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.

The action mechanism by which C1q/tumor necrosis factor-related protein-6 alleviates cerebral ischemia/reperfusion injury in diabetic mice

Studies have shown that C1q/tumor necrosis factor-related protein-6 (CTRP6) can alleviate renal ischemia/reperfusion injury in mice. However, its role in the brain remains poorly understood. To investigate the role of CTRP6 in cerebral ischemia/reperfusion injury associated with diabetes mellitus, a diabetes mellitus mouse model of cerebral ischemia/reperfusion injury was established by occlusion of the middle cerebral artery. To overexpress CTRP6 in the brain, an adeno-associated virus carrying CTRP6 was injected into the lateral ventricle. The result was that oxygen injury and inflammation in brain tissue were clearly attenuated, and the number of neurons was greatly reduced. In vitro experiments showed that CTRP6 knockout exacerbated oxidative damage, inflammatory reaction, and apoptosis in cerebral cortical neurons in high glucose hypoxia-simulated diabetic cerebral ischemia/reperfusion injury. CTRP6 overexpression enhanced the sirtuin-1 signaling pathway in diabetic brains after ischemia/reperfusion injury. To investigate the mechanism underlying these effects, we examined mice with depletion of brain tissue-specific sirtuin-1. CTRP6-like protection was achieved by activating the sirtuin-1 signaling pathway. Taken together, these results indicate that CTRP6 likely attenuates cerebral ischemia/reperfusion injury through activation of the sirtuin-1 signaling pathway.

Homer1a reduces inflammatory response after retinal ischemia/reperfusion injury

Elevated intraocular pressure(IOP)is one of the causes of retinal ischemia/reperfusion injury,which results in NRP3 inflammasome activation and leads to visual damage.Homerla is repo rted to play a protective role in neuroinflammation in the cerebrum.However,the effects of Homerla on NLRP3inflammasomes in retinal ischemia/reperfusion injury caused by elevated IOP remain unknown.In our study,animal models we re constructed using C57BL/6J and Homer1^(flox/-)/Homerla^(+/-)/Nestin-Cre^(+/-)mice with elevated IOP-induced retinal ischemia/repe rfusion injury.For in vitro expe riments,the oxygen-glucose deprivation/repe rfusion injury model was constructed with M uller cells.We found that Homerla ove rexpression amelio rated the decreases in retinal thickness and Muller cell viability after ischemia/reperfusion injury.Furthermore,Homerla knockdown promoted NF-κB P65^(Ser536)activation via caspase-8,NF-κB P65 nuclear translocation,NLRP3 inflammasome formation,and the production and processing of interleukin-1βand inte rleukin-18.The opposite results we re observed with Homerla ove rexpression.Finally,the combined administration of Homerla protein and JSH-23 significantly inhibited the reduction in retinal thickness in Homer1^(flox/-)Homer1a^(+/-)/Nestin-Cre^(+/-)mice and apoptosis in M uller cells after ischemia/reperfusion injury.Taken together,these studies demonstrate that Homer1a exerts protective effects on retinal tissue and M uller cells via the caspase-8/NF-KB P65/NLRP3 pathway after I/R 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.

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.

N-acetylserotonin alleviates retinal ischemia-reperfusion injury via HMGB1/RAGE/NF-κB pathway in rats

