Curcumin inhibits neuronal and vascular degeneration in retina after ischemia and reperfusion injury

Background

Neuron loss, glial activation and vascular degeneration are common sequelae of ischemia-reperfusion (I/R) injury in ocular diseases. The present study was conducted to explore the ability of curcumin to inhibit retinal I/R injury, and to investigate underlying mechanisms of the drug effects.

Methodology/Principal Findings

Different dosages of curcumin were administered. I/R injury was induced by elevating the intraocular pressure for 60 min followed by reperfusion. Cell bodies, brn3a stained cells and TUNEL positive apoptotic cells in the ganglion cell layer (GCL) were quantitated, and the number of degenerate capillaries was assessed. The activation of glial cells was measured by the expression level of GFAP. Signaling pathways including IKK-IκBα, JAK-STAT1/3, ERK/MAPK and the expression levels of β-tubulin III and MCP-1 were measured by western blot analysis. Pre-treatment using 0.01%–0.25% curcumin in diets significantly inhibited I/R-induced cell loss in GCL. 0.05% curcumin pre-treatment inhibited I/R-induced degeneration of retinal capillaries, TUNEL-positive apoptotic cell death in the GCL, brn3a stained cell loss, the I/R-induced up-regulation of MCP-1, IKKα, p-IκBα and p-STAT3 (Tyr), and down-regulation of β-tubulin III. This dose showed no effect on injury-induced GFAP overexpression. Moreover, 0.05% curcumin administered 2 days after the injury also showed a vaso-protective effect.

Conclusions/Significance

Curcumin protects retinal neurons and microvessels against I/R injury. The beneficial effects of curcumin on neurovascular degeneration may occur through its inhibitory effects on injury-induced activation of NF-κB and STAT3, and on over-expression of MCP-1. Curcumin may therefore serve as a promising candidate for retinal ischemic diseases.     

Hypothermia attenuates ischemia/reperfusion-induced endothelial cell apoptosis via alterations in apoptotic pathways and JNK signaling

Hypothermia is the most effective means of protecting the brain, heart and other organs during ischemia/reperfusion (I/R) injury. However, the precise mechanisms for hypothermia to inhibit I/R-induced endothelial cell apoptosis are not fully understood. In the present study, human umbilical endothelial cells (HUVECs) were exposed to ischemia followed by reperfusion under normothermia (37 °C) or hypothermia (33 °C). Our results showed that hypothermia markedly reduced I/R-induced endothelial cell apoptosis, the expression of cleaved caspase-3 and PARP. Moreover, hypothermia markedly reversed I/R-induced activation of Fas/caspase-8, the increase of Bax and decrease of Bcl-2. Furthermore, hypothermia inhibited JNK1/2 activation via MKP-1 induction. Together, these data demonstrate that hypothermia represses I/R-induced endothelial cell apoptosis by inhibiting both extrinsic- and intrinsic-dependent apoptotic pathways and activation of JNK1/2.     

Effects of antioxidant gene therapy on retinal neurons and oxidative stress in a model of retinal ischemia/reperfusion

Retinal ischemia/reperfusion (I/R) results in neuronal death and generation of reactive oxygen species. The aim of this study was to investigate the neuroprotective effect of manganese superoxide dismutase (SOD2) on retinal ganglion cells (RGCs) in an I/R-induced retinal injury model. One eye of each Wistar rat was pretreated with recombinant adeno-associated virus containing the SOD2 gene (AAV-SOD2) or recombinant AAV containing the GFP gene (AAV-GFP) by intravitreal injection 21 days before initiation of I/R injury. Retinal I/R injury was induced by elevating intraocular pressure for 1h, and reperfusion was established immediately afterward. The number of RGCs and the inner plexiform layer (IPL) thickness were measured by Fluorogold retrograde labeling and hematoxylin and eosin staining at 6 h, 24 h, 72 h, and 5 days after injury. Superoxide anion, the number of RGCs, IPL thickness, malondialdehyde (MDA) level, 8-hydroxy-2-deoxyguanosine (8-OHdG) level, MnSOD (manganese superoxide dismutase) activity, and nitrotyrosine level were measured by fluorescence staining, immunohistochemistry, and enzyme-linked immunosorbent analysis at 5 days after I/R injury. Severe RGC loss, reduced IPL thickness, reduced MnSOD activity, and increased superoxide ion, MDA, 8-OHdG, and nitrotyrosine production were observed after I/R injury. Administration of AAV-SOD2 significantly reduced the levels of superoxide ion, MDA, 8-OHdG, and nitrotyrosine and prevented the damage to RGCs and IPL. Delivery of the antioxidant gene inhibited I/R-induced RGC and IPL damage by reducing oxidative stress and nitrative stress, suggesting that MnSOD may be relevant for the neuroprotection of the inner retina from I/R-related diseases.     

