Protective effect of oxytocin on ovarian ischemia-reperfusion injury in rats

Oxytocin (OT), a neurohypophysial nonapeptide, plays dual role as a neurotransmitter/neuromodulator and a hormone. It has also well known protective properties against ischemia/reperfusion organ damage. This study investigated the effect of OT on experimentally induced ovarian torsion/de-torsion ischemia/reperfusion (I/R) injury in rats. Sprague-Dawley rats were assigned to five treatment groups (n = 7/group): Group 1, sham-operated; Group 2, torsion; Group 3, 80 IU/kg of OT administration 30 min prior to torsion; Group 4, torsion/de-torsion; and Group 5, torsion followed by 80 IU/kg of OT administration 30 min prior to de-torsion. OT administration significantly decreased the tissue malondialdehyde (MDA) levels in both the torsion and OT group (Group 3), and torsion/de-torsion OT group (Group 5) in comparison with the torsion-only group (Group 2) and torsion/de-torsion group (Group 4). Histopathological finding scores including follicular degeneration, edema, hemorrhage, vascular congestion, and infiltration by inflammatory cells were found to be significantly decreased in the torsion and OT group (Group 3), and torsion/de-torsion OT group (Group 5) when compared with the torsion-only group (Group 2) and torsion/de-torsion group (Group 4). In conclusion, these results, verified with histopathologic evaluation and biochemical assays, suggest a probable protective role for OT in ischemia and I/R injury in rat ovaries.     

Inhibition of endogenous thioredoxin-1 in the heart of transgenic mice does not confer cardioprotection in ischemic postconditioning

Thioredoxin-1 maintains the cellular redox status and decreases the infarct size in ischemia/reperfusion injury. However, whether the increase of thioredoxin-1 expression or its lack of activity modifies the protection conferred by ischemic postconditioning has not been yet elucidated. The aim was to evaluate if the thioredoxin-1 overexpression enhances the posctconditioning protective effect, and whether the lack of the activity abolishes the reduction of the infarct size. Wild type mice hearts, transgenic mice hearts overexpressing thioredoxin-1, and a dominant negative mutant (C32S/C35S) of thioredoxin-1 were used. The hearts were subjected to 30 min of ischemia and 120 min of reperfusion (Langendorff) (I/R group) or to postconditioning protocol (PostC group). The infarct size in the Wt-PostC group decreased in comparison to the Wt-I/R group (54.6 ± 2.4 vs. 39.2 ± 2.1%, p < 0.05), but this protection was abolished in DN-Trx1-PostC group (49.7 ± 1.1%). The ischemia/reperfusion and postconditioning in mice overexpressing thioredoxin-1 reduced infarct size at the same magnitude (35.9 ± 2.1 and 38.4 ± 1.3%, p < 0.05 vs. Wt-I/R). In Wt-PostC, Trx1-I/R and Trx1- PostC, Akt and GSK3β phosphorylation increased compared to Wt-I/R, without changes in DN-Trx1 groups. In conclusion, given that the cardioprotection conferred by thioredoxin-1 overexpression and postconditioning, is accomplished through the activation of the Akt/GSK3β survival pathway, no synergic effect was evidenced. Thioredoxin-1 plays a key role in the postconditioning, given that when this protein is inactive the cardioprotective mechanism was abolished. Thus, diverse comorbidities or situations modifying the thioredoxin activity, could explain the absence of this strong mechanism of protection in different clinical situations.     

