Apelin-13 protects the heart against ischemia-reperfusion injury through inhibition of ER-dependent apoptotic pathways in a time-dependent fashion

Endoplasmic reticulum (ER) stress is activated during and contributes to ischemia-reperfusion (I/R) injury. Attenuation of ER stress-induced apoptosis protects the heart against I/R injury. Using apelin, a ligand used to activate the apelin APJ receptor, which is known to be cardioprotective, this study was designed to investigate 1) the time course of changes in I/R injury after ER stress; 2) whether apelin infusion protects the heart against I/R injury via modulation of ER stress-dependent apoptosis signaling pathways; and 3) how phosphatidylinositol 3-kinase (PI3K)/Akt, endothelial nitric oxide synthase (eNOS), AMP-activated protein kinase (AMPK), and ERK activation are involved in the protection offered by apelin treatment. The results showed that, using an in vivo rat I/R model induced by 30 min of ischemia followed by reperfusion, infarct size (IS) increased from 2 h of reperfusion (34.85 ± 2.14%) to 12 h of reperfusion (48.98 ± 3.35, P < 0.05), which was associated with an abrupt increase in ER stress-dependent apoptosis activation, as evidenced by increased CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, and JNK activation (CHOP: 2.49-fold increase, caspase-12: 2.09-fold increase, and JNK: 3.38-fold increase, P < 0.05, respectively). Administration of apelin at 1 μg/kg not only completely abolished the activation of ER stress-induced apoptosis signaling pathways at 2 h of reperfusion but also significantly attenuated time-related changes at 24 h of reperfusion. Using pharmacological inhibition, we also demonstrated that PI3K/Akt, AMPK, and ERK activation were involved in the protection against I/R injury via inhibition of ER stress-dependent apoptosis activation. In contrast, although eNOS activation played a role in decreasing IS at 2 h of reperfusion, it failed to modify either IS or ER stress-induced apoptosis signaling pathways at 24 h after reperfusion.     

ERK/PP1a/PLB/SERCA2a and JNK Pathways Are Involved in Luteolin-Mediated Protection of Rat Hearts and Cardiomyocytes following Ischemia/Reperfusion

Luteolin has long been used in traditional Chinese medicine for treatment of various diseases. Recent studies have suggested that administration of luteolin yields cardioprotective effects during ischemia/reperfusion (I/R) in rats. However, the precise mechanisms of this action remain unclear. The aim of this study is to confirm that luteolin-mediated extracellular signal regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways are responsible for their cardioprotective effects during I/R. Wistar rats were divided into the following groups: (i) DMSO group (DMSO); (ii) I/R group (I/R); (iii) luteolin+I/R group (Lut+I/R); (iv) ERK1/2 inhibitor PD98059+I/R group (PD+I/R); (v) PD98059+luteolin+I/R group (PD+Lut+I/R); and (vi) JNK inhibitor SP600125+I/R group (SP+I/R). The following properties were measured: contractile function of isolated heart and cardiomyocytes; infarct size; the release of lactate dehydrogenase (LDH); the percentage of apoptotic cells; the expression levels of Bcl-2 and Bax; and phosphorylation status of ERK1/2, JNK, type 1 protein phosphatase (PP1a), phospholamban (PLB) and sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a). Our data showed that pretreatment with luteolin or SP600125 significantly improved the contraction of the isolated heart and cardiomyocytes, reduced infarct size and LDH activity, decreased the rate of apoptosis and increased the Bcl-2/Bax ratio. However, pretreatment with PD98059 alone before I/R had no effect on the above indexes. Further, these consequences of luteolin pretreatment were abrogated by co-administration of PD98059. We also found that pretreatment with PD98059 caused a significant increase in JNK expression, and SP600125 could cause ERK1/2 activation during I/R. In addition, we are the first to demonstrate that luteolin affects PP1a expression, which results in the up-regulation of the PLB, thereby relieving its inhibition of SERCA2a. These results showed that luteolin improves cardiomyocyte contractile function after I/R injury by an ERK1/2-PP1a-PLB-SERCA2a-mediated mechanism independent of JNK signaling pathway.     