AIM:To observe the effects of N-acetylserotonin(NAS)administration on retinal ischemia-reperfusion(RIR)injury in rats and explore the underlying mechanisms involving the high mobility group box 1(HMGB1)/receptor for advanced glycation end-products(RAGE)/nuclear factor-kappa B(NF-κB)signaling pathway.METHODS:A rat model of RIR was developed by increasing the pressure of the anterior chamber of the eye.Eighty male Sprague Dawley were randomly divided into five groups:sham group(n=8),RIR group(n=28),RIR+NAS group(n=28),RIR+FPS-ZM1 group(n=8)and RIR+NAS+FPS-ZM1 group(n=8).The therapeutic effects of NAS were examined by hematoxylin-eosin(H&E)staining,and retinal ganglion cells(RGCs)counting.The expression of interleukin 1 beta(IL-1β),HMGB1,RAGE,and nod-like receptor 3(NLRP3)proteins and the phosphorylation of nuclear factorkappa B(p-NF-κB)were analyzed by immunohistochemistry staining and Western blot analysis.The expression of HMGB1 protein was also detected by enzyme-linked immunosorbent assay(ELISA).RESULTS:H&E staining results showed that NAS significantly reduced retinal edema and increased the number of RGCs in RIR rats.With NAS therapy,the HMGB1 and RAGE expression decreased significantly,and the activation of the NF-κB/NLRP3 pathway was antagonized along with the inhibition of p-NF-κB and NLRP3 protein expression.Additionally,NAS exhibited an anti-inflammatory effect by reducing IL-1βexpression.The inhibitory of RAGE binding to HMGB1 by RAGE inhibitor FPS-ZM1 led to a significant decrease of p-NF-κB and NLRP3 expression,so as to the IL-1βexpression and retinal edema,accompanied by an increase of RGCs in RIR rats.CONCLUSION:NAS may exhibit a neuroprotective effect against RIR via the HMGB1/RAGE/NF-κB signaling pathway,which may be a useful therapeutic target for retinal disease.

Reperfusion after hypoxia-ischemia exacerbates brain injury with compensatory activation of the antiferroptosis system:based on a novel rat model

Hypoxic-ischemic encephalopathy,which predisposes to neonatal death and neurological sequelae,has a high morbidity,but there is still a lack of effective prevention and treatment in clinical practice.To better understand the pathophysiological mechanism underlying hypoxic-ischemic encephalopathy,in this study we compared hypoxic-ischemic reperfusion brain injury and simple hypoxic-ischemic brain injury in neonatal rats.First,based on the conventional RiceVannucci model of hypoxic-ischemic encephalopathy,we established a rat model of hypoxic-ischemic reperfusion brain injury by creating a common carotid artery muscle bridge.Then we performed tandem mass tag-based proteomic analysis to identify differentially expressed proteins between the hypoxic-ischemic reperfusion brain injury model and the conventional Rice-Vannucci model and found that the majority were mitochondrial proteins.We also performed transmission electron microscopy and found typical characteristics of ferroptosis,including mitochondrial shrinkage,ruptured mitochondrial membranes,and reduced or absent mitochondrial cristae.Further,both rat models showed high levels of glial fibrillary acidic protein and low levels of myelin basic protein,which are biological indicators of hypoxic-ischemic brain injury and indicate similar degrees of damage.Finally,we found that ferroptosis-related Ferritin(Fth1)and glutathione peroxidase 4 were expressed at higher levels in the brain tissue of rats with hypoxic-ischemic reperfusion brain injury than in rats with simple hypoxic-ischemic brain injury.Based on these results,it appears that the rat model of hypoxic-ischemic reperfusion brain injury is more closely related to the pathophysiology of clinical reperfusion.Reperfusion not only aggravates hypoxic-ischemic brain injury but also activates the anti-ferroptosis system.

Neuroprotective potential for mitigating ischemia-reperfusion-induced damage

Reperfusion following cerebral ischemia causes both structural and functional damage to brain tissue and could aggravate a patient's condition;this phenomenon is known as cerebral ischemia-reperfusion injury.Current studies have elucidated the neuroprotective role of the sirtuin protein family(Sirtuins)in modulating cerebral ischemia-reperfusion injury.However,the potential of utilizing it as a novel intervention target to influence the prognosis of cerebral ischemia-reperfusion injury requires additional exploration.In this review,the origin and research progress of Sirtuins are summarized,suggesting the involvement of Sirtuins in diverse mechanisms that affect cerebral ischemia-reperfusion injury,including inflammation,oxidative stress,blood-brain barrier damage,apoptosis,pyroptosis,and autophagy.The therapeutic avenues related to Sirtuins that may improve the prognosis of cerebral ischemia-reperfusion injury were also investigated by modulating Sirtuins expression and affecting representative pathways,such as nuclear factor-kappa B signaling,oxidative stress mediated by adenosine monophosphate-activated protein kinase,and the forkhead box O.This review also summarizes the potential of endogenous substances,such as RNA and hormones,drugs,dietary supplements,and emerging therapies that regulate Sirtuins expression.This review also reveals that regulating Sirtuins mitigates cerebral ischemia-reperfusion injury when combined with other risk factors.While Sirtuins show promise as a potential target for the treatment of cerebral ischemiareperfusion injury,most recent studies are based on rodent models with circadian rhythms that are distinct from those of humans,potentially influencing the efficacy of Sirtuinstargeting drug therapies.Overall,this review provides new insights into the role of Sirtuins in the pathology and treatment of cerebral ischemia-reperfusion injury.

