Postischemic Treatment Research Articles

Post-ischemic treatment of nalmefene hydrochloride attenuated lung ischemia-reperfusion injury in rats via the Sirt1/Nrf2/HO-1 pathway with inhibition of ferroptosis

Objective: To study the effect and mechanism of post-ischemic treatment of nalmefene in alleviating the lung ischemia-reperfusion injury by inhibiting ferroptosis through activation of the Sirt 1/Nrf 2/HO-1 axis. Methods: A total of 60 rats were randomly divided into six groups equally (n=10): the sham group, the model group(I/R), the nalmefene group, the nalmefene+EX527 group, the nalmefene+ML385 group, the nalmefene+Fe-citrate group (nalmefene+Fe group). The sham group without drug treatment was not treated with ischemia-reperfusion. The pulmonary ischemia-reperfusion model was established by occlusion of the left pulmonary hilum in the model group without drug treatment. After ischemic treatment, the nalmefene group was injected with nalmefene (15 μg/kg) via the tail vein at 5 minutes before reperfusion. The nalmefene+EX527 group, the nalmefene+ML385 group, and the nalmefene+Fe group were injected intraperitoneally with EX527 (5 mg/kg), ML385 (30 mg/kg), Fe-citrate(15 mg/kg), respectively, 2 h before moulding and then injected with nalmefene (15 μg/kg) via the tail vein at 5 minutes before reperfusion. All rats were sacrificed three hours after reperfusion, and the specimens from the upper lobe of the left lung tissue were preserved. The degree of lung tissue injury and the wet/dry weight ratio were assessed in each group of rats. Fe 2+, MDA, TNF-α, and IL-6 content, GSH activity and the expression levels of Sirt1, Nrf2, HO-1, ACSL4 and GPX4 were determined. Results: Compared with the sham group, the wet/dry weight ratio, lung tissue injury score, ACSL 4 expression level, Fe 2+, TNF-α, IL-6 and MDA content, Sirt 1, Nrf 2, HO-1 messenger RNA and protein expression levels were significantly increased (P<0.01), while GPX 4 expression level and GSH activity were significantly decreased in the model group (P<0.01). Compared with the model group, wet/dry weight ratio, lung tissue injury score, ACSL 4 expression level, Fe 2+, TNF-α, IL-6, and MDA content decreased significantly (P<0.01), Nrf 2, HO-1 messenger RNA and protein, GPX 4 expression, and GSH activity were significantly increased in the nalmefene group and the nalmefene+EX527 group (P<0.01). Sirt 1 messenger RNA and protein expression increased significantly in the nalmefene (P<0.01) and the nalmefene+EX527 groups (P>0.05). In the nalmefene+ML385 group, the wet/dry weight ratio, lung tissue injury score, TNF-α and IL-6 content were decreased significantly (P<0.01), while Sirt 1 messenger RNA and protein expression levels were significantly increased (P<0.01), but there were no significant changes in Nrf 2, HO-1 messenger RNA and protein expression levels, ACSL 4 and GPX 4 expression levels, Fe 2+, MDA content, and GSH activity (P>0.05). In the nalmefene+Fe group, wet/dry weight ratio, lung-injury score, TNF-α, IL-6, MDA content were decreased significantly (P<0.01), messenger RNA and protein expression levels of Sirt 1, Nrf 2, HO-1, and GSH activity were increased significantly (P<0.01), but there were no significant changes in Fe 2+content, ACSL 4 and GPX 4 expression levels (P>0.05). Compared with the nalmefene group, in the nalmefene+EX527 group, the nalmefene+ML385 group and the nalmefene+Fe group, wet/dry weight ratio, lung tissue damage score, ACSL 4 expression level, TNF-α, IL-6 and MDA content were significantly increased (P<0.01), the expression level of GPX 4 and GSH activity were significantly decreased (P<0.01). The expression levels of Sirt 1, Nrf 2, HO-1 messenger RNA and protein were significantly decreased in the nalmefene+EX527 group (P<0.01). The expression levels of Nrf 2, HO-1 messenger RNA and protein decreased significantly in the namemefene+ML385 group (P<0.01), but there was no significant change in Sirt 1 messenger RNA and protein expression level (P>0.05). Sirt 1, Nrf 2, HO-1 messenger RNA-protein expression levels did not change significantly in the nalmefene+Fe group (P>0.05). Conclusion: Post-ischemic treatment with nalmefene hydrochloride may alleviate pulmonary ischemia-reperfusion injury by inhibiting ferroptosis through activation of the Sirt 1/Nrf 2/HO-1 axis.

