Protective effect of labedipinedilol-A, a novel dihydropyridine-type calcium channel blocker, on myocardial apoptosis in ischemia-reperfusion injury

The effects of labedipinedilol-A, a novel dihydropyridine-type calcium channel blocker with alpha-/beta-adrenoceptor blocking activities, on myocardial infarct size, apoptosis and necrosis in the rat after myocardial ischemia/reperfusion (45 min/120 min) were investigated. Ten minutes prior to left coronary artery occlusion, rats were treated with vehicle or labedipinedilol-A (0.25 or 0.5 mg/kg, i.v.). In the vehicle group, myocardial ischemia-reperfusion induced creatine kinase (CK) release and caused cardiomyocyte apoptosis, as evidenced by DNA ladder formation and terminal dUTP deoxynucleotidyltransferase nick end-labeling (TUNEL) staining. Treatment with labedipinedilol-A (0.25 or 0.5 mg/kg) reduced infarct size significantly compared to vehicle group (18.75+/-0.65% and 8.27+/-0.29% vs. 41.72+/-0.73%, P<0.01). Labedipinedilol-A also reduced the CK, CK-MB, lactate dehydrogenase (LDH) and troponin T levels in blood. In addition, labedipinedilol-A (0.5 mg/kg) significantly decreased TUNEL positive cells from 19.21+/-0.52% to 9.73+/-0.81% (P<0.01), which is consistent with absence of DNA ladders in the labedipinedilol-A group. Moreover, labedipinedilol-A pretreatment also decreased calcium content in ischemic-reperfused myocardial tissue. In conclusion, these results demonstrate that labedipindielol-A, through reduction of calcium overload and apoptosis, exerts anti-infarct effect during myocardial ischemia-reperfusion and would be useful clinically in the prevention of acute myocardial infarction.     

COX-2 mediates morphine-induced delayed cardioprotection via an iNOS-dependent mechanism

Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) have been shown to be mediators of cardioprotection induced by ischemic preconditioning and opioids. However, it is not known whether COX-2 is involved in morphine-induced cardioprotection accompanied with iNOS. Therefore, we investigated the role of COX-2 in morphine-induced cardioprotection and the effect of iNOS on COX-2. Myocardial ischemia was induced by a 45-min coronary artery occlusion in mice. Infarct size (IS) as a percentage of the area at risk (AAR) was determined by triphenyltetrazolium chloride staining. The COX-2-selective inhibitor NS-398 was used to investigate the role of COX-2. Expression of COX-2 was assessed by Western blotting, and the myocardial prostaglandin (PG)E2 and 6-keto-PGF(1alpha) contents were measured using enzyme immunoassays. The iNOS-selective inhibitor SMT and iNOS gene-knockout mice were used to investigate the effect of iNOS on COX-2. IS/AAR was reduced significantly 1 and 24 h after morphine preconditioning. The infarct-sparing effect 24 h after morphine administration, but not the cardioprotection 1 h later, was completely abolished by NS-398. Marked enhancement of myocardial COX-2 expression was measured 24 h after morphine preconditioning associated with up-regulation of myocardial contents of PGE2 and 6-keto-PGF(1alpha). Neither the level of COX-2 nor the contents of PGE2 and 6-keto-PGF(1alpha) were enhanced 1 h later. Administration of SMT and targeted abrogation of iNOS gene blocked the enhancement of myocardial PGE2 and 6-keto-PGF(1alpha) 24 h after morphine administration but did not inhibit the up-regulation of COX-2 expression. We concluded that COX-2 mediates morphine-induced delayed cardioprotection via an iNOS-dependent pathway.     

Renal ischemia/reperfusion injury inhibits differentiation of dendritic cells derived from bone marrow monocytes in rats

Dendritic cells (DCs) are impacted by surgical injury, exercise, and other physiological stressors. This study aims to determine whether renal I/R injury affects 1) the differentiation of myeloid DCs from bone marrow monocytes (BMMos) and the maturation and activation state of these DCs and 2) DC infiltration of kidney. Sprague-Dawley rats were subjected to I/R injury or sham-operated. Creatinine clearance was monitored daily during the 14 d of reperfusion that followed the ischemic insult. At 2 and 14 d of reperfusion, the following were assessed 1) properties of BMMo-derived DCs (i.e., the amount of generated DCs, differentiation state markers [CD11c, CD80, CD86, and Ia], and functional state [MLR and amount of IL-12 produced]), and 2) the presence of DCs in the kidney. Numbers of BMMo-derived DCs were significantly decreased in the I/R injured group (compared with the sham-operated group) at 2 d but not 14 d. A comparison of the their functionality found mixed lymphocyte response [MLR] and IL-12 production were similar in the two groups at both time points. Also, immunohistochemistry showed infiltrating DCs in the outer medulla of the I/R injured kidney at 2 d but not 14 d of reperfusion. Thus, I/R stress reduces the number of DCs differentiated from BMMos but not the functional activity of these DCs. This decrease may reflect a stress-induced downshift in the capacity of BMMos to differentiate into DCs and a parallel upshift in the capacity of DCs to infiltrate the kidney.     

