Perilipin-2 mediates ferroptosis in oligodendrocyte progenitor cells and myelin injury after ischemic stroke

Differentiation of oligodendrocyte progenitor cells into mature myelin-forming oligodendrocytes contributes to remyelination.Failure of remyelination due to oligodendrocyte progenitor cell death can result in severe nerve damage.Ferroptosis is an iron-dependent form of regulated cell death caused by membrane rupture induced by lipid peroxidation,and plays an important role in the pathological process of ischemic stroke.However,there are few studies on oligodendrocyte progenitor cell ferroptosis.We analyzed transcriptome sequencing data from GEO databases and identified a role of ferroptosis in oligodendrocyte progenitor cell death and myelin injury after cerebral ischemia.Bioinformatics analysis suggested that perilipin-2(PLIN2)was involved in oligodendrocyte progenitor cell ferroptosis.PLIN2 is a lipid storage protein and a marker of hypoxia-sensitive lipid droplet accumulation.For further investigation,we established a mouse model of cerebral ischemia/reperfusion.We found significant myelin damage after cerebral ischemia,as well as oligodendrocyte progenitor cell death and increased lipid peroxidation levels around the infarct area.The ferroptosis inhibitor,ferrostatin-1,rescued oligodendrocyte progenitor cell death and subsequent myelin injury.We also found increased PLIN2 levels in the peri-infarct area that co-localized with oligodendrocyte progenitor cells.Plin2 knockdown rescued demyelination and improved neurological deficits.Our findings suggest that targeting PLIN2 to regulate oligodendrocyte progenitor cell ferroptosis may be a potential therapeutic strategy for rescuing myelin damage after cerebral ischemia.

T cell interactions with microglia in immune-inflammatory processes of ischemic stroke

The primary mechanism of secondary injury after cerebral ischemia may be the brain inflammation that emerges after an ischemic stroke,which promotes neuronal death and inhibits nerve tissue regeneration.As the first immune cells to be activated after an ischemic stroke,microglia play an important immunomodulatory role in the progression of the condition.After an ischemic stroke,peripheral blood immune cells(mainly T cells)are recruited to the central nervous system by chemokines secreted by immune cells in the brain,where they interact with central nervous system cells(mainly microglia)to trigger a secondary neuroimmune response.This review summarizes the interactions between T cells and microglia in the immune-inflammatory processes of ischemic stroke.We found that,during ischemic stroke,T cells and microglia demonstrate a more pronounced synergistic effect.Th1,Th17,and M1 microglia can co-secrete proinflammatory factors,such as interferon-γ,tumor necrosis factor-α,and interleukin-1β,to promote neuroinflammation and exacerbate brain injury.Th2,Treg,and M2 microglia jointly secrete anti-inflammatory factors,such as interleukin-4,interleukin-10,and transforming growth factor-β,to inhibit the progression of neuroinflammation,as well as growth factors such as brain-derived neurotrophic factor to promote nerve regeneration and repair brain injury.Immune interactions between microglia and T cells influence the direction of the subsequent neuroinflammation,which in turn determines the prognosis of ischemic stroke patients.Clinical trials have been conducted on the ways to modulate the interactions between T cells and microglia toward anti-inflammatory communication using the immunosuppressant fingolimod or overdosing with Treg cells to promote neural tissue repair and reduce the damage caused by ischemic stroke.However,such studies have been relatively infrequent,and clinical experience is still insufficient.In summary,in ischemic stroke,T cell subsets and activated microglia act synergistically to regulate inflammatory progression,mainly by secreting inflammatory factors.In the future,a key research direction for ischemic stroke treatment could be rooted in the enhancement of anti-inflammatory factor secretion by promoting the generation of Th2 and Treg cells,along with the activation of M2-type microglia.These approaches may alleviate neuroinflammation and facilitate the repair of neural tissues.