Soluble Urokinase Plasminogen Activator Receptor (suPAR) Promotes Cardioprotective Intracellular Signalling in Myocardial Ischaemia

Soluble urokinase Plasminogen Activator Receptor (suPAR) is an emerging biomarker of inflammation capable of predicting cardiovascular outcomes. However, the biological activity of suPAR on the heart is poorly understood. Potential cardiac intracellular signalling pathways triggered by suPAR were explored. Isolated Langendorff rat hearts undergoing global ischaemia received treatment with either suPAR infusion or perfusion buffer (controls). Proteins were extracted from hearts for each treatment group, and Troponin T in rat outflow perfusates following ischaemia were measured. The phosphorylation status of each kinase was characterised using a phospho-specific antibody and a ratio of phosphorylation to the total protein was calculated. Analysis between treatment groups was performed with unpaired t-test with Welch’s correction. Hearts receiving suPAR treatment after ischaemia showed a 1.4-fold increase in Akt phosphorylation, and a 1.6-fold increase in Akt phosphorylation if suPAR was given before ischaemia. The ratio of p-Akt/Akt was 1.83±0.14 (n=7) in the post-ischaemic suPAR treatment group vs 1.3±0.04 (n=6) in controls (p=0.007). Pre-ischaemic suPAR treatment gave a p-Akt/Akt ratio of 1.08±0.03 (n=8) vs 0.67±0.11 (n=5) in controls (p=0.01). Upstream signalling ERK1 and ERK2 phosphorylation also increased at 2.6-fold (p=0.009) and 1.9-fold (p=0.01), respectively, in suPAR treated heart vs controls. Finally, suPAR treated hearts obtained lower Troponin T values compared with controls after recovery from ischaemia (p<0.001). suPAR infusion promoted the phosphorylation of ERK and Akt kinases in isolated rat heart tissue and reduced related cardiac troponin release after a period of global no-flow ischaemia. These actions suggest that suPAR is a novel activator of the reperfusion injury salvage kinase (RISK) pathway.

Intrarenal Anti-Leptin Treatment Attenuates Ischemia and Reperfusion Injury

Introduction: Renal ischemia and reperfusion (IR) injury introduces cellular stress and is the main cause of acute kidney damage. Renal cells exposed to noxious stress induce the expression of the pleiotropic hormone leptin. As we have previously revealed a deleterious stress-related role for leptin expression, these results suggested that leptin is also involved in pathological renal remodeling. The systemic functions of leptin preclude the study of its local effects using conventional approaches. We have therefore designed a method to locally perturb leptin activity in specific tissues without affecting its systemic levels. This study explores whether local anti-leptin strategy is renoprotective in a post-IR porcine kidney model. Methods: We induced renal IR injury in pigs by exposing kidneys to ischemia and revascularization. Upon reperfusion, kidneys instantly received an intra-arterial bolus of either a leptin antagonist (LepA) or saline solution. Peripheral blood was sampled to assess systemic leptin, IL-6, creatinine, and BUN levels, and postoperative tissue samples were analyzed by hematoxylin and eosin histochemistry and immunohistochemistry. Results: Histology of IR/saline kidneys exhibited extensive necrosis of proximal tubular epithelial cells, as well as elevated levels of apoptosis markers and inflammation. In contrast, IR/LepA kidneys showed no signs of necrosis or inflammation with normal IL-6 and tall-like receptor 4 levels. LepA treatment led to upregulation in mRNA levels of leptin, leptin receptor, ERK1/2, STAT3, and transport molecule Na/H exchanger-3. Conclusions: Local, intrarenal postischemic LepA treatment at reperfusion prevented apoptosis and inflammation and was renoprotective. Selective intrarenal administration of LepA at reperfusion may provide a viable option for clinical implementation.

Therapeutic effects of a standardized-flavonoid Diospyros kaki L.f. leaf extract on transient focal cerebral ischemia-induced brain injury in mice.

This study aimed to investigate the neuroprotective and therapeutic effects of Diospyros kaki L.f. leaves (DK) on transient focal cerebral ischemic injury and underlying mechanisms using a middle cerebral artery occlusion (MCAO) model of mice. The animals received the MCAO operation on day 0. The daily administrations of DK (50 and 100mg/kg, p.o) and edaravone (6mg/kg, i.v), a reference drug with radical scavenging activity, were started 7days before (pre-treatment) or immediately after the MCAO operation (post-treatment) and continued during the experimental period. Histochemical, biochemical, and neurological changes and cognitive performance were evaluated. MCAO caused cerebral infarction and neuronal cell loss in the cortex, striatum, and hippocampus in a manner accompanied by spatial cognitive deficits. These neurological and cognitive impairments caused by MCAO were significantly attenuated by pre- and post-ischemic treatments with DK and edaravone, suggesting that DK, like edaravone, has therapeutic potential for cerebral ischemia-induced brain damage. DK and edaravone suppressed MCAO-induced changes in biomarkers for apoptosis (TUNEL-positive cell number and cleaved caspase-3 protein expression) and oxidative stress (glutathione and malondialdehyde contents) in the brain. Interestingly, DK, but not edaravone, mitigated an increase in blood-brain permeability and down-regulation of vascular endothelial growth factor protein expression caused by MCAO. Although the exact chemical constituents implicated in the effects of DK remain to be clarified, the present results indicate that DK exerts neuroprotective and therapeutic activity against transient focal cerebral ischemia-induced injury probably by suppressing oxidative stress, apoptotic process, and mechanisms impairing blood-brain barrier integrity in the brain.