Lycium barbarum polysaccharides reduce neuronal damage, blood-retinal barrier disruption and oxidative stress in retinal ischemia/reperfusion injury

Neuronal cell death, glial cell activation, retinal swelling and oxidative injury are complications in retinal ischemia/reperfusion (I/R) injuries. Lycium barbarum polysaccharides (LBP), extracts from the wolfberries, are good for "eye health" according to Chinese medicine. The aim of our present study is to explore the use of LBP in retinal I/R injury. Retinal I/R injury was induced by surgical occlusion of the internal carotid artery. Prior to induction of ischemia, mice were treated orally with either vehicle (PBS) or LBP (1 mg/kg) once a day for 1 week. Paraffin-embedded retinal sections were prepared. Viable cells were counted; apoptosis was assessed using TUNEL assay. Expression levels of glial fibrillary acidic protein (GFAP), aquaporin-4 (AQP4), poly(ADP-ribose) (PAR) and nitrotyrosine (NT) were investigated by immunohistochemistry. The integrity of blood-retinal barrier (BRB) was examined by IgG extravasations. Apoptosis and decreased viable cell count were found in the ganglion cell layer (GCL) and the inner nuclear layer (INL) of the vehicle-treated I/R retina. Additionally, increased retinal thickness, GFAP activation, AQP4 up-regulation, IgG extravasations and PAR expression levels were observed in the vehicle-treated I/R retina. Many of these changes were diminished or abolished in the LBP-treated I/R retina. Pre-treatment with LBP for 1 week effectively protected the retina from neuronal death, apoptosis, glial cell activation, aquaporin water channel up-regulation, disruption of BRB and oxidative stress. The present study suggests that LBP may have a neuroprotective role to play in ocular diseases for which I/R is a feature.     

Poly (ADP‐ribose) polymerase inhibition protects against myocardial ischaemia/reperfusion injury via suppressing mitophagy

Myocardial ischaemia/reperfusion (I/R) injury attenuates the beneficial effects of reperfusion therapy. Poly(ADP‐ribose) polymerase (PARP) is overactivated during myocardial I/R injury. Mitophagy plays a critical role in the development of myocardial I/R injury. However, the effect of PARP activation on mitophagy in cardiomyocytes is unknown. In this study, we found that I/R induced PARP activation and mitophagy in mouse hearts. Poly(ADP‐ribose) polymerase inhibition reduced the infarct size and suppressed mitophagy after myocardial I/R injury. In vitro, hypoxia/reoxygenation (H/R) activated PARP, promoted mitophagy and induced cell apoptosis in cardiomyocytes. Poly(ADP‐ribose) polymerase inhibition suppressed H/R‐induced mitophagy and cell apoptosis. Parkin knockdown with lentivirus vectors inhibited mitophagy and prevented cell apoptosis in H/R‐treated cells. Poly(ADP‐ribose) polymerase inhibition prevented the loss of the mitochondrial membrane potential (ΔΨm). Cyclosporin A maintained ΔΨm and suppressed mitophagy but FCCP reduced the effect of PARP inhibition on ΔΨm and promoted mitophagy, indicating the critical role of ΔΨm in H/R‐induced mitophagy. Furthermore, reactive oxygen species (ROS) and poly(ADP‐ribosylation) of CypD and TSPO might contribute to the regulation of ΔΨm by PARP. Our findings thus suggest that PARP inhibition protects against I/R‐induced cell apoptosis by suppressing excessive mitophagy via the ΔΨm/Parkin pathway.     