Inconsistent relation of MAPK activation to infarct size reduction by ischemic preconditioning in pigs

The importance of the activation of mitogen-activated protein kinases (MAPK) for the cardioprotection achieved by ischemic preconditioning (IP) is still controversial. We therefore measured infarct size and p38, extracellular signal-regulated kinase (ERK), and c-Jun NH(2)-terminal kinase (JNK) MAPK phosphorylation (by biopsies) in enflurane-anesthetized pigs. After 90 min low-flow ischemia and 120 min reperfusion, infarct size averaged 18.3 +/- 12.4 (SD)% (group 1, n = 14). At similar subendocardial blood flows, IP by 10 min ischemia and 15 min reperfusion (group 2, n = 14) reduced infarct size to 6.2 +/- 5.1% (P < 0.05). An inconsistent increase in p38, ERK, and p54 JNK phosphorylation (by Western blot) was found during IP; p46 JNK phosphorylation increased with the subsequent reperfusion. At 8 min of the sustained ischemia, p38, ERK, and p54 JNK phosphorylation were increased with no difference between groups (medians: p38: 207% of baseline in group 1 vs. 153% in group 2; ERK: 142 vs. 144%; p54 JNK: 171 vs. 155%, respectively). MAPK phosphorylation and reduction of infarct size by IP were not correlated, thus not supporting the concept of a causal role of MAPK in mediating cardioprotection by IP.     

Effects of Panax notoginseng saponins on pneumocyte apoptosis and c-Jun N-terminal kinase in lung ischemia/reperfusion injury

The aim of the present study is to investigate the effects of Panax notoginseng saponins (PNS) on pneumocyte apoptosis and apoptosis-related protein, as well as c-Jun N-terminal kinase (JNK) in lung ischemia/reperfusion (I/R) injury. Thirty Wistar rats were randomly divided into control group, I/R group and PNS group. The unilateral lung I/R model was replicated by obstruction of left lung hilus for 30 min and reperfusion for 120 min in vivo. The rats in PNS group were given intraperitoneal injection of PNS at 60 min before ischemia and 10 min before reperfusion. Some lung tissues sampled at the end of the experiment were assayed for wet/dry weight ratio (W/T). The expressions of phosphorylated JNK (p-JNK) and JNK protein were detected by Western blot. The expressions of Bcl-2, Bax and Caspase-3 protein were detected by immunocytochemistry techniques. The pneumocyte apoptotic index (AI) was detected by terminal deoxynuleotidy1 transferase mediated dUTP nick end labeling (TUNEL). The morphological and ultrastructure changes were observed under light microscope and electron microscope, and the injured alveolus rate (IAR) was counted as well. The results showed that compared to control group, I/R group showed increased expressions of p-JNK, Bcl-2, Bax and Caspase-3 protein (all P < 0.01), decreased ratio of Bcl-2/Bax (P < 0.05), and increased values of AI, W/T and IAR (all P < 0.01). Moreover, light microscope and electron microscope showed serious morphological and ultrastructure injury in I/R group. Compared to I/R group, PNS group showed markedly decreased expressions of p-JNK, Bax and Caspase-3 protein (all P < 0.01), increased expression of Bcl-2 protein and ratio of Bcl-2/Bax (both P < 0.01), and lower values of AI, W/T and IAR (all P < 0.01). Meanwhile, light morphological and ultrastructure injury was found to be alleviated in PNS group. These results suggest that PNS can protect lung tissue from I/R injury, and the mechanism may correlate with suppressing JNK signal pathway, up-regulating the ratio of Bcl-2/Bax which results in inhibition of Caspase-3 dependent apoptosis.     

Mitogen-activated protein kinases and their role in regulation of cellular processes

Mitogen-activated protein kinases (MAPKs) are evolutionary conserved enzymes connecting cell-surface receptors to critical regulatory targets within cells. The three major MAPK cascades are known, the extracellular signal-regulated protein kinase (ERK) cascade, c-Jun amino-terminal protein kinase/stress-activated protein kinase (JNK/SAPK) cascade and p38-MAPK cascade. This paper is focused on characterization of these MAPK cascades in terms of their distribution and biological role in some pathological processes (apoptosis, hypertrophy) with a special orientation on the role of MAPKs in cardiovascular system during ischemia/reperfusion.     