Therapeutic Benefits of Adropin in Aged Mice After Transient Ischemic Stroke via Reduction of Blood-Brain Barrier Damage.

Adropin is a peptide encoded by the energy homeostasis-associated gene (Enho) that is highly expressed in the brain. Aging and stroke are associated with reduced adropin levels in the brain and plasma. We showed that treatment with synthetic adropin provides long-lasting neuroprotection in permanent ischemic stroke. However, it is unknown whether the protective effects of adropin are observed in aged animals following cerebral ischemia/reperfusion. We hypothesized that adropin provides neuroprotection in aged mice subjected to transient middle cerebral artery occlusion. Aged (18-24 months old) male mice were subjected to 30 minutes of middle cerebral artery occlusion followed by 48 hours or 14 days of reperfusion. Sensorimotor (weight grip test and open field) and cognitive tests (Y-maze and novel object recognition) were performed at defined time points. Infarct volume was quantified by 2,3,5-triphenyltetrazolium chloride staining at 48 hours or Cresyl violet staining at 14 days post-middle cerebral artery occlusion. Blood-brain barrier damage, tight junction proteins, and MMP-9 (matrix metalloproteinase-9) were assessed 48 hours after middle cerebral artery occlusion by ELISA and Western blots. Genetic deletion of Enho significantly increased infarct volume and worsened neurological function, whereas overexpression of adropin dramatically reduced stroke volume compared to wild-type controls. Postischemic treatment with synthetic adropin peptide given at the onset of reperfusion markedly reduced infarct volume, brain edema, and significantly improved locomotor function and muscular strength at 48 hours. Delayed adropin treatment (4 hours after the stroke onset) reduced body weight loss, infarct volume, and muscular strength dysfunction, and improved long-term cognitive function. Postischemic adropin treatment significantly reduced blood-brain barrier damage. This effect was associated with reduced MMP-9 and preservation of tight junction proteins by adropin treatment. These data unveil a promising neuroprotective role of adropin in the aged brain after transient ischemic stroke via reducing neurovascular damage. These findings suggest that poststroke adropin therapy is a potential strategy to minimize brain injury and improve functional recovery in ischemic stroke patients.

Resveratrol promotes liver cell survival in mice liver-induced ischemia-reperfusion through unfolded protein response: a possible approach in liver transplantation

BackgroundIschemia-reperfusion (I/R) of the liver is a multifactorial condition that happens during transplantation and surgery. The deleterious effects of I/R result from the acute production of reactive oxygen species (ROS), which can trigger immediate tissue damage and induce a series of destructive cellular responses, including apoptosis organ failure and inflammation. The production of ROS in the I/R process can damage the antioxidant system and cause liver damage. Resveratrol has been shown to have antioxidant properties in several investigations. Here, we address the therapeutic effect of resveratrol on I/R-induced liver injury by focusing on unfolded protein response (UPR) signaling pathway.MethodsFive minutes before reperfusion, resveratrol was injected into the tail vein of mice. They were ischemic for 1 h and then re-perfused for 3 h before being slaughtered (I/R). The activity of liver enzymes and the expression levels of genes involved in the unfolded protein response pathway were used to measure the hepatic damage.ResultsOur results revealed that the low dose of resveratrol (0.02 and 0.2 mg/kg) post-ischemic treatment significantly reduced the ALT and AST levels. In addition, compared with the control group, the expression of UPR pathway genes GRP78, PERK, IRE1α, CHOP, and XBP1 was significantly reduced in the resveratrol group. In the mice that received lower doses of resveratrol (0.02 and 0.2 mg/kg), the histopathological changes induced by I/R were significantly improved; however, the highest dose (2 mg/kg) of resveratrol could not significantly protect and solve the I/R damage.ConclusionThe findings of this study suggest that hepatic ischemia occurs after liver transplantation and that receiving low-dose resveratrol treatment before reperfusion may promote graft survival through inhibition of UPR arms, especially PERK and IRE1α.

Targeted BRD4 protein degradation by dBET1 ameliorates acute ischemic brain injury and improves functional outcomes associated with reduced neuroinflammation and oxidative stress and preservation of blood–brain barrier integrity

Bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal domain (BET) protein family, plays a crucial role in regulating inflammation and oxidative stress that are tightly related to stroke development and progression. Consequently, BRD4 blockade has attracted increasing interest for associated neurological diseases, including stroke. dBET1 is a novel and effective BRD4 degrader through the proteolysis-targeting chimera (PROTAC) strategy. We hypothesized that dBET1 protects against brain damage and neurological deficits in a transient focal ischemic stroke mouse model by reducing inflammation and oxidative stress and preserving the blood–brain barrier (BBB) integrity. Post-ischemic dBET1 treatment starting 4 h after stroke onset significantly ameliorated severe neurological deficits and reduced infarct volume 48 h after stroke. dBET1 markedly reduced inflammation and oxidative stress after stroke, indicated by multiple pro-inflammatory cytokines and chemokines including IL-1β, IL-6, TNF-α, CCL2, CXCL1 and CXCL10, and oxidative damage markers 4-hydroxynonenal (4-HNE) and gp91phox and antioxidative proteins SOD2 and GPx1. Meanwhile, stroke-induced BBB disruption, increased MMP-9 levels, neutrophil infiltration, and increased ICAM-1 were significantly attenuated by dBET1 treatment. Post-ischemic dBET1 administration also attenuated ischemia-induced reactive gliosis in microglia and astrocytes. Overall, these findings demonstrate that BRD4 degradation by dBET1 improves acute stroke outcomes, which is associated with reduced neuroinflammation and oxidative stress and preservation of BBB integrity. This study identifies a novel role of BET proteins in the mechanisms resulting in ischemic brain damage, which can be leveraged to develop novel therapies.

Long‐Term Cardioprotective Potential of Exogenous Ubiquitin in Myocardial Ischemia/Reperfusion Injury

BackgroundCoronary heart disease (CHD) is a leading cause of death worldwide. Myocardial ischemia/reperfusion (I/R) injury is a clinical manifestation of CHD. Timely reperfusion of coronary vessels in an ischemic injury is essential to limit the infarct size. However, reperfusion itself can induce cardiac cell death, and lead to increased adverse remodeling after an I/R injury. Previously, our lab has shown that pretreatment with ubiquitin (UB; a highly evolutionarily conserved protein) decreases inflammatory response and preserves heart function 3 days post‐I/R. The objective of this study is to investigate the long‐term cardioprotective potential of UB post‐I/R. To enhance the clinical relevance, UB treatment was started at the time of reperfusion. It is hypothesized that UB treatment has long‐term cardioprotective potential, improves cardiac function, and attenuates cardiac remodeling post injury.MethodsC57BL/6 male mice underwent ligation of the left anterior descending coronary artery (LCA) for 45 min. Mice were then treated with UB (1 µg/g body weight, i.p. injection) or saline at time of reperfusion of LCA. Sham animals underwent the same surgery without the ligation of LCA. Mice were treated with UB or saline twice/day (i.p. injections) for 3 days followed by treatment with UB (1 μg/g/h)or saline using micro‐osmotic pumps for 28 days. Heart function was measured at 3, 7, 14 and 28 days post‐I/R using echocardiography. Mice were sacrificed at 28 days post‐I/R. Left ventricular (LV) tissue was used to measure remodeling parameters and serum samples were used to measure a panel of 23 cytokines/chemokines using multiplex assay.ResultsI/R+saline group exhibited a significant reduction in heart function as observed by decreased percent fractional shortening (%FS) and percent ejection fraction (%EF) vs sham (p&lt;0.05) at 3, 7, 14 and 28 days post‐I/R. However, I/R+UB group showed significantly higher %FS and %EF vs I/R+saline (p&lt;0.05) throughout the observation period. Increases in fibrosis (measured by trichrome staining), myocyte apoptosis (measured using TUNEL assay), and hypertrophy (measured using WGA staining) were significantly lower in I/R+UB vs I/R+saline (p&lt;0.05). Expression of MMP‐2 and TIMP‐4 was lower, while expression of MMP‐9 (active) was significantly higher in I/R+UB vs I/R+saline (p&lt;0.05). Serum levels of IL‐6, IL‐2 and G‐CSF were significantly lower in I/R+UB vs I/R+saline (p&lt;0.05).ConclusionPost‐ischemic UB treatment improves heart function, and associates with decreased myocardial remodeling parameters (fibrosis, apoptosis, and hypertrophy) as well as decreased serum cytokines/chemokines levels.

Anti-Protease Activity Deficient Secretory Leukocyte Protease Inhibitor (SLPI) Exerts Cardioprotective Effect against Myocardial Ischaemia/Reperfusion.

Inhibition of proteases shows therapeutic potential. Our previous studies demonstrated the cardioprotection by the Secretory Leukocyte Protease Inhibitor (SLPI) against myocardial ischaemia/reperfusion (I/R) injury. However, it is unclear whether the cardioprotective effect of SLPI seen in our previous works is due to the inhibition of protease enzymes. Several studies demonstrate that the anti-protease independent activity of SLPI could provide therapeutic benefits. Here, we show for the first time that recombinant protein of anti-protease deficient mutant SLPI (L72K, M73G, L74G) (mt-SLPI) could significantly reduce cell death and intracellular reactive oxygen species (ROS) production against an in vitro simulated I/R injury. Furthermore, post-ischaemic treatment of mt-SLPI is found to significantly reduce infarct size and cardiac biomarkers lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) activity, improve cardiac functions, attenuate I/R induced-p38 MAPK phosphorylation, and reduce apoptotic regulatory protein levels, including Bax, cleaved-Caspase-3 and total Capase-8, in rats subjected to an in vivo I/R injury. Additionally, the beneficial effect of mt-SLPI was not significantly different from the wildtype (wt-SLPI). In summary, SLPI could provide cardioprotection without anti-protease activity, which could be more clinically beneficial in terms of providing cardioprotection without interfering with basal serine protease activity.