CpG-ODN,the TLR9 agonist,attenuates myocardial ischemia/reperfusion injury: Involving activation of PI3K/Akt signaling

BackgroundToll-like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. The TLR9 ligand, CpG-ODN has been reported to improve cell survival. We examined effect of CpG-ODN on myocardial I/R injury.MethodsMale C57BL/6 mice were treated with either CpG-ODN, control-ODN, or inhibitory CpG-ODN (iCpG-ODN) 1 h prior to myocardial ischemia (60 min) followed by reperfusion. Untreated mice served as I/R control (n = 10/each group). Infarct size was determined by TTC straining. Cardiac function was examined by echocardiography before and after myocardial I/R up to 14 days.ResultsCpG-ODN administration significantly decreased infarct size by 31.4% and improved cardiac function after myocardial I/R up to 14 days. Neither control-ODN nor iCpG-ODN altered I/R-induced myocardial infarction and cardiac dysfunction. CpG-ODN attenuated I/R-induced myocardial apoptosis and prevented I/R-induced decrease in Bcl2 and increase in Bax levels in the myocardium. CpG-ODN increased Akt and GSK-3β phosphorylation in the myocardium. In vitro data suggested that CpG-ODN treatment induced TLR9 tyrosine phosphorylation and promoted an association between TLR9 and the p85 subunit of PI3K. Importantly, PI3K/Akt inhibition and Akt kinase deficiency abolished CpG-ODN-induced cardioprotection.ConclusionCpG-ODN, the TLR9 ligand, induces protection against myocardial I/R injury. The mechanisms involve activation of the PI3K/Akt signaling pathway.     

Overexpression of HSPA12B protects against cerebral ischemia/reperfusion injury via a PI3K/Akt-dependent mechanism

Background and purpose: HSPA12B is a newly discovered member of the Hsp70 family proteins. This study investigated the effects of HSPA12B on focal cerebral ischemia/reperfusion (I/R) injury in mice. Methods: Transgenic mice overexpressing human HSPA12B (Tg) and wild-type littermates (WT) were subjected to 60 min of middle cerebral artery occlusion to induce ischemia and followed by reperfusion (I/R). Neurological deficits, infarct volumes and neuronal death were examined at 6 and 24 hrs after reperfusion. Blood–brain-barrier (BBB) integrity and activated cellular signaling were examined at 3 hrs after reperfusion. Results: After cerebral I/R, Tg mice exhibited improvement in neurological deficits and decrease in infarct volumes, when compared with WT I/R mice. BBB integrity was significantly preserved in Tg mice following cerebral I/R. Tg mice also showed significant decreases in cell injury and apoptosis in the ischemic hemispheres. We observed that overexpression of HSPA12B activated PI3K/Akt signaling and suppressed JNK and p38 activation following cerebral I/R. Importantly, pharmacological inhibition of PI3K/Akt signaling abrogated the protection against cerebral I/R injury in Tg mice. Conclusions: The results demonstrate that HSPA12B protects the brains from focal cerebral I/R injury. The protective effect of HSPA12B is mediated though a PI3K/Akt-dependent mechanism. Our results suggest that HSPA12B may have a therapeutic potential against ischemic stroke.     

Protective effect of hesperidin against lung injury induced by intestinal ischemia/reperfusion in adult albino rats: Histological,immunohistochemical and biochemical study

Hesperidin is a naturally common flavonoid. It is an abundant and cheap by-product of citrus cultivation. It is reported to have antioxidative, anti-inflammatory and anticarcinogenic effects. This work was performed to investigate the possible protective role of hesperidin in ameliorating the effect of experimentally induced intestinal ischemia/reperfusion injury (I/R) on lung tissue, histologically, immunohistochemically and biochemically. Thirty male Wistar adult albino rats were randomized into three groups named: group I (control group); group II (I/R); and group III (I/R with hesperidin). Intestinal I/R was induced by occluding the superior mesenteric artery for 60 min, followed by 120 min of reperfusion period. Animals were given hesperidin orally 1 h before the onset of ischemia. At the end of the reperfusion period the lung tissues were extracted for histopathological examination and immunohistochemical detection of the distribution of inducible nitric oxide synthase (iNOS). Pulmonary edema was evaluated by lung tissue wet/dry weight ratios. The levels of malondialdehyde (MDA, a biomarker of oxidative damage), myeloperoxidase (MPO, an index of the degree of neutrophil accumulation) and glutathione (GSH, a biomarker of protective oxidative injury) were also determined in all dissected tissues. Pretreatment with hesperidin (in group III) alleviated lung morphological changes noticed in I/R group and the levels of MDA and MPO were significantly decreased whereas those of GSH were significantly increased. Immunohistochemical study revealed a significant decrease in the iNOS. Hesperidin also significantly alleviated the formation of pulmonary edema as evidenced by the decreased organ wet/dry weight ratios. Hesperidin exerts a protective effect against lung damage induced by intestinal I/R injury in rats by reducing oxidative stress.     