Caffeic acid phenethyl ester possesses potent cardioprotective effects in a rabbit model of acute myocardial ischemia-reperfusion injury

Although great achievements have been made in elucidating the molecular mechanisms contributing to acute myocardial ischemia/reperfusion (I/R) injury, an effective pharmacological therapy to protect cardiac tissues from serious damage associated with acute myocardial infarction, coronary arterial bypass grafting surgery, or acute coronary syndromes has not been developed. We examined the in vivo cardioprotective effects of caffeic acid phenethyl ester (CAPE), a natural product with potent anti-inflammatory, antitumor, and antioxidant activities. CAPE was systemically delivered to rabbits either 60 min before or 30 min after surgically inducing I/R injury. Infarct dimensions in the area at risk were reduced by >2-fold (P < 0.01) with CAPE treatment at either period. Accordingly, serum levels of normally cytosolic enzymes lactate dehydrogenase, creatine kinase (CK), MB isoenzyme of CK, and cardiac-specific troponin I were markedly reduced in both CAPE treatment groups (P < 0.05) compared with the vehicle-treated control group. CAPE-treated tissues displayed significantly less cell death (P < 0.05), which was in part due to inhibition of p38 mitogen-activated protein kinase activation and reduced DNA fragmentation often associated with caspase 3 activation (P < 0.05). In addition, CAPE directly blocked calcium-induced cytochrome c release from mitochondria. Finally, the levels of inflammatory proteins IL-1beta and TNF-alpha expressed in the area at risk were significantly reduced with CAPE treatment (P < 0.05). These data demonstrate that CAPE has potent cardioprotective effects against I/R injury, which are mediated, at least in part, by the inhibition of inflammatory and cell death responses. Importantly, protection is conferred when CAPE is systemically administered after the onset of ischemia, thus demonstrating potential efficacy in the clinical scenario.     

SP600125对大鼠肺缺血/再灌注损伤的保护作用及机制

目的:探讨SP600125-c-Jun氨基末端激酶(JNK)特异性抑制剂对大鼠肺缺血/再灌注损伤的保护作用及机制。方法:复制在体大鼠原位单肺缺血/再灌注模型,随机分3组(n=10):假手术对照组(Control组)、缺血再灌注组(I/R组)与缺血再灌注+SP600125干预组(SP600125组)。实验结束时取肺组织测湿/干重比(W/D)、肺泡损伤率(IAR);采用蛋白印迹法检测肺组织磷酸化JNK(p-JNK)、JNK蛋白的表达;免疫组化法检测肺组织Bcl-2、Bax、Caspase-3蛋白的表达;原位末端标记法检测肺组织细胞凋亡指数(AI);电镜观察肺组织超微结构的改变。结果:SP600125组肺组织p-JNK、Bax、caspase-3的蛋白表达显著低于I/R组(均P<0.01),Bcl-2的蛋白表达及Bcl-2/Bax的比值显著高于I/R组(均P<0.01),AI、W/D及IAR显著低于I/R组(均P<0.01),肺组织超微结构损伤不同程度减轻。结论:SP600125可能通过抑制JNK信号通路,上调Bcl-2/Bax的比值减少caspase-3依赖性的肺细胞凋亡,从而减轻肺缺血/再灌注损伤。     

Inhibition of c-Jun NH2-terminal kinase activity improves ischemia/reperfusion injury in rat lungs

Although c-Jun NH(2)-terminal kinase (JNK) has been implicated in the pathogenesis of transplantation-induced ischemia/reperfusion (I/R) injury in various organs, its significance in lung transplantation has not been conclusively elucidated. We therefore attempted to measure the transitional changes in JNK and AP-1 activities in I/R-injured lungs. Subsequently, we assessed the effects of JNK inhibition by the three agents including SP600125 on the degree of lung injury assessed by means of various biological markers in bronchoalveolar lavage fluid and histological examination including detection of apoptosis. In addition, we evaluated the changes in p38, extracellular signal-regulated kinase, and NF-kappaB-DNA binding activity. I/R injury was established in the isolated rat lung preserved in modified Euro-Collins solution at 4 degrees C for 4 h followed by reperfusion at 37 degrees C for 3 h. We found that AP-1 was transiently activated during ischemia but showed sustained activation during reperfusion, leading to significant lung injury and apoptosis. The change in AP-1 was generally in parallel with that of JNK, which was activated in epithelial cells (bronchial and alveolar), alveolar macrophages, and smooth muscle cells (bronchial and vascular) on immunohistochemical examination. The change in NF-kappaB qualitatively differed from that of AP-1. Protein leakage, release of lactate dehydrogenase and TNF-alpha into bronchoalveolar lavage fluid, and lung injury were improved, and apoptosis was suppressed by JNK inhibition. In conclusion, JNK plays a pivotal role in mediating lung injury caused by I/R. Therefore, inhibition of JNK activity has potential as an effective therapeutic strategy for preventing I/R injury during lung transplantation.     