Cell polarization in ischemic stroke: molecular mechanisms and advances

Ischemic stroke is a cerebrovascular disease associated with high mortality and disability rates. Since the inflammation and immune response play a central role in driving ischemic damage, it becomes essential to modulate excessive inflammatory reactions to promote cell survival and facilitate tissue repair around the injury site. Various cell types are involved in the inflammatory response, including microglia, astrocytes, and neutrophils, each exhibiting distinct phenotypic profiles upon stimulation. They display either proinflammatory or anti-inflammatory states, a phenomenon known as ‘cell polarization.’ There are two cell polarization therapy strategies. The first involves inducing cells into a neuroprotective phenotype in vitro, then reintroducing them autologously. The second approach utilizes small molecular substances to directly affect cells in vivo. In this review, we elucidate the polarization dynamics of the three reactive cell populations(microglia, astrocytes, and neutrophils) in the context of ischemic stroke, and provide a comprehensive summary of the molecular mechanisms involved in their phenotypic switching. By unraveling the complexity of cell polarization, we hope to offer insights for future research on neuroinflammation and novel therapeutic strategies for ischemic stroke.

Overexpression of low-density lipoprotein receptor prevents neurotoxic polarization of astrocytes via inhibiting NLRP3 inflammasome activation in experimental ischemic stroke

Neurotoxic astrocytes are a promising therapeutic target for the attenuation of cerebral ischemia/reperfusion injury.Low-density lipoprotein receptor,a classic cholesterol regulatory receptor,has been found to inhibit NLR family pyrin domain containing protein 3(NLRP3)inflammasome activation in neurons following ischemic stroke and to suppress the activation of microglia and astrocytes in individuals with Alzheimer’s disease.However,little is known about the effects of low-density lipoprotein receptor on astrocytic activation in ischemic stroke.To address this issue in the present study,we examined the mechanisms by which low-density lipoprotein receptor regulates astrocytic polarization in ischemic stroke models.First,we examined low-density lipoprotein receptor expression in astrocytes via immunofluorescence staining and western blotting analysis.We observed significant downregulation of low-density lipoprotein receptor following middle cerebral artery occlusion reperfusion and oxygen-glucose deprivation/reoxygenation.Second,we induced the astrocyte-specific overexpression of low-density lipoprotein receptor using astrocyte-specific adeno-associated virus.Low-density lipoprotein receptor overexpression in astrocytes improved neurological outcomes in middle cerebral artery occlusion mice and reversed neurotoxic astrocytes to create a neuroprotective phenotype.Finally,we found that the overexpression of low-density lipoprotein receptor inhibited NLRP3 inflammasome activation in oxygen-glucose deprivation/reoxygenation injured astrocytes and that the addition of nigericin,an NLRP3 agonist,restored the neurotoxic astrocyte phenotype.These findings suggest that low-density lipoprotein receptor could inhibit the NLRP3-meidiated neurotoxic polarization of astrocytes and that increasing low-density lipoprotein receptor in astrocytes might represent a novel strategy for treating cerebral ischemic stroke.

The miR-9-5p/CXCL11 pathway is a key target of hydrogen sulfide-mediated inhibition of neuroinflammation in hypoxic ischemic brain injury

We previously showed that hydrogen sulfide(H2S)has a neuroprotective effect in the context of hypoxic ischemic brain injury in neonatal mice.However,the precise mechanism underlying the role of H2S in this situation remains unclear.In this study,we used a neonatal mouse model of hypoxic ischemic brain injury and a lipopolysaccharide-stimulated BV2 cell model and found that treatment with L-cysteine,a H2S precursor,attenuated the cerebral infarction and cerebral atrophy induced by hypoxia and ischemia and increased the expression of miR-9-5p and cystathionineβsynthase(a major H2S synthetase in the brain)in the prefrontal cortex.We also found that an miR-9-5p inhibitor blocked the expression of cystathionineβsynthase in the prefrontal cortex in mice with brain injury caused by hypoxia and ischemia.Furthermore,miR-9-5p overexpression increased cystathionine-β-synthase and H2S expression in the injured prefrontal cortex of mice with hypoxic ischemic brain injury.L-cysteine decreased the expression of CXCL11,an miR-9-5p target gene,in the prefrontal cortex of the mouse model and in lipopolysaccharide-stimulated BV-2 cells and increased the levels of proinflammatory cytokines BNIP3,FSTL1,SOCS2 and SOCS5,while treatment with an miR-9-5p inhibitor reversed these changes.These findings suggest that H2S can reduce neuroinflammation in a neonatal mouse model of hypoxic ischemic brain injury through regulating the miR-9-5p/CXCL11 axis and restoringβ-synthase expression,thereby playing a role in reducing neuroinflammation in hypoxic ischemic brain injury.