Ischemia-reperfusion-induced calpain activation and SERCA2a degradation are attenuated by exercise training and calpain inhibition

The Ca2+-activated protease calpain has been shown to play a deleterious role in the heart during ischemia-reperfusion (I/R). We tested the hypothesis that exercise training would minimize I/R-induced calpain activation and provide cardioprotection against I/R-induced injury. Hearts from adult male rats were isolated in a working heart preparation, and myocardial injury was induced with 25 min of global ischemia followed by 45 min of reperfusion. In sedentary control rats, I/R significantly increased calpain activity and impaired cardiac performance (cardiac work during reperfusion = 24% of baseline). Compared with sedentary animals, exercise training prevented the I/R-induced rise in calpain activity and improved cardiac work (recovery = 80% of baseline). Similar to exercise, pharmacological inhibition of calpain activity resulted in comparable cardioprotection against I/R injury (recovery = 86% of baseline). The exercise-induced protection against I/R-induced calpain activation was not due to altered myocardial protein levels of calpain or calpastatin. However, exercise training was associated with increased myocardial antioxidant enzyme activity (Mn-SOD, catalase) and a reduction in oxidative stress. Importantly, exercise training also prevented the I/R-induced degradation of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a. These findings suggest that increases in endogenous antioxidants may diminish the free radical-mediated damage and/or degradation of Ca2+ handling proteins (such as SERCA2a) typically observed after I/R. In conclusion, these results support the concept that calpain activation is an important component of I/R-induced injury and that exercise training provides cardioprotection against I/R injury, at least in part, by attenuating I/R-induced calpain activation.     

Inhibition of MMP-2 activation and release as a novel mechanism for HDL-induced cardioprotection

High density lipoproteins (HDL) protect the heart against ischemia/reperfusion (I/R) injury, and matrix metalloproteinase-2 (MMP-2) directly contributes to cardiac contractile dysfunction after I/R. To investigate the possible involvement of MMP-2 inhibition in HDL-mediated cardioprotection, isolated rat hearts underwent 20 min of low-flow ischemia and 30 min of reperfusion. Plasma-derived and synthetic HDL attenuated the I/R-induced cardiac MMP-2 activation and release in a dose-dependent way. The attenuation of I/R-induced MMP-2 activation by HDL correlated with the reduction of post-ischemic contractile dysfunction and cardiomyocyte necrosis. These results indicate prevention of MMP-2 activation as a novel mechanism for HDL-mediated cardioprotection.     

Bererine induces peripheral lymphocytes immune regulations to realize its neuroprotective effects in the cerebral ischemia/reperfusion mice

Previous studies have shown that Bererine (Ber) has significant neuroprotective effects and the present article aimed to investigate its mechanism from the aspect of peripheral immune system. We evaluated the effects of Ber 24 h after cerebral ischemia/reperfusion (I/R) on histologic injury in BALB/C mice and NOD-SCID (severe combined immunodeficient) mice lacking T and B cells. Infarct volume, neurological scores and brain water content were strikingly reduced by Ber in BALB/C mice versus NOD-SCID mice. Which suggested that Ber induces peripheral immune regulations to realize its neuroprotective effects in the cerebral I/R mice. Then we tested the lymphocytes from BALB/C lymph nodes (LNs) with their survival, activation, proliferation, cell cycle, apoptosis and differentiation induced by cytokine secretion to provide direct evidences that Ber realized its neuroprotective effects by regulating I/R-induced peripheral lymphocytes early immunoactivation and following immunotolerance and to better understand the importance of peripheral immune system following I/R insults.     

Glaucocalyxin B protects against oxygen-glucose-deprivation/reperfusion-induced neuronal injury in PC-12 cells