SAPKs regulation of ischemic preconditioning

The role of stress-activated protein kinases (SAPKs), c-Jun NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase, in preconditioning (PC) was examined with the use of isolated rat hearts subjected to four cyclic episodes of 5-min ischemia and 10-min reperfusion followed by 30-min ischemia and 2-h reperfusion (I/R). A group of hearts was preperfused with 100 microM curcumin, a c-Jun and JNK1 inhibitor, or 5 microM SB 203580, a p38 MAP kinase inhibitor. Another group of hearts was preperfused with 20 microM anisomycin, a stimulator for both JNK and p38 MAP kinases. I/R increased the protein levels of JNK1, c-Jun, and p38 MAP kinase. PC also enhanced the induction of these kinases, but subsequent I/R-mediated increase was blocked by PC. Curcumin blocked I/R- and PC-mediated increase in JNK1 and c-Jun protein levels, whereas it had no effects on p38 MAP kinase. SB 203580, on the other hand, was equally effective in reducing the p38 MAP kinase activation but exerted no effects on JNK1 and c-Jun induction. I/R-mediated increased myocardial infarction was reduced by any of the following compounds: anisomycin, curcumin, and SB 203580. The cardioprotective effects of PC were abolished by either curcumin or SB 203580. The results demonstrate that PC is mediated by a signal-transduction pathway involving both JNK1 and p38 MAP kinase. Activation of SAPKs, although transient, is obligatory for PC.     

Epigallocatechin-3-gallate enhances ischemia/reperfusion-induced apoptosis in human umbilical vein endothelial cells via AKT and MAPK pathways

Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, has been shown to promote apoptosis in cancer cells. However, the role of EGCG in endothelial cells following ischemia/reperfusion (I/R) injury remains unclear. In the present study, we investigated the mechanisms by which EGCG enhances I/R-induced cell growth inhibition and apoptosis in human umbilical vein endothelial cells (HUVECs). Our results showed that EGCG treatment caused cell proliferation inhibition during I/R injury, and this effect was associated with increased p27 and p21 levels and reduced cyclin D1 level. Moreover, treatment of cells with EGCG resulted in increase of caspase-3 and Bax and decrease of Bcl-2, enhancing I/R-induced apoptosis. Interestingly, EGCG decreased I/R-induced phosphorylation of AKT and its downstream substrates Foxo1 and Foxo3a and ERK1/2. In contrast, EGCG increased JNK1/2 and c-Jun phosphorylation. Furthermore, both wortamannin (PI3K inhibitor) and U0126 (MEK1/2 inhibitor) markedly enhanced EGCG-induced apoptosis during I/R, whereas SP600125 (JNK inhibitor) attenuated the action of EGCG. Taken together, our study for the first time suggest that EGCG is able to enhance growth arrest and apoptosis of HUVECs during I/R injury, at least in part, through inhibition of AKT and ERK1/2 and activation of JNK1/2 signaling pathways.     

Testosterone is responsible for enhanced susceptibility of males to ischemic renal injury

Female mice are much more resistant to ischemia/reperfusion (I/R)-induced kidney injury when compared with males. Although estrogen administration can partially reduce kidney injury associated with I/R, we demonstrated that the presence of testosterone, more than the absence of estrogen, plays a critical role in gender differences in susceptibility of the kidney to ischemic injury. Testosterone administration to females increases kidney susceptibility to ischemia. Dihydrotestosterone, which can not be aromatized to estrogen, has effects equal to those of testosterone. Castration reduces the I/R-induced kidney injury. In contrast, ovariectomy does not affect kidney injury induced by ischemia in females. Testosterone reduces ischemia-induced activation of nitric oxide synthases (NOSs) and Akt and the ratio of extracellular signal related kinase (ERK) to c-jun N-terminal kinase (JNK) phosphorylation. Pharmacological (Nomega-nitro-L-arginine) or genetic (endothelial NOS or inducible NOS) inhibition of NOSs in females enhances kidney susceptibility to ischemia. Nitric oxide increases Akt phosphorylation and protects Madin-Darby canine kidney epithelial cells from oxidant stress. Antagonists of androgen or estrogen receptors do not affect the gender differences. In conclusion, testosterone inhibits the post-ischemic activation of NOSs and Akt and the ratio of ERK to JNK phosphorylation through non-androgen receptor-medicated mechanisms, leading to increased inflammation and increased functional injury to the kidney. These findings provide a new paradigm for the design of therapies for ischemia/reperfusion injury and may be important to our understanding of the pathophysiology of acute renal failure in pregnancy where plasma androgen levels are elevated.     