Effect of Qingre Huayu Decoction on Autophagy and Apoptosis of Neurons in Rats with Ischemic Brain Injury by Regulating Ca^(2+)/CaMKKβ-AMPK-mTOR Pathway

[Objectives]To explore the neuroprotective mechanism of Qingre Huayu Decoction on rats with acute cerebral ischemia injury.[Methods]SD rats were divided into sham operation group,ischemia model group,low,medium and high dose groups of Qingre Huayu De-coction,with 10 rats in each group.Referring to the MCAO operation model,both the sham operation group and the model group were given normal saline by gavage,and the Qingre Huayu Decoction group was given different doses of Qingre Huayu Decoction by gavage.After the op-eration,the rats were scored for neurological deficit,neurons were stained with HE,apoptotic cells were detected with TUNEL,and the levels of autophagy and apoptotic proteins in the Ca^(2+)/CaMKKβ-AMPK-mTORpathway in brain tissue were detected with Western-blot.[Results]Compared with the model group,the neurological function score of Qingre Huayu Decoction Group decreased significantly(P<0.05),the pathological damage of neurons in Qingre Huavu Decoction Group decreased.the proportion of apoptosis-positive cells detected by TUNEL de-creased(P<0.05),and the expression of CaMKKβand AMPK increased,expression of mTOR decreased,expression of Beclin-1 and LC3 increased,and expression of Caspase-3decreased in Qingre Huayu Decoction Group(P<0.05).[Conclusions]Qingre Huayu Decoction may play a neuroprotective role by activating Ca^(2+)/CaMKKβ-AMPK-mTOR pathway and regulating the level of apoptosis and autophagy.

Treatment with β-sitosterol ameliorates the effects of cerebral ischemia/reperfusion injury by suppressing cholesterol overload, endoplasmic reticulum stress, and apoptosis

β-Sitosterol is a type of phytosterol that occurs naturally in plants.Previous studies have shown that it has anti-oxidant,anti-hyperlipidemic,anti-inflammatory,immunomodulatory,and anti-tumor effects,but it is unknown whetherβ-sitosterol treatment reduces the effects of ischemic stroke.Here we found that,in a mouse model of ischemic stroke induced by middle cerebral artery occlusion,β-sitosterol reduced the volume of cerebral infarction and brain edema,reduced neuronal apoptosis in brain tissue,and alleviated neurological dysfunction;moreover,β-sitosterol increased the activity of oxygen-and glucose-deprived cerebral cortex neurons and reduced apoptosis.Further investigation showed that the neuroprotective effects ofβ-sitosterol may be related to inhibition of endoplasmic reticulum stress caused by intracellular cholesterol accumulation after ischemic stroke.In addition,β-sitosterol showed high affinity for NPC1L1,a key transporter of cholesterol,and antagonized its activity.In conclusion,β-sitosterol may help treat ischemic stroke by inhibiting neuronal intracellular cholesterol overload/endoplasmic reticulum stress/apoptosis signaling pathways.

Acupuncture activates signal transduction pathways related to brain-tissue restoration after ischemic injury

A middle cerebral artery occlusion-model was established in rats using the improved thread embolism method.Rats were treated with acupuncture at either Dazhui（DU14）,Renzhong（DU26）, Baihui（DU20）,or a non-meridian point.Detection with protein-chip technology showed that the level of protein phosphorylation in both groups was upregulated or downregulated depending on the signaling pathway compared with the model group that did not receive acupuncture.Analysis of proteins showing downregulated phosphorylation revealed that five signaling pathways were activated in the acupuncture-treatment group,while only two were activated in the acupuncture-control group.In contrast,analysis of proteins showing upregulated phosphorylation revealed only one pathway was activated in the acupuncture-treatment group,whereas four pathways were activated in the acupuncture-control group.Furthermore,the number of activated proteins in the acupuncture-treatment group was not only higher than the acupuncture-control group,but unlike the acupuncture-control group,the majority of activated proteins were key proteins in the signaling pathways.Our findings indicate that acupuncture at specific points can activate multiple signaling pathways to promote the restoration of brain tissue following ischemic injury,and that this is based on a combination of effects resulting from multiple pathways,targets,and means.