Oxidative stress has been implicated in the development of cerebral ischemia/reperfusion (I/R) injury. Glaucocalyxin B (GLB), one of five ent-kauranoid diterpenoids, was reported to possess neuroprotective activity. However, the effect of GLB on oxygen-glucose-deprivation/reperfusion (OGD/R)-induced cell injury in PC-12 cells has not been explored. PC-12 cells was treated with various concentrations of GLB (0, 2.5, 5 and 10 μM), and cell viability was detected using the MTT assay. PC-12 cells were pretreated with the indicated concentration of GLB (2.5-10 μM, 2 hours pretreatment), and were maintained under OGD for 3 hours, followed by 24 hours of reoxygenation. Cell viability was assessed using the MTT assay. The levels of superoxide dismutase, malondialdehyde, and glutathione peroxidase were detected using commercially available ELISA Kits. Intracellular reactive oxygen species level was measured using the fluorescent probe 2′,7′-dichlorofluorescein diacetate. The levels of Bcl-2, Bax, p-Akt, Akt, p-mTOR, mTOR were detected using Western blot. Our results revealed that GLB significantly protected PC12 cells against OGD/R-induced cell injury. In addition, GLB efficiently inhibited oxidative stress and cell apoptosis in OGD/R-stimulated PC-12 cells. Mechanistic studies revealed that pretreatment with GLB could induce the activation of Akt/mTOR signaling pathway resulting in protection of OGD-treated PC12 cells. In conclusion, our data indicate for the first time that GLB protects against OGD/R-induced neuronal injury in PC-12 cells. The mechanism of the protective effect of GLB is partially associated with activation of the Akt/mTOR signaling pathway. Thus, GLB may be a potential agent for protection against cerebral I/R injury.     

sRAGE抑制缺血/再灌注引起的心肌细胞自噬

可溶性晚期糖基化终末产物受体（soluble receptor for advanced glycation end products,sRAGE）作为内源性保护物质,能够拮抗心肌缺血/再灌注(ischemia/reperfusion,I/R）损伤发生,重要机制是减轻心肌细胞凋亡。而近年来随着细胞死亡研究的深入,细胞自噬被认为是一种新的细胞程序性死亡。sRAGE是否可以抑制缺血/再灌引起的心肌细胞自噬尚未见报道。本文研究证明,sRAGE可抑制缺血/再灌注引起的心肌细胞自噬。以心肌细胞缺氧/复氧模拟心肌细胞缺血/再灌注模型,蛋白质印迹检测自噬门户蛋白beclin-1的表达,激光共聚焦显微镜检测自噬小体及自噬溶酶体的形成。心肌再灌注期间,心肌细胞自噬小体增加,而自噬溶酶体下降。细胞内自噬小体堆积,说明心肌细胞缺血/再灌注使自噬小体与溶酶体结合受损,清除发生障碍。与缺血/再灌注（I/R）组比较,缺血/再灌+sRAGE（I/R+sRAGE）组的自噬流减弱。此外,自噬门户蛋白beclin-1也表达下降。以上结果从细胞形态学和蛋白水平两方面说明,sRAGE抑制了I/R引起的心肌细胞自噬。换言之,sRAGE可以直接作用于心肌细胞拮抗缺血/再灌注损伤,其保护性作用可能与抑制心肌细胞自噬有关。     

Mitochondrial oxidant stress triggers cell death in simulated ischemia-reperfusion

To clarify the relationship between reactive oxygen species (ROS) and cell death during ischemia-reperfusion (I/R), we studied cell death mechanisms in a cellular model of I/R. Oxidant stress during simulated ischemia was detected in the mitochondrial matrix using mito-roGFP, a ratiometric redox sensor, and by Mito-Sox Red oxidation. Reperfusion-induced death was attenuated by over-expression of Mn-superoxide dismutase (Mn-SOD) or mitochondrial phospholipid hydroperoxide glutathione peroxidase (mito-PHGPx), but not by catalase, mitochondria-targeted catalase, or Cu,Zn-SOD. Protection was also conferred by chemically distinct antioxidant compounds, and mito-roGFP oxidation was attenuated by NAC, or by scavenging of residual O₂ during the ischemia (anoxic ischemia). Mitochondrial permeability transition pore (mPTP) oscillation/opening was monitored by real-time imaging of mitochondrial calcein fluorescence. Oxidant stress caused release of calcein to the cytosol during ischemia, a response that was inhibited by chemically diverse antioxidants, anoxia, or over-expression of Mn-SOD or mito-PHGPx. These findings suggest that mitochondrial oxidant stress causes oscillation of the mPTP prior to reperfusion. Cytochrome c release from mitochondria to the cytosol was not detected until after reperfusion, and was inhibited by anoxic ischemia or antioxidant administration during ischemia. Although DNA fragmentation was detected after I/R, no evidence of Bax activation was detected. Over-expression of the anti-apoptotic protein Bcl-X_L in cardiomyocytes did not confer protection against I/R-induced cell death. Moreover, murine embryonic fibroblasts with genetic depletion of Bax and Bak, or over-expression of Bcl-X_L, failed to show protection against I/R. These findings indicate that mitochondrial ROS during ischemia triggers mPTP activation, mitochondrial depolarization, and cell death during reperfusion through a Bax/Bak-independent cell death pathway. Therefore, mitochondrial apoptosis appears to represent a redundant death pathway in this model of simulated I/R. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Role of P2X7 purinoceptors in neuroprotective mechanism of ischemic postconditioning in mice

