-3 expression at the in- jury site was detected using immunofluorescent staining. Bax and Bcl-2 expression at the injury site was assessed using western blot assay. In the 35-minute moxibustion group, infarct volume was decreased, neuronal apoptosis was reduced, caspase-9, caspase-3 and Bax expres- sion was lower, and Bcl-2 expression was increased, compared with the 15-minute moxibustion group. Our findings show that 35-minute moxibustion has a greater anti-apoptotic effect than 15-minute moxibustion after focal cerebral ischemia/reperfusion injury.

Lateral intracerebroventricular injection of Apelin-13 inhibits apoptosis after cerebral ischemia/reperfusion injury

Apelin- 13 inhibits neuronal apoptosis caused by hydrogen peroxide, yet apoptosis following cerebral ischemia-reperfusion injury has rarely been studied. In this study, Apelin-13 （0.1 μg/g） was injected into the lateral ventricle of middle cerebral artery occlusion model rats. TTC, TUNEL, and immuno- histochemical staining showed that compared with the cerebral ischemia/reperfusion group, infarct volume and apoptotic cell number at the ischemic penumbra region were decreased in the Apelin-13 treatment group. Additionally, Apelin-13 treatment increased Bcl-2 immtmoreactivity and decreased caspase-3 immunoreactivity, Our findings suggest that Apelin-13 is neuroprotective against cerebral ischemia/reperfusion injury through inhibition of neuronal apoptosis.

Neuroprotective effects of total saponins from Rubus parvifolius L. on cerebral ischemia/reperfusion injury in rats

This study examines the neuroprotective effects and mechanisms of action of total saponins from Rubus parvifolius L. （TSRP） on focal cerebral ischemia and reperfusion injury in rats. Focal cerebral ischemia and reperfusion injury was performed in rats using the suture method. The results indicate that intragastric injection of TSRP, at 5, 10 and 20 mg/kg, could decrease neurological impairment, reduce cerebral infarct volume, diminish pathological changes, and significantly inhibit the apoptosis of neurons surrounding the ischemic area. In addition, TSRP upregulated the expression of the anti-apoptotic factor Bcl-2, at the protein and mRNA levels, and it downregulated the expression of the pro-apoptotic factor Bax, at the protein and mRNA levels. These findings indicate that TSRP protects against cerebral ischemia/reperfusion injury, and that it may do so by regulating the expression of Bcl-2 and Bax.