The cardioprotective effect of different doses of vasopressin (AVP) against ischemia–reperfusion injuries in the anesthetized rat heart

The aim of the present study was to investigate the protective effect of various doses of exogenous vasopressin (AVP) against ischemia–reperfusion injury in anesthetized rat heart. Anesthetized rats were randomly divided into seven groups (n = 4–13) and all of them subjected to prolonged 30 min regional ischemia and 120 min reperfusion. Group I served as saline control with ischemia, in treatment groups II, III, IV and V, respectively different doses of AVP (0.015, 0.03, 0.06 and 1.2 μg/rat) were infused within 10 min prior to ischemia, in group VI, an AVP-selective V1 receptor antagonist (SR49059, 1 mg/kg, i.v.) was administrated prior to effective dose of AVP injection and in group VII, SR49059 (1 mg/kg, i.v.) was only administrated prior to ischemia. Various doses of AVP significantly prevented the decrease in heart rate (HR) at the end of reperfusion compared to their baseline and decreased infarct size, biochemical parameters [LDH (lactate dehydrogenase), CK-MB (creatine kinase-MB) and MDA (malondialdehyde) plasma levels], severity and incidence of ventricular arrhythmia, episodes and duration of ventricular tachycardia (VT) as compared to control group. Blockade of V1 receptors by SR49059 attenuated the cardioprotective effect of AVP on ventricular arrhythmias and biochemical parameters, but partially returned infarct size to control. AVP 0.03 μg/rat was known as effective dose. Our results showed that AVP owns a cardioprotective effect probably via V1 receptors on cardiac myocyte against ischemia/reperfusion injury in rat heart in vivo.     

Cellular and molecular mechanisms of 17β-estradiol postconditioning protection against gastric mucosal injury induced by ischemia/reperfusion in rats

AimsTo investigate the protective effects of 17β-estradiol postconditioning against ischemia/reperfusion (I–R)-induced gastric mucosal injury in rats.Main methodsThe animal model of gastric ischemia/reperfusion was established by clamping of the celiac artery for 30 min and reperfusion for 30 min, 1 h, 3 h, 6 h, 12 h or 24 h. 17β-estradiol at doses of 5, 50 or 100 μg/kg (rat) was administered via peripheral veins 2 min before reperfusion. In a subgroup of rats, the estrogen receptor antagonist fulvestrant (Ful, 2 mg/kg) was intravenously injected prior to 17β-estradiol administration. Histological and immunohistochemical methods were employed to assess the gastric mucosal injury index and gastric mucosal cell apoptosis and proliferation. The malondialdehyde (MDA) concentration, superoxide dismutase (SOD) activity, xanthine oxidase (XOD) activity and hydroxyl free radical (–OH) inhibitory ability were determined by colorimetric assays. Subsequently, the expression of Bcl-2 and Bax in rat gastric mucosa was examined by western blotting.Key findings17β-estradiol dose-dependently inhibited gastric I–R (GI–R) injury, and 17β-estradiol (50 μg/kg) markedly attenuated GI–R injury 1 h after reperfusion. 17β-estradiol inhibited gastric mucosal cell apoptosis and promoted gastric mucosal cell proliferation in addition to increasing SOD activity and –OH inhibitory ability and decreasing the MDA content and XOD activity. The Bax protein level increased 1 h after GI–R and was markedly reduced by intravenous administration of 17β-estradiol. In contrast, the level of Bcl-2 protein decreased 1 h after GI–R and was restored to normal levels by intravenous administration of 17β-estradiol. These effects of 17β-estradiol were inhibited by pretreatment with fulvestrant.Significance17β-estradiol postconditioning should be investigated further as a possible strategy against gastric mucosal injury.     