Cerebral ischemia–reperfusion (I–R) injury is one of the primary causes of ischemic stroke. Ischemic postconditioning (iPoCo) is evolving as an important adaptive technique to contain I–R injury. Some recent studies have shown neuroprotective effect of iPoCo. However, neuroprotective mechanism of iPoCo is not clear. So, the present study has been undertaken to investigate the possible role of P2X7 purinoceptors in neuroprotective mechanism of iPoCo in mice. Bilateral carotid artery occlusion for 12 min followed by R for 24 h produced a significant rise in cerebral infarct size and neurological severity score (NSS) along with impairment of memory and motor coordination. iPoCo, involving three episodes of 10-s carotid artery occlusion with intermittent R of 10 s applied just after ischemic insult of 12 min, produced a significant decrease in cerebral infarct size and NSS along with reversal of I–R-induced impairment of memory and motor coordination. iPoCo induced neuroprotective effects were significantly abolished by pretreatment with selective purinergic P2X7 receptor blocker Brilliant Blue G (40 mg/kg intraperitoneal). It may be concluded that neuroprotective effect of iPoCo probably involves in activation of purinergic P2X7 receptors.     

Cardiomyocyte protective effects of thyroid hormone during hypoxia/reoxygenation injury through activating of IGF-1-mediated PI3K/Akt signalling

Ischaemia/reperfusion (I/R) injury is a common clinical condition that results in apoptosis and oxidative stress injury. Thyroid hormone was previously reported to elicit cardiac myocyte hypertrophy and promote cardiac function after cardiac injury. We used an in vivo mouse model of I/R injury and in vitro primary cardiomyocyte culture assays to investigate the effects of thyroid hormone on cardiomyocytes during hypoxia/reoxygenation (H/R) injury. The results showed that T3 pretreatment in vivo significantly improved left ventricular function after I/R injury. In vitro, T3 pretreatment decreased cell apoptosis rate, inhibited caspase-3 activity and decreased the Bax/Bcl-2 ration induced by H/R injury. T3 pretreatment significantly attenuated the loss of mitochondrial membrane potential. Furthermore, it was observed that T3 diminished the expression of NCX1 protein and decreased SERCA2a protein expression in H/R-induced cardiomyocytes, and T3 prevented intracellular Ca²⁺ increase during H/R injury. Also, T3 increased the expression of IGF-1, and PI3K/Akt signalling in cardiomyocytes under H/R-induced injury, and that the protective effect of T3 against H/R-induced injury was blocked by the PI3K inhibitor LY294002. IGF-1 receptor (IGF-1R) inhibitor GSK1904529A significantly inhibited the expression of IGF-1R and PI3K/Akt signalling. In summary, T3 pretreatment protects cardiomyocytes against H/R-induced injury by activating the IGF-1-mediated PI3K/Akt signalling pathway.     

Inhibition of the activity of poly (ADP-ribose) polymerase reduces heart ischaemia/reperfusion injury via suppressing JNK-mediated AIF translocation

Poly (ADP-ribose) polymerase (PARP) has been proposed to play an important role in the pathogenesis of heart ischaemia/reperfusion (I/R) injury. However, the mechanisms of PARP-mediated heart I/R injury in vivo are still not thoroughly understood. Therefore, in this study, we investigate the effect of PARP inhibition on heart I/R injury and try to elucidate the underlying mechanisms. Studies were performed with I/R rats' hearts in vivo. Ischaemia followed by reperfusion caused a significant increase in Poly (ADP-ribose) (PAR), c-Jun NH2-terminal kinase (JNK) and apoptosis-inducing factor (AIF) activity. Administration of 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ), an inhibitor of PARP, decreased myocardial infarction size from 61.11+/-7.46%[0] to 38.83+/-5.67% (P<0.05) and cells apoptosis from 35+/-5.3% to 20+/-4.1% (P<0.05) and simultaneously improved the cardiac function. Western blot analysis showed that administration of DPQ reduced the activation of JNK and attenuated mitochondrial-nuclear translocation of AIF. Additionally, administration of SP600125, an inhibitor of JNK, attenuated mitochondrial-nuclear translocation of AIF. The results of the present study demonstrated that the inhibition of PARP was able to reduce heart I/R injury in vivo. Our results also suggested that JNK may be downstream of PARP activation and be required for PARP-mediated AIF translocation. Inhibition of the activity of PARP may reduce heart I/R injury via suppressing AIF translocation mediated by JNK.     