A feasible strategy for focal cerebral ischemiareperfusion injury: remote ischemic postconditioning

It is difficult to control the degree of ischemic postconditioning in the brain and other isch- emia-sensitive organs. Remote ischemic postconditioning could protect some ischemia-sensitive organs through measures on terminal organs. In this study, a focal cerebral ischemia-reperftlsion injury model was established using three cycles of remote ischernic postconditioning, each cycle consisted of 10-minute occlusion of the femoral artery and 10-minute opening. The results showed that, remote ischemic postconditioning significantly decreased the percentage of the in- farct area and attenuated brain edema. In addition, inflammatory nuclear factor-KB expression was significantly lower, while anti-apoptotic Bcl-2 expression was significantly elevated in the ce- rebral cortex on the ischemic side. Our findings indicate that remote ischemic postconditioning attenuates focal cerebral ischemia/reperfusion injury, and that the neuroprotective mechanism is mediated by an anti-apoptotic effect and reduction of the inflammatory response.

Dabrafenib,an inhibitor of RIP3 kinase-dependent necroptosis,reduces ischemic brain injury

Ischemic brain injury triggers neuronal cell death by apoptosis via caspase activation and by necroptosis through activation of the receptor-interacting protein kinases （RIPK） associated with the tumor necrosis factor-alpha （TNF-a）/death receptor. Recent evidence shows RIPK inhibitors are neuroprotective and al- leviate ischemic brain injury in a number of animal models, however, most have not yet undergone clinical trials and safety in humans remains in question. Dabrafenib, originally identified as a B-raf inhibitor that is currently used to treat melanoma, was later revealed to be a potent RIPK3 inhibitor at micromolar con- centrations. Here, we investigated whether Dabrafenib would show a similar neuroprotective effect in mice subjected to ischemic brain injury by photothrombosis. Dabrafenib administered intraperitoneally at 10 mg/ kg one hour after photothrombosis-induced focal ischemic injury significantly reduced infarct lesion size in C57BL6 mice the following day, accompanied by a markedly attenuated upregulation of TNF-u. However, subsequent lower doses （5 mg/kg/day） failed to sustain this neuroprotective effect after 4 days. Dabrafenib bl ocked lipopolysaccharides-induced activation of TNF-ct in bone marrow-derived macrophages, suggesting that Dabrafenib may attenuate TNF-ct-induced necroptotic pathway after ischemic brain injury. Since Dab- rafenib is already in clinical use for the treatment of melanoma, it might be repurposed for stroke therapy.

Curcumin protects against ischemic spinal cord injury The pathway effect

Inducible nitric oxide synthase and N-methyI-D-aspartate receptors have been shown to participate in nerve cell injury during spinal cord ischemia. This study observed a protective effect of curcumin on ischemic spinal cord injury. Models of spinal cord ischemia were established by ligating the lumbar artery from the left renal artery to the bifurcation of the abdominal aorta. At 24 hours after model establishment, the rats were intraperitoneally injected with curcumin, Reverse transcrip- tion-polymerase chain reaction and immunohistochemical results demonstrated that after spinal cord ischemia, inducible nitric oxide synthase and N-methyI-D-aspartate receptor mRNA and protein expression significantly increased. However, curcumin significantly decreased inducible nitric oxide synthase and N-methyI-D-aspartate receptor mRNA and protein expression in the ischemic spinal cord. Tadov scale results showed that curcumin significantly improved motor function of the rat hind limb after spinal cord ischemia. The results demonstrate that curcumin exerts a neuroprotective ef- fect against ischemic spinal cord injury by decreasing inducible nitric oxide synthase and N-methyI-D-aspartate receptor expression.