Abstract WMP113: Protective Effects Of Adropin In Aged Mice Following Permanent And Transient Ischemic Stroke

Background: Adropin is a peptide encoded by the energy homeostasis associated gene ( Enho ) highly expressed in the brain. Our unpublished data revealed robust effects of adropin on reducing neurovascular damage and behavioral impairments in young mice following stroke. However, it is unknown whether these protective effects of adropin are observed in aged animals after stroke. Hypothesis: Considering that aging is the most critical risk factor for stroke, we hypothesized that adropin provides neuroprotection in aged mice subjected to ischemic stroke. Methods: Aged (18-24 months old) male adropin knockout ( Enho -/- ), adropin overexpressing transgenics (AdrTg), and their corresponding control littermates were subjected to permanent middle cerebral artery occlusion (pMCAO). Adhesive removal, nesting behavior, and novel object recognition test were conducted before and up to 14 days after MCAO. Infarct volume was quantified by MRI at 14 days post-MCAO. In a separate experiment, we utilized aged male Enho -/- and AdrTg mice subjected to 30 min of transient MCAO (tMCAO). To investigate the potential therapeutic benefit of adropin in stroke, aged wild-type mice were randomly selected to receive a bolus injection of vehicle or synthetic adropin (900 nmol/kg; i.v.) at the onset of ischemia. Neurobehavioral tests were performed before and at 48h after stroke, and infarct size was measured by TTC staining at 48h. Results: Adropin deficiency consistently increased infarct volume and exacerbated neurological impairments in permanent and transient MCAO mice compared to Enho -/- . In contrast, transgenic overexpression of adropin significantly improved neurological function compared to WT mice. Also, AdrTg mice displayed better recovery of recognition memory function, whereas Enho -/- mice exhibited worse stroke outcomes compared to wild-type littermates following pMCAO. Similarly, treatment with synthetic adropin peptide resulted in a smaller stroke volume and better motor function recovery than vehicle-treated mice following tMCAO. Conclusions: These findings consistently show that transgenic overexpression of adropin or post-ischemic treatment with adropin peptide is neuroprotective in aged mice subjected to ischemic stroke.

Hypothermia Induced by Oxcarbazepine after Transient Forebrain Ischemia Exerts Therapeutic Neuroprotection through Transient Receptor Potential Vanilloid Type 1 and 4 in Gerbils.

In the present study, we investigated the neuroprotective effect of post-ischemic treatment with oxcarbazepine (OXC; an anticonvulsant compound) against ischemic injury induced by transient forebrain ischemia and its mechanisms in gerbils. Transient ischemia was induced in the forebrain by occlusion of both common carotid arteries for 5 min under normothermic conditions (37 ± 0.2 °C). The ischemic gerbils were treated with vehicle, hypothermia (whole-body cooling; 33.0 ± 0.2 °C), or 200 mg/kg OXC. Post-ischemic treatments with vehicle and hypothermia failed to attenuate and improve, respectively, ischemia-induced hyperactivity and cognitive impairment (decline in spatial and short-term memory). However, post-ischemic treatment with OXC significantly attenuated the hyperactivity and the cognitive impairment, showing that OXC treatment significantly reduced body temperature (to about 33 °C). When the hippocampus was histopathologically examined, pyramidal cells (principal neurons) were dead (lost) in the subfield Cornu Ammonis 1 (CA1) of the gerbils treated with vehicle and hypothermia on Day 4 after ischemia, but these cells were saved in the gerbils treated with OXC. In the gerbils treated with OXC after ischemia, the expression of transient receptor potential vanilloid type 1 (TRPV1; one of the transient receptor potential cation channels) was significantly increased in the CA1 region compared with that in the gerbils treated with vehicle and hypothermia. In brief, our results showed that OXC-induced hypothermia after transient forebrain ischemia effectively protected against ischemia–reperfusion injury through an increase in TRPV1 expression in the gerbil hippocampal CA1 region, indicating that TRPV1 is involved in OXC-induced hypothermia.

Methyl palmitate protects heart against ischemia/reperfusion-induced injury through G-protein coupled receptor 40-mediated activation of the PI3K/AKT pathway

This study aimed to investigate whether methyl palmitate (MP) exerts cardioprotective effect against the ischemia/reperfusion (I/R) injury and its mechanisms underlying. The cultured adult cardiomyocytes were treated with vehicle or lactic acid ischemic buffer (pH 6.8) during hypoxia/reoxygenation. In addition, the cardioprotective effect of MP was evaluated using the ex vivo heart model of I/R injury. Here, we found that MP significantly reduced the I/R-induced cardiomyocyte death. Treatment with GW1100 (a GPR40-antagonist) or wortmannin (a phosphatidylinositol 3-kinase, PI3K, specific inhibitor) significantly attenuated the level of phospho-AKT (p-AKT) and abolished the MP-induced cardioprotection against the I/R-induced injury. Using the ex vivo I/R model, we also demonstrated that pretreatment with MP significantly reduced the size of myocardial infarction and the levels of cleaved-caspase 3 and MDA, and increased the protein levels of GPR40 and p-AKT induced by I/R. The cardioprotective effect of MP was evaluated also using the in vivo heart model of I/R injury. We demonstrated that post-ischemic treatment with MP significantly attenuated the size of myocardial infarction and the serum level of CK-MB induced by in vivo I/R model. Taken together, our data suggest that MP could provide significant cardioprotection against the I/R injury, and the underlying mechanisms by which MP prevented the cardiomyocyte death might be mediated through the GPR40-activated PI3K/AKT signaling pathways. These findings suggest the potential applications of MP in the treatment of I/R-induced heart injury.