Impaired autophagy contributes to hepatocellular damage during ischemia/reperfusion: Heme oxygenase-1 as a possible regulator

Liver ischemia and reperfusion (I/R) injury is characterized by oxidative stress that is accompanied by alterations of the endogenous defensive system. Emerging evidence suggests a protective role for autophagy induced by multiple stressors including reactive oxygen species. Meanwhile, heme oxygenase-1 (HO-1) has long been implicated in cytoprotection against oxidative stress in vitro and in vivo. Therefore, we investigated the impact of autophagy in the pathogenesis of liver I/R and its molecular mechanisms, particularly its linkage to HO-1. By using transmission electron microscopic analysis and biochemical autophagic flux assays with microtubule-associated protein 1 light chain 3-II, and beclin-1, representative autophagy markers, and p62, a selective substrate for autophagy, we found that reperfusion reduced autophagy both in the rat liver and in primary cultured hepatocytes. When autophagy was further inhibited with chloroquine or wortmannin, I/R-induced hepatocellular injury was aggravated. While livers that underwent I/R showed increased levels of mammalian target of rapamaycin and calpain 1 and 2, inhibition of calpain 1 and 2 induced an autophagic response in hepatocytes subjected to hypoxia/reoxygenation. HO-1 increased autophagy, and HO-1 reduced I/R-induced calcium overload in hepatocytes and prevented calpain 2 activation both in vivo and in vitro. Taken together, these findings suggest that the impaired autophagy during liver I/R, which is mediated by calcium overload and calpain activation, contributes to hepatocellular damage and the HO-1 system protects the liver from I/R injury through enhancing autophagy.     

Carboxy terminus of heat shock protein (HSP) 70-interacting protein (CHIP) inhibits HSP70 in the heart

Heat shock protein (HSP) 70 plays a critical role in protecting the heart from various stressor-induced cell injuries; the mechanism remains to be further understood. The present study aims to elucidate the effect of a probiotics-derived protein, LGG-derived protein p75 (LGP), in alleviating the ischemia/reperfusion (I/R)-induced heart injury. We treated rats with the I/R with or without preadministration with LGP. The levels of HSP70 and carboxy terminus of HSP70-interacting protein (CHIP) in the heart tissue were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The effect of CHIP on suppression of HSP70 and the effect of LGP on suppression of CHIP were investigated with an I/R rat model and a cell culture model. The results showed that I/R-induced infarction in the heart could be alleviated by pretreatment with LGP. HSP70 was detected in na?ve rat heart tissue extracts. I/R treatment significantly suppressed the level of HSP70 and increased the levels of CHIP in the heart. A complex of CHIP/HSP70 was detected in heart tissue extracts. The addition of recombinant CHIP to culture inhibited HSP70 in heart cells. LGP was bound CHIP in heart cells and prevented the CHIP from binding HSP70. In summary, I/R can suppress HSP70 and increase CHIP in heart cells. CHIP can suppress HSP70 that can be prevented by pretreatment with LGP. The results imply that CHIP may be a potential target in the prevention of I/R-induced heart cell injury.     