Blood microRNAs as potential diagnostic and prognostic markers in cerebral ischemic injury

MicroRNAs are a family of small, genome-encoded endogenous RNAs that are transcribed but are not translated into proteins. They serve essential roles in virtually every aspect of brain function, including neurogenesis, neural development, and cellular responses leading to changes in synaptic plasticity. They are also implicated in neurodegeneration and neurological disorders, in responses to hypoxia and ischemia, and in ischemic tolerance induced by ischemic preconditioning. In recent developments, miRNA expres- sion profiling has been examined in stroke, and these studies indicate that miRNAs have emerged as key mediators in ischemic stroke biology. Both increased and decreased miRNA levels may be needed either as prevention or treatment of stroke. Novel approaches are being developed to get miRNA related therapeu- tics into the brain across an intact blood-brain barrier, including chemical modification, use of targeting molecules and methods to disrupt the blood-brain barrier.

Correlation between muscular strength and basal nuclei ischemic/hemorrhagic stroke-induced corticospinal tract injury,as detected by diffusion tensor imaging and tractography

BACKGROUND： Conventional neuroimaging diagnosis does not assist with the monitoring or evaluation of basal nuclei ischemic and hemorrhagic stroke, or motor functional recovery. Magnetic resonance, diffusion tensor imaging, and diffusion tensor tractography have all been used to observe features of cerebral white matter fibrous structures. In addition, diffusion tensor tractography is the only non-invasive imaging method to display the corticospinal tract in vivo. OBJECTIVE： To evaluate the impairment degree of corticospinal tract induced by basal nuclei ischemic and hemorrhagic stroke through the use of magnetic resonance, diffusion tensor imaging, and diffusion tensor tractography, and to analyze the correlation to muscular strength. DESIGN, TIME AND SETTING： A retrospective case analysis was performed at the Department of Medical Imaging, Neurology and Neurosurgery, Fuzhou General Hospital of Nanjing Military Area Command of Chinese PLA between November 2002 and June 2008. PARTICIPANTS： A total of 15 patients with acute or subacute cerebral ischemic stroke and nine with hemorrhagic stroke in the basal nuclei were selected. METHODS： Magnetic resonance, diffusion tensor imaging, and diffusion tensor tractography results and data were analyzed. Fractional anisotropy and directionally encoded color maps were obtained. Three-dimensional tractography of bilateral corticospinal tract was created, and corticospinal tract integrity was graded. Fractional anisotropy of infarct region and corresponding contralateral normal regions were measured, and hematoma volume in hemorrhagic stroke patients was determined. Hand motor function ability was evaluated using Brunstorm criteria. MAIN OUTCOME MEASURES： Fractional anisotropy of infarct region and corresponding contralateral normal regions; hematoma volume in hemorrhagic stroke patients; correlation between muscular strength and corticospinal tract impairment degree in ischemic stroke and hemorrhagic stroke patients before and after treatment. RESULTS： In ischemic stroke patients, the fractional anisotropy value was significantly lower in the infarct area of white matter than in the normal hemisphere （P 〈 0.01）. The impairment degree of corticospinal tract negatively correlated with muscular strength of the corresponding hand （r = -0.97 P 〈 0.01）. The hematoma volume of hemorrhagic stroke patients significantly negatively correlated with Spearman test results for muscular strength of the corresponding hand （r = -0.88, P 〈 0.01）. CONCLUSION： Corticospinal tract impairment severity negatively correlated with muscular strength and motor functional recovery, which suggested that diffusion tensor imaging and diffusion tensor tractography could be used to evaluate corticospinal tract motor function.