Post-Ischemic Treatment of Recombinant Human Secretory Leukocyte Protease Inhibitor (rhSLPI) Reduced Myocardial Ischemia/Reperfusion Injury.

Myocardial ischemia/reperfusion (I/R) injury is a major cause of mortality and morbidity worldwide. Among factors contributing to I/R injury, proteolytic enzymes could also cause cellular injury, expand the injured area and induce inflammation, which then lead to cardiac dysfunction. Therefore, protease inhibition seems to provide therapeutic benefits. Previous studies showed the cardioprotective effect of secretory leukocyte protease inhibitor (SLPI) against myocardial I/R injury. However, the effect of a post-ischemic treatment with SLPI in an in vivo I/R model has never been investigated. In the present study, recombinant human (rh) SLPI (rhSLPI) was systemically injected during coronary artery occlusion or at the onset of reperfusion. The results show that post-ischemic treatment with rhSLPI could significantly reduce infarct size, Lactate Dehydrogenase (LDH) and Creatine kinase-MB (CK-MB) activity, inflammatory cytokines and protein carbonyl levels, as well as improving cardiac function. The cardioprotective effect of rhSLPI is associated with the attenuation of p38 MAPK phosphorylation, Bax, caspase-3 and -8 protein levels and enhancement of pro-survival kinase Akt and ERK1/2 phosphorylation. In summary, this is the first report showing the cardioprotective effects against myocardial I/R injury of post-ischemic treatments with rhSLPI in vivo. Thus, these results suggest that SLPI could be used as a novel therapeutic strategy to reduce myocardial I/R injury.

Design, synthesis and biological evaluation of brain penetrant benzazepine-based histone deacetylase 6 inhibitors for alleviating stroke-induced brain infarction.

Histone deacetylase 6 (HDAC6) has become a promising therapeutic target for central nervous system diseases due to its more complex protein structure and biological functions. However, low brain penetration of reported HDAC6 inhibitors limits its clinical application in neurological disorders. Therefore, the benzazepine, a brain-penetrant rigid fragment, was utilized to design a series of selective HDAC6 inhibitors to improve brain bioavailability. Various synthetic strategies were applied to assemble the tetrahydro-benzazepine ring, and 22 compounds were synthesized. Among them, compound 5 showed low nanomolar potency and strong isozyme selectivity for the inhibition of HDAC6 (IC50=1.8nM, 141-fold selectivity over HDAC1) with efficient binding patterns like coordination with the zinc ion and π-π stacking effect. Western blot results showed it could efficiently transport into SH-SY5Y cells and selectively enhance the acetylation level of α-tubulin with a moderate effect on Histone H3. Notably, pharmacokinetic studies demonstrated that compound 5 (brain/plasma ratio of 2.30) had an excellent ability to penetrate the blood-brain barrier of C57 mice. In male rats with transient middle cerebral artery occlusion (MCAO), compound 5 significantly reduced the cerebral infarction from 21.22% to 11.47% and alleviated neurobehavioral deficits in post-ischemic treatment, which provided a strong rationale for pursuing HDAC6-based therapies for ischemic stroke.

CD200 Change Is Involved in Neuronal Death in Gerbil Hippocampal CA1 Field Following Transient Forebrain Ischemia and Postischemic Treatment with Risperidone Displays Neuroprotection without CD200 Change.

It has been reported that CD200 (Cluster of Differentiation 200), expressed in neurons, regulates microglial activation in the central nervous system, and a decrease in CD200 expression causes an increase in microglial activation and neuronal loss. The aim of this study was to investigate time-dependent changes in CD200 expression in the hippocampus proper (CA1, 2, and 3 fields) after transient forebrain ischemia for 5 min in gerbils. In this study, 5-min ischemia evoked neuronal death (loss) of pyramidal neurons in the CA1 field, but not in the CA2/3 fields, at 5 days postischemia. In the sham group, CD200 expression was found in pyramidal neurons of the CA1 field, and the immunoreactivity in the group with ischemia was decreased at 6 h postischemia, dramatically increased at 12 h postischemia, decreased (to level found at 6 h postischemia) at 1 and 2 days postischemia, and significantly increased again at 5 days postischemia. At 5 days postischemia, CD200 immunoreactivity was strongly expressed in microglia and GABAergic neurons. However, in the CA3 field, the change in CD200 immunoreactivity in pyramidal neurons was markedly weaker than that in the CA1 field, showing there was no expression of CD 200 in microglia and GABAergic neurons. In addition, treatment of 10 mg/kg risperidone (an atypical antipsychotic drug) after the ischemia hardly changed CD200 immunoreactivity in the CA1 field, showing that CA1 pyramidal neurons were protected from the ischemic injury. These results indicate that the transient ischemia-induced change in CD200 expression may be associated with specific and selective neuronal death in the hippocampal CA1 field following transient forebrain ischemia.