Selective ischemic-hemisphere targeting Ginkgolide B liposomes with improved solubility and therapeutic efficacy for cerebral ischemia-reperfusion injury

Cerebral ischemia-reperfusion injury(CI/RI)remains the main cause of disability and death in stroke patients due to lack of effective therapeutic strategies.One of the main issues related to CI/RI treatment is the presence of the blood-brain barrier(BBB),which affects the intracerebral delivery of drugs.Ginkgolide B(GB),a major bioactive component in commercially available products of Ginkgo biloba,has been shown significance in CI/RI treatment by regulating inflammatory pathways,oxidative damage,and metabolic disturbance,and seems to be a candidate for stroke recovery.However,limited by its poor hydrophilicity and lipophilicity,the development of GB preparations with good solubility,stability,and the ability to cross the BBB remains a challenge.Herein,we propose a combinatorial strategy by conjugating GB with highly lipophilic docosahexaenoic acid(DHA)to obtain a covalent complex GB-DHA,which can not only enhance the pharmacological effect of GB,but can also be encapsulated in liposomes stably.The amount of finally constructed Lipo@GB-DHA targeting to ischemic hemisphere was validated 2.2 times that of free solution in middle cerebral artery occlusion(MCAO)rats.Compared to the marketed ginkgolide injection,Lipo@GB-DHA significantly reduced infarct volume with better neurobehavioral recovery in MCAO rats after being intravenously administered both at 2 h and 6 h post-reperfusion.Low levels of reactive oxygen species(ROS)and high neuron survival in vitro was maintained via Lipo@GB-DHA treatment,while microglia in the ischemic brain were polarized from the pro-inflammatory M1 phenotype to the tissue-repairing M2 phenotype,which modulate neuroinflammatory and angiogenesis.In addition,Lipo@GB-DHA inhibited neuronal apoptosis via regulating the apoptotic pathway and maintained homeostasis by activating the autophagy pathway.Thus,transforming GB into a lipophilic complex and loading it into liposomes provides a promising nanomedicine strategy with excellent CI/RI therapeutic efficacy and industrialization prospects.

Network-pharmacology-based research on protective effects and underlying mechanism of Shuxin decoction against myocardial ischemia/reperfusion injury with diabetes

BACKGROUND Patients with diabetes mellitus are at higher risk of myocardial ischemia/reperfusion injury(MI/RI).Shuxin decoction(SXT)is a proven recipe modification from the classic herbal formula"Wu-tou-chi-shi-zhi-wan"according to the traditional Chinese medicine theory.It has been successfully used to alleviate secondary MI/RI in patients with diabetes mellitus in the clinical setting.However,the underlying mechanism is still unclear.AIM To further determine the mechanism of SXT in attenuating MI/RI associated with diabetes.METHODS This paper presents an ensemble model combining network pharmacology and biology.The Traditional Chinese Medicine System Pharmacology Database was accessed to select key components and potential targets of the SXT.In parallel,therapeutic targets associated with MI/RI in patients with diabetes were screened from various databases including Gene Expression Omnibus,DisGeNet,Genecards,Drugbank,OMIM,and PharmGKB.The potential targets of SXT and the therapeutic targets related to MI/RI in patients with diabetes were intersected and subjected to bioinformatics analysis using the Database for Annotation,Visualization and Integrated Discovery.The major results of bioinformatics analysis were subsequently validated by animal experiments.RESULTS According to the hypothesis derived from bioinformatics analysis,SXT could possibly ameliorate lipid metabolism disorders and exert anti-apoptotic effects in MI/RI associated with diabetes by reducing oxidized low density lipoprotein(LDL)and inhibiting the advanced glycation end products(AGE)-receptor for AGE(RAGE)signaling pathway.Subsequent animal experiments confirmed the hypothesis.The treatment with a dose of SXT(2.8 g/kg/d)resulted in a reduction in oxidized LDL,AGEs,and RAGE,and regulated the level of blood lipids.Besides,the expression of apoptosis-related proteins such as Bax and cleaved caspase 3 was down-regulated,whereas Bcl-2 expression was up-regulated.The findings indicated that SXT could inhibit myocardial apoptosis and improve cardiac function in MI/RI in diabetic rats.CONCLUSION This study indicated the active components and underlying molecular therapeutic mechanisms of SXT in MI/RI with diabetes.Moreover,animal experiments verified that SXT could regulate the level of blood lipids,alleviate cardiomyocyte apoptosis,and improve cardiac function through the AGE-RAGE signaling pathway.