Scutellarin protects oxygen/glucose-deprived astrocytes and reduces focal cerebral ischemic injury

Scutellarin, a bioactive flavone isolated from Scutellaria baicalensis, has anti-inflammatory, anti-neurotoxic, anti-apoptotic and anti-oxida- tive effects and has been used to treat cardiovascular and cerebrovascular diseases in China. However, the mechanisms by which scutellarin mediates neuroprotection in cerebral ischemia remain unclear. The interaction between scutellarin and nicotinamide adenine dinucleotide phosphate oxidase 2 （NOX2） was assessed by molecular docking study, which showed that scutellarin selectively binds to NOX2 with high affinity. Cultures of primary astrocytes isolated from the cerebral cortex of neonatal Sprague-Dawley rats were pretreated with 2, 10 or 50 μM scutellarin for 30 minutes. The astrocytes were then subjected to oxygen/glucose deprivation by incubation for 2 hours in glucose-free Dulbecco＇s modified Eagle＇s medium in a 95% N2/5% CO2 incubator, followed by simulated reperfusion for 22 hours. Cell viability was assessed by cell counting kit-8 assay. Expression levels of NOX2, connexin 43 and caspase-3 were assessed by western blot assay. Reactive oxygen species were measured spectrophotometrically. Pretreatment with 10 or 50 μM scutellarin substantially increased viability, reduced the expression of NOX2 and caspase-3, increased the expression of connexin 43, and diminished the levels of reactive oxygen, species in astrocytes subjected to ischemia-＇reperfusion. We also assessed the effects of scutellarin in vivo in the rat transient middle cerebral artery occlusion model of cerebral ischemia-reperfusion injury. Rats were given intraperitoneal injection of 100 mg/kg scutellarin 2 hours before surgery. The Bederson scale was used to assess neurological deficit, and 2,3,5-triphenyltetrazolium chloride staining was used to measure infarct size. Western blot assay was used to assess expression of NOX2 and connexin 43 in brain tissue. Enzyme-linked immunosorbent assay was used to detect 8-hydroxydeoxyguanosine （8-OHdG）, 4-hydroxy-2-nonenal （4-HNE） and 3-nitrotyrosin （3-NT） in brain tissue. Immunofluorescence double staining was used to determine the co-expression of caspase-3 and NeuN. Pretreatment with scutellarin im- proved the neurological function of rats with focal cerebral ischemia, reduced infarct size, diminished the expression of NOX2, reduced levels of 8-OHdG, 4-HNE and 3-NT, and reduced the number of cells co-expressing caspase-3 and NeuN in the injured brain tissue. Furthermore, we examined the effect of the NOX2 inhibitor apocynin. Apocynin substantially increased connexin 43 expression in vivo and in vitro. Collectively, our findings suggest that scutellarin protects against ischemic injury in vitro and in vivo by downregulating NOX2, upregulating connexin 43, decreasing oxidative damage, and reducing apoptotic cell death.

Ischemic post-conditioning to counteract intestinal ischemia/reperfusion injury

Intestinal ischemia is a severe disorder with a variety of causes.Reperfusion is a common occurrence during treatment of acute intestinal ischemia but the injury resulting from ischemia/reperfusion(IR)may lead toeven more serious complications from intestinal atrophy to multiple organ failure and death.The susceptibility of the intestine to IR-induced injury(IRI)appears from various experimental studies and clinical settings such as cardiac and major vascular surgery and organ transplantation.Where as oxygen free radicals,activation of leukocytes,failure of microvascular perfusion,cellular acidosis and disturbance of intracellular homeo-stasis have been implicated as important factors inthe pathogenesis of intestinal IRI,the mechanisms underlying this disorder are not well known.To date,increasing attention is being paid in animal studies to potential pre-and post-ischemia treatments that protect against intestinal IRI such as drug interference with IR-induced apoptosis and inflammation processes and ischemic pre-conditioning.However,better insight is needed into the molecular and cellular events associated with reperfusion-induced damage to develop effective clinical protection protocols to combat this disorder.In this respect,the use of ischemic post-conditioning in combination with experimentally prolonged acidosis blocking deleterious reperfusion actions may turn out to have particular clinical relevance.