Clinacanthus nutans Mitigates Neuronal Death and Reduces Ischemic Brain Injury: Role of NF-κB-driven IL-1β Transcription.

Neuroinflammation has been shown to exacerbate ischemic brain injury, and is considered as a prime target for the development of stroke therapies. Clinacanthus nutans Lindau (C. nutans) is widely used in traditional medicine for treating insect bites, viral infection and cancer, due largely to its anti-oxidative and anti-inflammatory properties. Recently, we reported that an ethanol extract from the leaf of C. nutans could protect the brain against ischemia-triggered neuronal death and infarction. In order to further understand the molecular mechanism(s) for its beneficial effects, two experimental paradigms, namely, in vitro primary cortical neurons subjected to oxygen-glucose deprivation (OGD) and in vivo rat middle cerebral artery (MCA) occlusion, were used to dissect the anti-inflammatory effects of C. nutans extract. Using promoter assays, immunofluorescence staining, and loss-of-function (siRNA) approaches, we demonstrated that transient OGD led to marked induction of IL-1β, IL-6 and TNFα, while pretreatment with C. nutans suppressed production of inflammatory cytokines in primary neurons. C. nutans inhibited IL-1β transcription via preventing NF-κB/p65 nuclear translocation, and siRNA knockdown of either p65 or IL-1β mitigated OGD-mediated neuronal death. Correspondingly, post-ischemic treatment of C. nutans attenuated IκBα degradation and decreased IL-1β, IL-6 and TNFα production in the ischemic brain. Furthermore, IL-1β siRNA post-ischemic treatment reduced cerebral infarct, thus mimicking the beneficial effects of C. nutans. In summary, our findings demonstrated the ability for C. nutans to suppress NF-κB nuclear translocation and inhibit IL-1β transcription in ischemic models. Results further suggest the possibility for using C. nutans to prevent and treat stroke patients.

Genetic Deletion or Pharmacological Inhibition of Cyclooxygenase-2 Reduces Blood-Brain Barrier Damage in Experimental Ischemic Stroke.

Cyclooxygenase (COX)-2 and matrix metalloproteinase (MMP)-9 are two crucial mediators contributing to blood-brain barrier (BBB) damage during cerebral ischemia. However, it is not known whether MMP-9 activation is involved in COX-2-mediated BBB disruption in ischemic stroke. In this study, we hypothesized that genetic deletion or pharmacological inhibition of COX-2 reduces BBB damage by reducing MMP-9 activity in a mouse model of ischemic stroke. Male COX-2 knockout (COX-2−/−) and wild-type (WT) mice were subjected to 60 min of middle cerebral artery occlusion (MCAO) followed by 24 h of reperfusion. Genetic deletion of COX-2 or post-ischemic treatment with CAY10404, a highly selective COX-2 inhibitor, significantly reduced BBB damage and hemorrhagic transformation, as assessed by immunoglobulin G (IgG) extravasation and brain hemoglobin (Hb) levels, respectively. Immunoblotting analysis showed that tight junction proteins (TJPs) zonula occludens (ZO)-1 and occludin as well as junctional adhesion molecule-A (JAM-A) and the basal lamina protein collagen IV were dramatically reduced in the ischemic brain. Stroke-induced loss of these BBB structural proteins was significantly attenuated in COX-2−/− mice. Similarly, stroke-induced loss of ZO-1 and occludin was significantly attenuated by CAY10404 treatment. Ischemia-induced increase in MMP-9 protein levels in the ipsilateral cerebral cortex was significantly reduced in COX-2−/− mice. Stroke induced a dramatic increase in MMP-9 enzymatic activity in the ischemic cortex, which was markedly reduced by COX-2 gene deficiency or pharmacological inhibition with CAY10404. Levels of myeloperoxidase (MPO, an indicator of neutrophil infiltration into the brain parenchyma), neutrophil elastase (NE), and lipocalin-2 (LCN2, also known as neutrophil gelatinase-associated lipocalin), measured by western blot and specific ELISA kits, respectively, were markedly increased in the ischemic brain. Increased levels of markers for neutrophil infiltration were significantly reduced in COX-2−/− mice compared with WT controls following stroke. Altogether, neurovascular protective effects of COX-2 blockade are associated with reduced BBB damage, MMP-9 expression/activity and neutrophil infiltration. Our study shows for the first time that MMP-9 is an important downstream effector contributing to COX-2-mediated neurovascular damage in ischemic stroke. Targeting the COX-2/MMP-9 pathway could represent a promising strategy to reduce neuroinflammatory events in order to preserve the BBB integrity and ameliorate ischemic stroke injury.