Cystamine prevents ischemia-reperfusion injury by inhibiting polyamination of RhoA

Transglutaminase2 (TGase2) activates Rho-associated kinase (ROCK), an important mediator of ischemia-reperfusion (IR) injury, through polyamination of RhoA. Cystamine, an oxidized dimer of cysteamine inhibits the transamidation activity of TGase2. We examined whether addition of cystamine to an organ preservation solution protects rat cardiomyocyte cells (H9C2) from cell death in IR injury. H9C2 cells were stored under hypoxic conditions at 4 °C in laboratory-made preservation solution (SNU) or SNU solution supplemented with cystamine (SNU-C1), and cell preservation in the two solutions was compared by measuring the release of lactate dehydrogenase. The cells were preserved more effectively in SNU-C1 than in SNU solution. Cystamine inhibited the intracellular activity of TGase2 which increased during cold storage or reoxygenation. The inhibition of TGase2 by cystamine reduced the polyamination of RhoA, the interaction between RhoA and ROCK2, and F-actin formation. Cystamine also prevented the activation of caspases during cold storage. These results suggest that addition of cystamine to the organ preservation solution significantly enhances cardiomyocytes preservation apparently by inhibiting TGase2-mediated RhoA-ROCK pathway and that TGase2 may play an important role in IR injury by regulating ROCK.     

Role of UCH-L1/ubiquitin in acute testicular ischemia-reperfusion injury

The most significant complication of testicular torsion is loss of the testis, which may lead to impaired fertility. Molecular mechanisms how spermatogenesis impairs owing to testicular torsion remain unknown. This investigation, by using mouse model of testicular torsion, was undertaken to gain insight into the cellular and molecular mechanism underlying torsion-induced germ cell loss. Male mice were subjected to 2 h ischemia-inducing torsion, and testes were examined at 24, 48, and 72 h after the repair of torsion (reperfusion). Ischemia-reperfusion (IR) of the testes resulted in germ cell, mostly in spermatogonia, apoptosis, which was revealed by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) technique. At 24 h after torsion repair germ cell apoptosis reached peak, then decreased until 72 h repair. Western blots showed that apoptotic proteins (p53, Caspase-3 and -9) gradually were upregulated at 48 h reperfusion, however, anti-apoptotic proteins (Bcl-2 and BDNF) were downregulated in the relevant IR treatment. IR injury induced CHOP protein appearance with maximum expression at 24 h of reperfusion. Furthermore, the germ cell apoptosis triggered downregulation of ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1) at both mRNA and protein levels. To test further whether ubiquitination was involved in IR stress, both mono- and poly-ubiquitin levels in IR stress condition were examined, which showed that both mono- and poly-ubiquitin expression significantly impaired. These results provide evidences of UCH-L1/ubiquitination signaling to the testis IR injury in vivo.     

Differential patterns of peroxynitrite mediated apoptosis in proximal tubular epithelial cells following ATP depletion recovery

Ischemia-reperfusion injury (IRI) is characterized by ATP depletion in the ischemic phase, followed by a rapid increase in reactive oxygen species, including peroxynitrite in the reperfusion phase. In this study, we examined the role of peroxynitrite on cytotoxicity and apoptosis in an in vitro model of ATP depletion-recovery. Porcine proximal tubular epithelial (LLC-PK₁) cells were ATP depleted for either 2 h (2/2) or 4 h (4/2) followed by recovery in serum free medium for 2 h. A subset of cells was treated with 100 μM of the peroxynitrite scavenger, iron (III) tetrakis (N-methyl-4′pyridyl) porphyrin pentachloride (FeTMPyP) 30 min prior to and during treatment/recovery. Treatment with FeTMPyP reduced cytotoxicity and superoxide levels at both the 2/2 and 4/2 time points, however FeTMPyP decreased nitric oxide only at the 2/2 time point. FeTMPyP also partially blocked caspase-3 and caspase-8 activation at both 2/2 and 4/2 time points. At the 4/2 time point, FeTMPyP also partially inhibited the ATP depletion mediated increase in tumor necrosis factor alpha (TNF-α) and decreased Bax and FasL gene expression. These data show that peroxynitrite induces apoptosis by activation of multiple pathways depending on length and severity of insult following ATP depletion-recovery.     