Ischemic preconditioning protects against ischemic brain injury

In this study, we hypothesized that an increase in integrin αβand its co-activator vascular endothelial growth factor play important neuroprotective roles in ischemic injury. We performed ischemic preconditioning with bilateral common carotid artery occlusion for 5 minutes in C57BL/6J mice. This was followed by ischemic injury with bilateral common carotid artery occlusion for 30 minutes. The time interval between ischemic preconditioning and lethal ischemia was 48 hours. Histopathological analysis showed that ischemic preconditioning substantially diminished damage to neurons in the hippocampus 7 days after ischemia. Evans Blue dye assay showed that ischemic preconditioning reduced damage to the blood-brain barrier 24 hours after ischemia. This demonstrates the neuroprotective effect of ischemic preconditioning. Western blot assay revealed a significant reduction in protein levels of integrin αβ, vascular endothelial growth factor and its receptor in mice given ischemic preconditioning compared with mice not given ischemic preconditioning 24 hours after ischemia. These findings suggest that the neuroprotective effect of ischemic preconditioning is associated with lower integrin αβand vascular endothelial growth factor levels in the brain following ischemia.

Remote ischemic perconditioning prevents liver transplantation-induced ischemia/reperfusion injury in rats: Role of ROS/RNS and e NOS

AIM To investigate the underlying mechanisms of the protective role of remote ischemic perconditioning (RIPerC) in rat liver transplantation. METHODS Sprague-Dawley rats were subjected to sham, orthotopic liver transplantation (OLT), ischemic postconditioning (IPostC) or RIPerC. After 3 h reperfusion, blood samples were taken for measurement of alanine aminotransferase, aspartate aminotransferase, creatinine (Cr) and creatinine kinase-myocardial band (CK-MB). The liver lobes were harvested for the following measurements: reactive oxygen species (ROS), H2O2, mitochondrial membrane potential (Delta psi m) and total nitric oxide (NO). These measurements were determined using an ROS/H2O2, JC1 and Total NOx Assay Kit, respectively. Endothelial NO synthase (eNOS) was analyzed by reverse transcription-polymerase chain reaction (RTPCR) and western blotting, and peroxynitrite was semiquantified by western blotting of 3-nitrotyrosine. RESULTS Compared with the OLT group, the grafts subjected to RIPerC showed significantly improved liver and remote organ functions (P < 0.05). ROS (P < 0.001) including H2O2 (P < 0.05) were largely elevated in the OLT group as compared with the sham group, and RIPerC (P < 0.05) reversed this trend. The collapse of Delta psi m induced by OLT ischemia/reperfusion (I/R) injury was significantly attenuated in the RIPerC group (P < 0.001). A marked increase of NO content and phosphoserine eNOS, both in protein and mRNA levels, was observed in liver graft of the RIPerC group as compared with the OLT group (P < 0.05). I/R-induced 3-nitrotyrosine content was significantly reduced in the RIPerC group as compared with the OLT group (P < 0.05). There were no significant differences between the RIPerC and IPostC groups for all the results except Cr. The Cr level was lower in the RIPerC group than in the IPostC group (P < 0.01). CONCLUSION Liver graft protection by RIPerC is similar to or better than that of IPostC, and involves inhibition of oxidative stress and up-regulation of the PI3K/Akt/eNOS/NO pathway.

S100B protein in serum is elevated after global cerebral ischemic injury

BACKGROUND:S100B protein in patients with cardiac arrest,hemorrhagic shock and other causes of global cerebral ischemic injury will be dramatically increased.Ischemic brain injury may elevate the level of serum S100 B protein and the severity of brain damage.METHODS:This article is a critical and descriptive review on S100 B protein in serum after ischemic brain injury.We searched Pubmed database with key words or terms such as "S100B protein", "cardiac arrest", "hemorrhagic shock" and "ischemia reperfusion injury" appeared in the last five years.RESULTS:S100B protein in patients with cardiac arrest,hemorrhagic shock and other causes of ischemic brain injury will be dramatically increased.Ischemic brain injury elevated the level of serum S100 B protein,and the severity of brain damage.CONCLUSION:The level of S100 B protein in serum is elevated after ischemic brain injury,but its mechanism is unclear.