PERK (Protein Kinase RNA-Like ER Kinase) Branch of the Unfolded Protein Response Confers Neuroprotection in Ischemic Stroke by Suppressing Protein Synthesis.

Background and Purpose- Ischemic stroke impairs endoplasmic reticulum (ER) function, causes ER stress, and activates the unfolded protein response. The unfolded protein response consists of 3 branches controlled by ER stress sensor proteins, which include PERK (protein kinase RNA-like ER kinase). Activated PERK phosphorylates eIF2α (eukaryotic initiation factor 2 alpha), resulting in inhibition of global protein synthesis. Here, we aimed to clarify the role of the PERK unfolded protein response branch in stroke. Methods- Neuron-specific and tamoxifen-inducible PERK conditional knockout (cKO) mice were generated by cross-breeding Camk2a-CreERT2 with Perkf/f mice. Transient middle cerebral artery occlusion was used to induce stroke. Short- and long-term stroke outcomes were evaluated. Protein synthesis in the brain was assessed using a surface-sensing-of-translation approach. Results- After tamoxifen-induced deletion of Perk in forebrain neurons was confirmed in PERK-cKO mice, PERK-cKO and control mice were subjected to transient middle cerebral artery occlusion and 3 days or 3 weeks recovery. PERK-cKO mice had larger infarcts and worse neurological outcomes compared with control mice, suggesting that PERK-induced eIF2α phosphorylation and subsequent suppression of translation protects neurons from ischemic stress. Indeed, better stroke outcomes were observed in PERK-cKO mice that received postischemic treatment with salubrinal, which can restore the ischemia-induced increase in phosphorylated eIF2α in these mice. Finally, our data showed that post-treatment with salubrinal improved functional recovery after stroke. Conclusions- Here, we presented the first evidence that postischemic suppression of translation induced by PERK activation promotes recovery of neurological function after stroke. This confirms and further extends our previous observations that recovery of ER function impaired by ischemic stress critically contributes to stroke outcome. Therefore, future research should include strategies to improve stroke outcome by targeting unfolded protein response branches to restore protein homeostasis in neurons.

Treadmill exercise improves neurological function by inhibiting autophagy and the binding of HMGB1 to Beclin1 in MCAO juvenile rats

AimsTreadmill exercise is a beneficial treatment following childhood stroke. Thus, studies focusing on the neuroprotective mechanism of exercise training during postischemic treatment in children with ischemic stroke are urgently needed. We evaluated the effects of treadmill exercise on autophagy after cerebral ischemia in young rats. Main methodsRats (23–25 days old) underwent cerebral ischemia-reperfusion (CI/R) surgery. The experimental animals were divided into 5 groups, and some groups received either treadmill exercise, a rapamycin (RAPA) injection or combination therapy for 3 or 7 days. We performed a series of experimental tests including neurological scoring, hematoxylin-eosin staining (H&E), Nissl staining, triphenyl tetrazolium chloride (TTC) staining, Western blot analysis (WB), immunofluorescence (IF), enzyme-linked immunosorbent assay (ELISA), transmission electron microscopy (TEM) and Terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) fluorescence. Key findingsThe experimental data indicated that treadmill exercise inhibited autophagy in the ischemic penumbra, inhibited high mobility group box 1 (HMGB1) translocation and binding to Beclin1, reduced apoptosis, reduced infarct volumes, and aided in functional recovery. However, RAPA promoted the opposite effects of treadmill exercise. SignificanceWe found that treadmill exercise improves the neurological deficits induced by CI/R by inhibiting autophagy and HMGB1 binding to Beclin1.

Postischemic supplementation of folic acid improves neuronal survival and regeneration in vitro

Supplementation of folic acid (FA) is beneficial to several neurological diseases because it promotes notch signaling and neurogenesis and reduces blood homocysteine levels. We hypothesized that postischemic supplementation of FA is beneficial for neuronal survival and regeneration. The objective of the present study was to determine the postischemic neuroprotective and neuroregenerative efficacy of FA supplementation and its effects on various cellular processes in vitro. This work benefited from the use of FA and glucose-free media to better assess the ischemic neuroprotection provided by FA supplementation. The postischemic supplementation of FA significantly improved cell viability, and the improvement was primarily by obstructing the oxygen-glucose deprivation (OGD)–activated apoptosis. Furthermore, postischemic treatment with FA significantly reduced the mitochondrial membrane depolarization and the formation of acidic organelles triggered by OGD. Moreover, FA's effect on neuroregeneration following OGD was evaluated by measuring the cell proliferation and neurite outgrowth length. Treatment with FA enhanced cell proliferation and neurite outgrowth significantly. Thus, these results revealed some of the mechanisms by which FA supplementation provided neuroprotection and neuroregeneration following ischemic injury and highlighted the need for further research into the potential of folic acid as a clinical drug for ischemic stroke.