Block of P2X7 receptors could partly reverse the delayed neuronal death in area CA1 of the hippocampus after transient global cerebral ischemia

Transient global ischemia (which closely resembles clinical situations such as cardiac arrest, near drowning or severe systemic hypotension during surgical procedures), often induces delayed neuronal death in the brain, especially in the hippocampal CA1 region. The mechanism of ischemia/reperfusion (I/R) injury is not fully understood. In this study, we have shown that the P2X7 receptor antagonist, BBG, reduced delayed neuronal death in the hippocampal CA1 region after I/R injury; P2X7 receptor expression levels increased before delayed neuronal death after I/R injury; inhibition of the P2X7 receptor reduced I/R-induced microglial microvesicle-like components, IL-1β expression, P38 phosphorylation, and glial activation in hippocampal CA1 region after I/R injury. These results indicate that antagonism of the P2X7 receptor and signaling pathways of microglial MV shedding, such as src-protein tyrosine kinase, P38 MAP kinase and A-SMase, might be a promising therapeutic strategy for clinical treatment of transient global cerebral I/R injury.     

Tissue kallikrein protects neurons from hypoxia/reoxygenation-induced cell injury through Homer1b/c

Previous studies have demonstrated that human tissue kallikrein (TK) gene delivery protects against mouse cerebral ischemia/reperfusion (I/R) injury through bradykinin B2 receptor (B2R) activation. We have also reported that exogenous TK administration can suppress glutamate- or acidosis-induced neurotoxicity through the extracellular signal-regulated kinase1/2 (ERK1/2) pathway. To further explore the neuroprotection mechanisms of TK, in the present study we performed immunoprecipitation analysis and identified a scaffolding protein Homer1b/c using MALDI-TOF MS analysis. Here, we tested the hypothesis that TK reduces cell injury induced by oxygen and glucose deprivation/reoxygenation (OGD/R) through activating Homer1b/c. We found that TK increased the expression of Homer1b/c in a concentration- and time-dependent manner. Moreover, TK facilitated the translocation of Homer1b/c to the plasma membrane under OGD/R condition by confocal microscope assays. We also observed that overexpression of Homer1b/c showed the neuroprotection against OGD/R-induced cell injury by enhancing cell survival, reducing LDH release, caspase-3 activity and cell apoptosis. However, the knockdown of Homer1b/c by small interfering RNA showed the opposite effects, indicating that Homer1b/c had protective effects against OGD/R-induced neuronal injury. More interestingly, TK exerted its much more significantly neuroprotective effects after Homer1b/c overexpression, whereas it exerted its reduced effects after Homer1b/c knockdown. In addition, TK pretreatment increased the phosphorylation of the ERK1/2 and Akt-GSK3β through Homer1b/c activation. The beneficial effects of Homer1b/c were abolished by the ERK1/2 or PI3K antagonist. Therefore, we propose novel signaling mechanisms involved in the anti-hypoxic function of TK through activation of Homer1b/c-ERK1/2 and Homer1b/c-PI3K-Akt signaling pathways.     

C-phycocyanin protects against ischemia-reperfusion injury of heart through involvement of p38 MAPK and ERK signaling

We previously showed that C-phycocyanin (PC), an antioxidant biliprotein pigment of Spirulina platensis (a blue-green alga), effectively inhibited doxorubicin-induced oxidative stress and apoptosis in cardiomyocytes. Here we investigated the cardioprotective effect of PC against ischemia-reperfusion (I/R)-induced myocardial injury in an isolated perfused Langendorff heart model. Rat hearts were subjected to 30 min of global ischemia at 37 degrees C followed by 45 min of reperfusion. Hearts were perfused with PC (10 microM) or Spirulina preparation (SP, 50 mg/l) for 15 min before the onset of ischemia and throughout reperfusion. After 45 min of reperfusion, untreated (control) hearts showed a significant decrease in recovery of coronary flow (44%), left ventricular developed pressure (21%), and rate-pressure product (24%), an increase in release of lactate dehydrogenase and creatine kinase in coronary effluent, significant myocardial infarction (44% of risk area), and TdT-mediated dUTP nick end label-positive apoptotic cells compared with the preischemic state. PC or SP significantly enhanced recovery of heart function and decreased infarct size, attenuated lactate dehydrogenase and creatine kinase release, and suppressed I/R-induced free radical generation. PC reversed I/R-induced activation of p38 MAPK, Bax, and caspase-3, suppression of Bcl-2, and increase in TdT-mediated dUTP nick end label-positive apoptotic cells. However, I/R also induced activation of ERK1/2, which was enhanced by PC treatment. Overall, these results for the first time showed that PC attenuated I/R-induced cardiac dysfunction through its antioxidant and antiapoptotic actions and modulation of p38 MAPK and ERK1/2.