Interferon-γpriming enhances the therapeutic effects of menstrual blood-derived stromal cells in a mouse liver ischemia-reperfusion model

BACKGROUND Mesenchymal stem cells(MSCs)have been used in liver transplantation and have certain effects in alleviating liver ischemia-reperfusion injury(IRI)and regulating immune rejection.However,some studies have indicated that the effects of MSCs are not very significant.Therefore,approaches that enable MSCs to exert significant and stable therapeutic effects are worth further study.AIM To enhance the therapeutic potential of human menstrual blood-derived stromal cells(MenSCs)in the mouse liver ischemia-reperfusion(I/R)model via interferon-γ(IFN-γ)priming.METHODS Apoptosis was analyzed by flow cytometry to evaluate the safety of IFN-γpriming,and indoleamine 2,3-dioxygenase(IDO)levels were measured by quantitative real-time reverse transcription polymerase chain reaction,western blotting,and ELISA to evaluate the efficacy of IFN-γpriming.In vivo,the liver I/R model was established in male C57/BL mice,hematoxylin and eosin and TUNEL staining was performed and serum liver enzyme levels were measured to assess the degree of liver injury,and regulatory T cell(Treg)numbers in spleens were determined by flow cytometry to assess immune tolerance potential.Metabolomics analysis was conducted to elucidate the potential mechanism underlying the regulatory effects of primed MenSCs.In vitro,we established a hypoxia/reoxygenation(H/R)model and analyzed apoptosis by flow cytometry to investigate the mechanism through which primed MenSCs inhibit apoptosis.Transmission electron microscopy,western blotting,and immunofluorescence were used to analyze autophagy levels.RESULTS IFN-γ-primed MenSCs secreted higher levels of IDO,attenuated liver injury,and increased Treg numbers in the mouse spleens to greater degrees than untreated MenSCs.Metabolomics and autophagy analyses proved that primed MenSCs more strongly induced autophagy in the mouse livers.In the H/R model,autophagy inhibitors increased the level of H/R-induced apoptosis,indicating that autophagy exerted protective effects.In addition,primed MenSCs decreased the level of H/R-induced apoptosis via IDO and autophagy.Further rescue experiments proved that IDO enhanced the protective autophagy by inhibiting the mammalian target of rapamycin(mTOR)pathway and activating the AMPK pathway.CONCLUSION IFN-γ-primed MenSCs exerted better therapeutic effects in the liver I/R model by secreting higher IDO levels.MenSCs and IDO activated the AMPK-mTOR-autophagy axis to reduce IRI,and IDO increased Treg numbers in the spleen and enhanced the MenSC-mediated induction of immune tolerance.Our study suggests that IFN-γ-primed MenSCs may be a novel and superior MSC product for liver transplantation in the future.

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.

A molecular probe carrying anti-tropomyosin 4 for early diagnosis of cerebral ischemia/reperfusion injury

In vivo imaging of cerebral ischemia/reperfusion injury remains an important challenge.We injected porous Ag/Au@SiO_(2) bimetallic hollow nanoshells carrying anti-tropomyosin 4 as a molecular probe into mice with cerebral ischemia/reperfusion injury and observed microvascular changes in the brain using photoacoustic imaging with ultrasonography.At each measured time point,the total photoacoustic signal was significantly higher on the affected side than on the healthy side.Twelve hours after reperfusion,cerebral perfusion on the affected side increased,cerebrovascular injury worsened,and anti-tropomyosin 4 expression increased.Twenty-four hours after reperfusion and later,perfusion on the affected side declined slowly and stabilized after 1 week;brain injury was also alleviated.Histopathological and immunohistochemical examinations confirmed the brain injury tissue changes.The nanoshell molecular probe carrying anti-tropomyosin 4 has potential for use in early diagnosis of cerebral ischemia/reperfusion injury and evaluating its progression.