Protective effect of ischemic preconditioning on hepatic ischemia-reperfusion injury by advancing the expressive phase of survivin in rats

BACKGROUND: Survivin is a new and important gene in the regulation of apoptosis. It is very important to explore the effect of the expression of survivin protein caused by ischemia-reperfusion (IR) injury. The effect of IR injury caused by ischemic preconditioning (IP) on the liver in rats and the relation between the protective effect of IP and the expression of survivin are unclear. METHODS: One hundred and fifty male Wistar rats (weighing 190-210 g, aged 6-7 weeks) were divided into three groups at random: ischemic preconditioning (IP), ischemia-reperfusion (IR) and sham-operation (SO). Sample specimens were collected from each group at 6, 12, 24, 48, and 72 hours after reperfusion. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by an automatic biochemical analyzer. Malondialdehyde (MDA) in liver tissue was measured. Pathological changes in the liver and immunohistochemical staining for survivin were determined with an optical microscope. RESULTS: The ALT levels in the IP and IR groups after reperfusion at each time were higher than those in the SO group (P<0.05), whereas after reperfusion for 6 and 12 hours, the ALT levels in the IP group were lower than those in the IR group (P<0.05). The AST levels in all IP and IR groups were higher than those in the SO group (P<0.05), whereas after reperfusion for 12, 24, 48 and 72 hours, the AST levels in the IP group were lower than those in the IR group (P<0.05). The MDA concentrations after reperfusion in the IP group were lower than those in the IR group (P<0.05), though the MDA concentrations in the IP and IR groups increased in contrast to those in the SO group after reperfusion at each time (P<0.05). After reperfusion for 12, 24, 48 and 72 hours, the number of survivin-positive cells was larger in the IP and IR groups than in the SO group (P<0.05). After reperfusion for 12, 24, and 48 hours the number of survivin-positive cells in the IP group increased compared with that in the IR group (P<0.05). CONCLUSIONS: IR increases the protein expression of survivin in liver tissue. IP inhibits the accumulation of MDA, advances the expressive phase of survivin protein in hepatic tissue, and improves liver function.

Mitofusin-2 mediated mitochondrial Ca2+ uptake 1/2 induced liver injury in rat remote ischemic perconditioning liver transplantation and alpha mouse liver-12 hypoxia cell line models

AIM To investigate the protective mechanism of mitofusin-2 (Mfn2) in rat remote ischemic perconditioning (RIC) models and revalidate it in alpha mouse liver-12 (AML-12) hypoxia cell lines. METHODS Sprague-Dawley rats were divided into three groups (n = 6 each): sham, orthotopic liver transplantation and RIC. After operation, blood samples were collected to test alanine aminotransferase and aspartate aminotransferase. The liver lobes were harvested for histopathological examination, western blotting (WB) and quantitative real-time (qRT)-PCR. AML-12 cell lines were then subjected to normal culture, anoxic incubator tank culture (hypoxia) and anoxic incubator tank culture with Mfn2 knockdown (hypoxia + Si), and data of qRT-PCR, WB, mitochondrial membrane potential (Delta psi m), apoptosis, endoplasmic reticulum Ca2+ concentrations and mitochondrial Ca2+ concentrations were collected. RESULTS Both sham and normal culture groups showed no injury during the experiment. The RIC group showed amelioration of liver function compared with the orthotopic liver transplantation group (P < 0.05). qRTPCR and WB confirmed that Mfn2-mitochondrial Ca2+ uptake 1/2 (MICUs) axis was changed (P < 0.005). In AML-12 cell lines, compared with the hypoxia group, the hypoxia + Si group attenuated the collapse of..m and apoptosis (P < 0.005). The endoplasmic reticulum Ca2+ decrease and mitochondrial Ca2+ overloading observed in the hypoxia group were also attenuated in the hypoxia + Si group (P < 0.005). Finally, qRT-PCR and WB confirmed the Mfn2-MICUs axis change in all the groups (P < 0.005). CONCLUSION Mfn2 participates in liver injury in rat RIC models and AML-12 hypoxia cell lines by regulating the MICUs pathway.