Activation of the JNK pathway is important for cardiomyocyte death in response to simulated ischemia

Multiple signaling pathways, including the c-Jun N-terminal kinase (JNK) pathway, are activated in myocardial ischemia and reperfusion (MI/R) and correlate with cell death. However, the role of the JNK pathway in MI/R-induced cell death is poorly understood. In a rabbit model, we found that ischemia followed by reperfusion resulted in JNK activation which could be detected in cytosol as well as in mitochondria. To address the functional role of the JNK activation, we examined the consequences of blockade of JNK activation in isolated cardiomyocytes under conditions of simulated ischemia. The JNK activity was stimulated approximately sixfold by simulated ischemia and reperfusion (simulated MI). When a dominant negative mutant of JNK kinase-2 (dnJNKK2), an upstream regulator of JNK, and JNK-interacting protein-1 (JIP-1) were expressed in myocytes by recombinant adenovirus, the activation of JNK by simulated MI was reduced 53%. Furthermore, the TNFalpha-activated JNK activity in H9c2 cells was completely abolished by dnJNKK2 and JIP-1. In correlation, when dnJNKK2 and JIP-1 were expressed in cardiomyocytes, both constructs significantly reduced cell death after simulated MI compared to vector controls. We conclude that activation of the JNK cascade is important for cardiomyocyte death in response to simulated ischemia.     

Role of ERK1/2 in the anti-apoptotic and cardioprotective effects of nitric oxide after myocardial ischemia and reperfusion

Objective: Experimental results from cultured cells suggest that there is cross-talk between nitric oxide (NO) and extracellular signal-regulated kinase (ERK) in their anti-apoptotic effect. However, the cross-talk between these two molecules in either direction has not been confirmed in the whole organ or whole animal level. The aim of the present study was to determine whether ERK may play a role in the anti-apoptotic and cardioprotective effects of NO in myocardial ischemia/reperfusion (MI/R). Methods: Isolated perfused mouse hearts were subjected to 20 min of global ischemia and 120 min of reperfusion and treated with vehicle or an NO donor (SNAP, 10 μM) during reperfusion. To determine the role of ERK1/2 in the anti-apoptotic and cardioprotective effects of NO, hearts were pre-treated (10 min before ischemia) with U0126, a selective MEK1/2 inhibitor (1 μM). Results: Treatment with SNAP exerted significant cardioprotective effects as evidenced by reduced cardiac apoptosis (TUNEL and caspase 3 activity, p < 0.01), and improved cardiac functional recovery (p < 0.01). In addition, treatment with SNAP resulted in a 2.5-fold increase in ERK activation when compared with heart receiving vehicle. Pre-treatment with U0126 slightly increased post-ischemic myocardial apoptosis but had no significant effect on cardiac functional recovery in this isolated perfused heart model. However, treatment with U0126 completely blocked SNAP-induced ERK activation and markedly, although not completely, inhibited the cardioprotection exerted by SNAP. Conclusion: These results demonstrate that nitric oxide exerts its anti-apoptotic and cardioprotective effects, at least in part, by activation of ERK in ischemic/reperfused heart. The first two authors contribute equally to this study.     

USP49 inhibits ischemia–reperfusion-induced cell viability suppression and apoptosis in human AC16 cardiomyocytes through DUSP1–JNK1/2 signaling