Eph receptor A4 regulates motor neuron ferroptosis in spinal cord ischemia/reperfusion injury in rats

Previous studies have shown that the receptor tyrosine kinase Eph receptor A4(EphA4) is abundantly expressed in the nervous system. The EphA4 signaling pathway plays an important role in regulating motor neuron ferroptosis in motor neuron disease. To investigate whether EphA4 signaling is involved in ferroptosis in spinal cord ischemia/reperfusion injury, in this study we established a rat model of spinal cord ischemia/reperfusion injury by clamping the left carotid artery and the left subclavian artery. We found that spinal cord ischemia/reperfusion injury increased EphA4 expression in the neurons of anterior horn, markedly worsened ferroptosis-related indicators, substantially increased the number of mitochondria exhibiting features consistent with ferroptosis, promoted deterioration of motor nerve function, increased the permeability of the blood-spinal cord barrier, and increased the rate of motor neuron death. Inhibition of EphA4 largely rescued these effects. However, intrathecal administration of the ferroptosis inducer Erastin counteracted the beneficial effects conferred by treatment with the EphA4 inhibitor. Mass spectrometry and a PubMed search were performed to identify proteins that interact with EphA4, with the most notable being Beclin1 and Erk1/2. Our results showed that inhibition of EphA4 expression reduced binding to Beclin1, markedly reduced p-Beclin1, and reduced Beclin1-XCT complex formation. Inhibition of EphA4 also reduced binding to p-Erk1/2 and markedly decreased the expression of c-Myc, transferrin receptor 1, and p-Erk1/2. Additionally, we observed co-localization of EphA4 and p-Beclin1 and of EphA4 and p-ERK1/2 in neurons in the anterior horn. In conclusion, EphA4 participates in regulating ferroptosis of spinal motor neurons in the anterior horn in spinal cord ischemia/reperfusion injury by promoting formation of the Beclin1-XCT complex and activating the Erk1/2/c-Myc/transferrin receptor 1 axis.

Atorvastatin Alleviates Myocardial Ischemia-Reperfusion Injury via miR-26a-5p/FOXO1

Purpose: Ischemia-reperfusion (I/R) injury exacerbates myocardial cell death (including apoptosis and necrosis), leading to complications such as arrhythmias, myocardial stenosis, microvascular obstruction and heart failure, and it is particularly important to seek new strategies to mitigate reperfusion injury. In this paper, we will investigate whether atorvastatin can alleviate myocardial ischemia-reperfusion injury and verify its molecular mechanism. Methods: We successfully constructed a hypoxia-reperfusion (H/R) H9c2 cell model and transfected miR-26a-5p mimic, miR-26a-5p inhibitor and its negative control NC-mimic or NC-inhibitor into H9c2 cells using a transfection kit. The expression of miR-26a-5p and FOXO1 were detected by RT-qPCR assay, the expression of related proteins by Western blot assay, the cell viability of H9c2 cells by CCK-8 assay, the apoptosis rate of H9c2 cells by flow cytometry, the CK and LDH activity in cells by CK and LDH assay kits. The targeting relationship between miR-26a-5p and FOXO1 was verified by dual luciferase reporter gene assay. Results: MiR-26a-5p expression was decreased in H/R-induced cells and FOXO1 expression was increased in H/R-induced cells. Atorvastatin alleviated H/R injury in cardiomyocytes and was most effective at a concentration of 1 μM. Atorvastatin alleviated H/R injury in cardiomyocytes by upregulating miR-26a-5p expression, miR-26a-5p and FOXO1 were negatively regulated by targeting. Conclusion: Atorvastatin can alleviate H/R injury in cardiomyocytes by regulating miR-26a-5p/FOXO1.