Enhanced expression of P2X4 purinoceptors in pyramidal neurons of the rat hippocampal CA1 region may be involved ischemia-reperfusion injury

Ischemic stroke is the most serious disease that harms human beings. In principle, its treatment is to restore blood flow supply as soon as possible. However, after the blood flow is restored, it will lead to secondary brain injury, that is, ischemia-reperfusion injury. The mechanism of ischemia-reperfusion injury is very complicated. This study showed that P2X4 receptors in the pyramidal neurons of rat hippocampus were significantly upregulated in the early stage of ischemia-reperfusion injury. Neurons with high expression of P2X4 receptors are neurons that are undergoing apoptosis. Intraventricular injection of the P2X4 receptor antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) and PSB-12062 can partially block neuronal apoptosis, to promote the survival of neurons, indicating that ATP through P2X4 receptors is involved in the process of cerebral ischemia-reperfusion injury. Therefore, identifying the mechanism of neuronal degeneration induced by extracellular ATP via P2X4 receptors after ischemia-reperfusion will likely find new targets for the treatment of ischemia-reperfusion injury, and will provide a useful theoretical basis for the treatment of ischemia-reperfusion injury.     

Calcitonin gene-related peptide-mediated cardioprotection of postconditioning in isolated rat hearts

Previous studies have demonstrated that endogenous calcitonin gene-related peptide (CGRP) plays an important role in mediation of ischemic preconditioning. In the present study, we tested whether CGRP is also involved in mediation of the protective effects of postconditioning in isolated rat hearts. Sixty minutes of left coronary artery occlusion and followed by 60 min of reperfusion caused a significant decrease in cardiac function and a significant increase in creatine kinase (CK) release and infarct size. Postconditioning with three cycles of 1-min ischemia and 1-min reperfusion produced a marked improvement of cardiac function and decreased CK release and infarct size, concomitantly with an increase in the release of CGRP release in coronary effluent. However, the cardioprotection afforded by postconditioning was abolished by CGRP 8-37 (10^− 7 M), a selective CGRP receptor antagonist, or pretreatment with capsaicin (50 mg/kg, s.c.), which depletes transmitters in sensory nerves. Exogenous CGRP (5 × 10^− 9 M) administration of CGRP reappeared postconditioning-like cardioprotection in the rats pretreated with capsaicin. These results suggest that the protective effects of ischemic postconditioning are related to stimulation of endogenous CGRP release in rat hearts.     

对大鼠压疮缺血- 再灌注损伤模型构建及应用的思考

目的:为构建大鼠压疮缺血-再灌注损伤模型提供简易模型装置及有效的造模方法。方法:自制简易压疮缺血-再灌注损伤模型装置,对120只雌性大鼠腿部近膝关节骨隆突处进行为期3日的压疮造模,5个循环/日,每个循环分别实现120 min的缺血期及30 min的再灌注期,分别于造模第1 d、2 d、3 d及造模结束第1 d观察大鼠病灶创面的颜色、形态、水肿、结痂、渗出以及大鼠行为学状况、并于造模结束第1 d统计存活率及成模率。结果:造模第1 d,Ⅰ期压疮100只,Ⅱ期压疮17只,死亡3只;造模第2 d,Ⅰ期压疮26只,Ⅱ期压疮84只,死亡7只;造模第3 d,Ⅰ期压疮11只,Ⅱ期压疮95只,死亡4只;造模结束第1 d,Ⅰ期压疮5只,Ⅱ期压疮101只,死亡0只。120只实验大鼠,共14只大鼠死亡,造模存活率达88.33%,Ⅱ期压疮造模成功率达84.17%。结论:本造模装置可有效制备大鼠压疮缺血-再灌注动物模型,具有接近临床,操作简便,无需麻醉,Ⅱ期压疮成模率较高、避免铁片植入带来的皮肤负损伤等优势,这将为皮肤压疮、乃至慢性损伤组织的机制研究、修复及愈合相关研究提供重要理论和实验依据。     

Leptin fluctuates in intestinal ischemia-reperfusion injury as inflammatory cytokine

As leptin is an active mediator mainly secreted by adipose tissue and is closely related with energy metabolism, we evaluate both the changes of leptin levels in serum and adipose tissue with a concise radioimmunoassay and the changes of leptin mRNA expression in adipose tissue with RT-PCR, during the severe metabolic impediment in rat intestinal ischemia-reperfusion (I/R) injury. Results show that not only leptin levels in serum and adipose tissue but also its mRNA expression in adipose tissue undergo a fluctuation according to different injury times. Therefore, we conclude that leptin has a time-dependent response to acute inflammatory stimuli and acts as an anti-inflammatory cytokine.