Dual-specificity protein phosphatases (DUSP) also known as mitogen-activated protein kinase (MAPK) phosphatases (MKPs) can dephosphorylate MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38. DUSP1-mediated JNK dephosphorylation has been found to play an antiapoptotic role against cardiac ischemia–reperfusion (I/R) injury. However, the regulation of DUSP1–JNK pathway remains unclear. In the current study, ubiquitin-specific peptidase 49 (USP49) expression in human AC16 cardiomyocytes following I/R injury was measured by real-time polymerase chain reaction and western blot analysis. Cell viability, apoptosis, the Bax, Bcl-2, and DUSP1 expression, and the activity of MAPKs in AC16 cardiomyocytes following indicated treatment was measured by CCK-8, flow cytometry, and western blot analysis. The direct interaction between USP49 and DUSP1 was measured by coimmunoprecipitation and ubiquitination analysis. The effect of USP49 on apoptosis and JNK activity in rat cardiomyocytes following I/R injury was also measured by TUNEL and western blot analysis. Here, we found that USP49 expression was time-dependently increased in AC16 cardiomyocytes following I/R. I/R-induced cell apoptosis and JNK1/2 activation both in in vivo and in vitro reversed by USP49 overexpression in AC16 cardiomyocytes. Inhibiting JNK1/2 activation significantly inhibited USP49 knockdown-induced the cell viability inhibition, apoptosis and the JNK1/2 activation in AC16 cardiomyocytes. Moreover, USP49 positively regulated DUSP1 expression through deubiquitinating DUSP1. Overall, our findings establish USP49 as a novel regulator of DUSP1–JNK1/2 signaling pathway with a protective role in cardiac I/R injury.     

Hypothermia-induced cardioprotection using extended ischemia and early reperfusion cooling

Optimal timing of therapeutic hypothermia for cardiac ischemia is unknown. Our prior work suggests that ischemia with rapid reperfusion (I/R) in cardiomyocytes can be more damaging than prolonged ischemia alone. Also, these cardiomyocytes demonstrate protein kinase C (PKC) activation and nitric oxide (NO) signaling that confer protection against I/R injury. Thus we hypothesized that hypothermia will protect most using extended ischemia and early reperfusion cooling and is mediated via PKC and NO synthase (NOS). Chick cardiomyocytes were exposed to an established model of 1-h ischemia/3-h reperfusion, and the same field of initially contracting cells was monitored for viability and NO generation. Normothermic I/R resulted in 49.7 +/- 3.4% cell death. Hypothermia induction to 25 degrees C was most protective (14.3 +/- 0.6% death, P < 0.001 vs. I/R control) when instituted during extended ischemia and early reperfusion, compared with induction after reperfusion (22.4 +/- 2.9% death). Protection was completely lost if onset of cooling was delayed by 15 min of reperfusion (45.0 +/- 8.2% death). Extended ischemia/early reperfusion cooling was associated with increased and sustained NO generation at reperfusion and decreased caspase-3 activation. The NOS inhibitor N(omega)-nitro-L-arginine methyl ester (200 microM) reversed these changes and abrogated hypothermia protection. In addition, the PKCepsilon inhibitor myr-PKCepsilon v1-2 (5 microM) also reversed NO production and hypothermia protection. In conclusion, therapeutic hypothermia initiated during extended ischemia/early reperfusion optimally protects cardiomyocytes from I/R injury. Such protection appears to be mediated by increased NO generation via activation of protein kinase Cepsilon; nitric oxide synthase.     

Nitric oxide mediates cardiac protection of tissue kallikrein by reducing inflammation and ventricular remodeling after myocardial ischemia/reperfusion

We assessed the role of nitric oxide (NO) and the kinin B2 receptor in mediating tissue kallikrein's actions in intramyocardial inflammation and cardiac remodeling after ischemia/reperfusion (I/R) injury. Adenovirus carrying the human tissue kallikrein gene was delivered locally into rat hearts 4 days prior to 30-minute ischemia followed by 24-hour or 7-day reperfusion with or without administration of icatibant, a kinin B2 receptor antagonist, or N(omega)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor. Kallikrein gene delivery improved cardiac contractility and diastolic function, reduced infarct size at 1 day after I/R without affecting mean arterial pressure. Kallikrein treatment reduced macrophage/monocyte and neutrophil accumulation in the infarcted myocardium in association with reduced intercellular adhesion molecule-1 levels. Kallikrein increased cardiac endothelial nitric oxide synthase phosphorylation and NO levels and decreased superoxide formation, TGF-beta1 levels and Smad2 phosphorylation. Furthermore, kallikrein reduced I/R-induced JNK, p38MAPK, IkappaB-alpha phosphorylation and nuclear NF-kappaB activation. In addition, kallikrein improved cardiac performance, reduced infarct size and prevented ventricular wall thinning at 7 days after I/R. The effects of kallikrein on cardiac function, inflammation and signaling mediators were all blocked by icatibant and L-NAME. These results indicate that tissue kallikrein through kinin B2 receptor and NO formation improves cardiac function, prevents inflammation and limits left ventricular remodeling after myocardial I/R by suppression of oxidative stress, TGF-beta1/Smad2 and JNK/p38MAPK signaling pathways and NF-kappaB activation.     