Electroacupuncture reduces injury to the blood-brain barrier following cerebral ischemia/reperfusion injury

This study used electroacupuncture at Renzhong （DU26） and Baihui （DU20） in a rat model of cerebral ischemia/reperfusion injury. Neurological deficit scores, western blotting, and reverse transcription-PCR results demonstrated that electroacupuncture markedly reduced neurological deficits, decreased corpus striatum aquaporin-4 protein and mRNA expression, and relieved damage to the blood-brain barrier in a rat model of cerebral ischemia/reperfusion injury. These results suggest that electroacupuncture most likely protects the blood-brain barrier by regulating aquaporin-4 expression following cerebral ischemia/reperfusion injury.

Matrix metalloproteinase-9 expression and blood brain barrier permeability in the rat brain after cerebral ischemia/reperfusion injury

BACKGROUND： The integrity of the blood brain barrier （BBB） plays an important role in the patho-physiological process of cerebral ischemia/reperfusion injury. It has been recently observed that metalloproteinase-9 （MMP-9） is closely related to cerebral ischemia/reperfusion injury OBJECTIVE： This study was designed to observe MMP-9 expression in the rat brain after cerebral ischemia/reperfusion injury and to investigate its correlation to BBB permeability. DESIGN, TIME AND SETTING： This study, a randomized controlled animal experiment, was performed at the Institute of Neurobiology, Central South University between September 2005 and March 2006. MATERIALS： Ninety healthy male SD rats, aged 3-4 months, weighing 200-280 g, were used in the present study. Rabbit anti-rat MMP-9 polyclonal antibody （Boster, Wuhan, China） and Evans blue （Sigma, USA） were also used. METHODS： All rats were randomly divided into 9 groups with 10 rats in each group： normal control group, sham-operated group, and ischemia for 2 hours followed by reperfusion for 3, 6, 12 hours, 1, 2, 4 and 7 days groups. In the ischemia/reperfusion groups, rats were subjected to ischemia/reperfusion injury by suture occlusion of the right middle cerebral artery. In the sham-operated group, rats were merely subjected to vessel dissociation. In the normal control group, rats were not modeled. MAIN OUTCOME MEASURES： BBB permeability was assessed by determining the level of effusion of Evans blue. MMP-9 expression was detected by an immunohistochemical method. RESULTS： All 90 rats were included in the final analysis. BBB permeability alteration was closely correlated to ischemia/reperfusion time. BBB permeability began to increase at ischemia/reperfusion for 3 hours, then it gradually reached a peak level at ischemia/reperfusion for 1 day, and thereafter it gradually decreased. MMP-9 expression began to increase at ischemia/reperfusion for 3 hours, then gradually reached its peak level 2 days after perfusion, and thereafter it gradually decreased. CONCLUSION： MMP-9 expression increases in rat brain tissue after focal cerebral ischemia/reperfusion injury, which correlates with increased permeability of the BBB.

Propofol protects against blood-spinal cord barrier disruption induced by ischemia/reperfusion injury

Propofol has been shown to exert neuroprotective effects on the injured spinal cord.However,the effect of propofol on the blood-spinal cord barrier（BSCB） after ischemia/reperfusion injury（IRI） is poorly understood.Therefore,we investigated whether propofol could maintain the integrity of the BSCB.Spinal cord IRI（SCIRI） was induced in rabbits by infrarenal aortic occlusion for 30 minutes.Propofol,30 mg/kg,was intravenously infused 10 minutes before aortic clamping as well as at the onset of reperfusion.Then,48 hours later,we performed histological and m RNA/protein analyses of the spinal cord.Propofol decreased histological damage to the spinal cord,attenuated the reduction in BSCB permeability,downregulated the m RNA and protein expression levels of matrix metalloprotease-9（MMP-9） and nuclear factor-κB（NF-κB）,and upregulated the protein expression levels of occludin and claudin-5.Our findings suggest that propofol helps maintain BSCB integrity after SCIRI by reducing MMP-9 expression,by inhibiting the NF-κB signaling pathway,and by maintaining expression of tight junction proteins.