Inhibition of MLK3-MKK4/7-JNK1/2 pathway by Akt1 in exogenous estrogen-induced neuroprotection against transient global cerebral ischemia by a non-genomic mechanism in male rats

Numerous studies have demonstrated the neuroprotective effects of estrogen in experimental cerebral ischemia. To investigate molecular mechanisms of estrogen neuroprotection in global ischemia, immunoblotting, immunohistochemistry and Nissel-staining analysis were used. Our results showed that chronic pretreatment with beta-estradiol 3-benzoate (E2) enhanced Akt1 activation and reduced the activation of mixed-lineage kinase 3 (MLK3), mitogen-activated protein kinase kinase 4/7 (MKK4/7), and c-Jun N-terminal kinase 1/2 (JNK1/2) in the hippocampal CA1 subfield during reperfusion after 15 min of global ischemia. In addition, E2 reduced downstream JNK nuclear and non-nuclear components, c-Jun and Bcl-2 phosphorylation and Fas ligand protein expression induced by ischemia/reperfusion. Administration of phosphoinositide 3-kinase (PI3K) inhibitor LY 294,002 prevented both activation of Akt1 and inhibition of MLK3, MKK4/7 and JNK1/2. The interaction between ERalpha and the p85 subunit of PI3K was also examined. E2 and antiestrogen ICI 182,780 promoted and prevented this interaction, respectively. Furthermore, ICI 182,780 blocked both the activation of Akt1 and the inhibition of MLK3, MKK4/7 and JNK1/2. Photomicrographs of cresyl violet-stained brain sections showed that E2 reduced CA1 neuron loss after 5 days of reperfusion, which was abolished by ICI 182,780 and LY 294,002. Our data indicate that in response to estrogen, ERalpha interacts with PI3K to activate Akt1, which may inhibit the MLK3-MKK4/7-JNK1/2 pathway to protect hippocampal CA1 neurons against global cerebral ischemia in male rats.     

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.     

Acute rosiglitazone treatment is cardioprotective against ischemia-reperfusion injury by modulating AMPK, Akt, and JNK signaling in nondiabetic mice

Rosiglitazone (RGZ), a peroxisome proliferator-activated receptor (PPAR)-γ agonist, has been demonstrated to possess cardioprotective properties during ischemia-reperfusion. However, this notion remains controversial as recent evidence has suggested an increased risk in cardiac events associated with long-term use of RGZ in patients with type 2 diabetes. In this study, we tested the hypothesis that acute RGZ treatment is beneficial during I/R by modulating cardioprotective signaling pathways in a nondiabetic mouse model. RGZ (1 μg/g) was injected intravenously via the tail vein 5 min before reperfusion. Myocardial infarction was significantly reduced in mice treated with RGZ compared with vehicle controls (8.7% ± 1.1% vs. 20.2% ± 2.5%, P < 0.05). Moreover, isolated hearts were subjected to 20 min of global, no-flow ischemia in an ex vivo heart perfusion system. Postischemic recovery was significantly improved with RGZ treatment administered at the onset of reperfusion compared with vehicle (P < 0.001). Immunoblot analysis data revealed that the levels of both phospho-AMP-activated protein kinase (Thr(172)) and phospho-Akt (Ser(473)) were significantly upregulated when RGZ was administered 5 min before reperfusion compared with vehicle. On the other hand, inflammatory signaling [phospho-JNK (Thr(183)/Tyr(185))] was significantly downregulated as a result of RGZ treatment compared with vehicle (P < 0.05). Intriguingly, pretreatment with the selective PPAR-γ inhibitor GW-9662 (1 μg/g iv) 10 min before reperfusion significantly attenuated these beneficial effects of RGZ on the ischemic heart. Taken together, acute treatment with RGZ can reduce ischemic injury in a nondiabetic mouse heart via modulation of AMP-activated protein kinase, Akt, and JNK signaling pathways, which is dependent on PPAR-γ activation.