The effect of hyperglycemia on blood brain barrier of rats with focal cerebral ischemia/reperfusion injury

Objective： To determine whether hyperglycemia could aggravate the microvascular damage in ischemic stroke. Methods： Hyperglycemia model was made by injection of streptozocin through subcutaneous injection in wistar rats. Using the suture model, the rats were subjected to 3h of focal ischemia and different times of reperfusion, including 6,12,24,48,96h and 7d. TIC dyeing was used to Show the infarction area of rats. The infarctive volume of rats were calculated by computer imaging analysis system;Matrix metalloproteinase （MMP-2） and （MMP-9）were detected by immunohistochemistly and in situ hybridization histochemistly in Wistar rats. Results： The infarctive volume was siginificantly larger in hyperglycemic rats than that of nonhyperglycemic rats. The level of MMP-2, MMP-9 expression in the group of hyperglycemic rats was higher than that of nonhyperglycemic rats. Conclusion： Hyperglycemia aggravated the injury of focal ischmia-repeffusion in wistar rats and the higher expression of MMP-2,MMP-9 might be one of the mechanism in aggravation of focal ischemia/repeffusion injury.

Effects of kallikrein gene transfer on penumbral microvascular proliferation and on regional cerebral blood flow following cerebral ischemia/reperfusion injury

BACKGROUND： Recent findings have demonstrated that the kallikrein-kinin system （KKS） participates in the pathological process of cerebral ischemia/reperfusion injury. Kallikrein gene transfer exhibits neural protective effects following cerebral infarction. OBJECTIVE： To observe the effects of kallikrein gene transfer on vascular proliferation in the peripheral infarct focus and on regional cerebral blood flow （rCBF） following cerebral ischemia/reperfusion injury. DESIGN, TIME AND SETTING： The completely randomized, controlled experiment was performed at the Lin Baixin Laboratory Center, the Second Affiliated Hospital of Sun Yat-sun University between September 2007 and April 2008. MATERIALS： pUCI9-HTK plasmid was constructed and maintained in the Laboratory for Neurology, the Second Affiliated Hospital of Sun Yat-sen University, China. Mouse anti-human kallikrein 1 monoclonal antibody was purchased from R＆D Systems, USA. METHODS Ninety healthy, male, Sprague Dawley rats were used. Middle cerebral artery occlusion （MCAO） was established in all rats to induce cerebral ischemia/reperfusion injury. Following MCAO establishment, all rats were randomly divided into three groups （n = 30）： blank control, saline, and pAdCMV-HTK. The saline and pAdCMV-HTK groups were stereotactically micro-injected with 5μL of physiological saline or with pAdCMV-HTK [multiplicity of infection （MOI） = 20], respectively, into the ischemic penumbra. In the blank control group, only sham injection was performed. MAIN OUTCOME MEASURES： At 12, 24, and 72 hours after treatment, cerebral infarction volume was measured by 2, 3, 5-triphenyltetrazolium chloride （TTC） staining. Exogenous HTK expression, as well as regional vascular endothelial growth factor （VEGF） expression, was detected by immunohistochemistry. rCBF was examined by 14C-iodoantipyrine micro tracing. In addition, neurological severity score （NSS） was performed. Higher scores indicated more severe neurological deficits. RESULTS： NSS results demonstrated that compared with the saline and the blank control groups, the pAdCMV-HTK group exhibited lower NSSs 24 hours after pAdCMV-HTK injection （P 〈 0.05）. The NSSs were further decreased after 72 hours （P 〈 0.01）. Cerebral infarction volume at 24 hours, and in particular at 72 hours after treatment, was significantly reduced in the pAdCMV-HTK group compared with the blank control and saline groups （P 〈 0.05）. The rCBF in the area surrounding the infarction lesion was slightly decreased in all groups compared with the contralateral area. At 24 and 72 hours following treatment, the rCBF in the peripheral infarction lesion was significantly elevated in the pAdCMV-HTK group compared with the blank control and saline groups （P 〈 0.05）. Immunohistochemistry results revealed that VEGF-positive cells were primarily found in the cortex and in some white matter surrounding the cerebral infarction lesion. In addition, the expression of VEGF in the pAdCMV-HTK group was significantly higher compared with that in the blank control and saline groups at 12, 24, and 72 hours following treatment （P 〈 0.05）. CONCLUSION： Following cerebral ischemia/reperfusion, kallikrein gene transfer can promote vascular proliferation in the brain tissue surrounding the infarction lesion, improve rCBF, and reduce infarction volume, thereby exhibiting protective effects to attenuate neurological deficits.