κ-Opioid receptor stimulation modulates TLR4/NF-κB signaling in the rat heart subjected to ischemia–reperfusion

It is well documented that the Toll-like receptor 4 (TLR4)/NF-κB signaling mediates early inflammation during myocardial ischemia and reperfusion. Our previous study has demonstrated that κ-opioid receptor stimulation with U50,488H produces cardioprotective and anti-inflammatory effects. The aim of the present study was to investigate whether κ-opioid receptor stimulation could modulate the TLR4/NF-κB signaling and reduce neutrophil accumulation and TNF-α induction in an ischemia–reperfusion injured rat heart model. Rats were randomly exposed to sham operation, myocardial ischemia and reperfusion (MI/R), and MI/R + U50,488H in the absence or presence of Nor-BNI, a selective κ-opioid receptor antagonist. The results demonstrated that after MI/R, the expressions of myocardial TLR4 and NF-κB increased significantly both in ischemia area and risking area. Compared with MI/R, κ-opioid receptor stimulation with U50,488H significantly attenuated the expressions of TLR4 and NF-κB. At the mean time, it also reduced myeloperoxidase (MPO) levels, both serum and myocardial TNF-α production, myocardial infarct sizes (INF/AAR%) and myocardial apoptosis induced by MI/R, all the effects of U50,488H were abolished by Nor-BNI. These data provide evidence for the first time that κ-opioid receptor stimulation inhibits TLR4/NF-κB signaling in the rat heart subjected to MI/R.     

Caffeic acid attenuates rat liver reperfusion injury through sirtuin 3-dependent regulation of mitochondrial respiratory chain

Sirtuin 3 (Sirt3) plays critical roles in regulating mitochondrial oxidative metabolism. However, whether Sirt3 is involved in liver ischemia and reperfusion (I/R) injury remains elusive. Caffeic acid (CA) is a natural antioxidant derived from Salvia miltiorrhiza. Whether CA protects against liver I/R injury through regulating Sirt3 and the mitochondrial respiratory chain (MRC) is unclear. This study investigated the effect of CA on liver I/R injury, microcirculatory disturbance, and potential mechanisms, particularly focusing on Sirt3-dependent MRC. Liver I/R of male Sprague-Dawley rats was established by occlusion of portal area vessels for 30 min followed by 120 min of reperfusion. CA (15 mg/kg/h) was continuously infused via the femoral vein starting 30 min before ischemia. After I/R, Sirt3 expression, and MRC activity decreased, acetylation of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 and succinate dehydrogenase complex, subunit A, flavoprotein variant provoked, and the liver microcirculatory disturbance and injury were observed. Treatment with CA attenuated liver injury, inhibited Sirt3 down-expression, and up-regulated MRC activity. CA attenuated rat liver microcirculatory disturbance and oxidative injury through regulation of Sirt3 and the mitochondrial respiratory chain.     

Mesenchymal stem cell-derived exosomes increase ATP levels,decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury

We have previously identified exosomes as the paracrine factor secreted by mesenchymal stem cells. Recently, we found that the key features of reperfusion injury, namely loss of ATP/NADH, increased oxidative stress and cell death were underpinned by proteomic deficiencies in ischemic/reperfused myocardium, and could be ameliorated by proteins in exosomes. To test this hypothesis in vivo, mice (C57Bl6/J) underwent 30 min ischemia, followed by reperfusion (I/R injury). Purified exosomes or saline was administered 5 min before reperfusion. Exosomes reduced infarct size by 45% compared to saline treatment. Langendorff experiments revealed that intact but not lysed exosomes enhanced viability of the ischemic/reperfused myocardium. Exosome treated animals exhibited significant preservation of left ventricular geometry and contractile performance during 28 days follow-up. Within an hour after reperfusion, exosome treatment increased levels of ATP and NADH, decreased oxidative stress, increased phosphorylated-Akt and phosphorylated-GSK-3β, and reduced phosphorylated-c-JNK in ischemic/reperfused hearts. Subsequently, both local and systemic inflammation were significantly reduced 24 h after reperfusion. In conclusion, our study shows that intact exosomes restore bioenergetics, reduce oxidative stress and activate pro-survival signaling, thereby enhancing cardiac function and geometry after myocardial I/R injury. Hence, mesenchymal stem cell-derived exosomes are a potential adjuvant to reperfusion therapy for myocardial infarction.     

Niacin bound chromium treatment induces myocardial Glut-4 translocation and caveolar interaction via Akt,AMPK and eNOS phosphorylation in streptozotocin induced diabetic rats after ischemia-reperfusion injury

Diabetes, one of the major risk factors of metabolic syndrome culminates in the development of Ischemic Heart Disease (IHD). Refined diets that lack micronutrients, mainly trivalent chromium (Cr³⁺) have been identified as the contributor in the rising incidence of diabetes. We investigated the effect of niacin-bound chromium (NBC) during ischemia/reperfusion (IR) injury in streptozotocin induced diabetic rats. Rats were randomized into: Control (Con); Diabetic (Dia) and Diabetic rats fed with NBC (Dia + NBC). After 30 days of treatment, the isolated hearts were subjected to 30 min of global ischemia followed by 2 h of reperfusion. NBC treatment demonstrated significant increase in left ventricular functions and significant reduction in infarct size and cardiomyocyte apoptosis in Dia + NBC compared with Dia. Increased Glut-4 translocation to the lipid raft fractions was also observed in Dia + NBC compared to Dia. Reduced Cav-1 and increased Cav-3 expression along with phosphorylation of Akt, eNOS and AMPK might have resulted in increased Glut-4 translocation in Dia + NBC. Our results indicate that the cardioprotective effect of NBC is mediated by increased activation of AMPK, Akt and eNOS resulting in increased translocation of Glut-4 to the caveolar raft fractions thereby alleviating the effects of IR injury in the diabetic myocardium.     

Rb1 postconditioning attenuates liver warm ischemia-reperfusion injury through ROS-NO-HIF pathway

AimsGinsenoside Rb1 could prevent ischemic neuronal death and focal cerebral ischemia, but its roles to liver warm I/R injury remain to be defined. We determined if Rb1 would attenuate warm I/R injury in mice.Main methodsMice were divided into sham, I/R, Rb1 + I/R (Rb1 postconditioning, 20 mg/kg, i.p. after ischemia), sham + L-NAME, I/R + L-NAME, and Rb1 + I/R + L-NAME groups using 60 min of the liver median and left lateral lobes ischemia. Serum levels of alanine aminotransferase (ALT) were measured and morphology changes of livers were evaluated. Contents of nitric oxide (NO) and nitric oxide synthase (NOS), malondialdehye (MDA) and activity of superoxide dismutase (SOD) were measured. Expressions of Akt, p-Akt, iNOS, HIF-1alpha, tumor necrosis factor-a (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) were also determined by western blot or immunohistochemistry.Key findingsRb1 postconditioning attenuated the dramatically functional and morphological injuries. The levels of ALT were significantly reduced in Rb1 group (p < 0.05). Rb1 upregulated the concentrations of NO, iNOS in serum, iNOS, and activity of SOD in hepatic tissues (p < 0.05), while it dramatically reduced the concentration of MDA (p < 0.05). Protein expressions of p-Akt, iNOS and HIF-1alpha were markedly enhanced in Rb1 group. Protein and mRNA expressions of TNF-α and ICAM-1 were markedly suppressed by Rb1 (p < 0.05).SignificanceWe found that Rb1 postconditioning could protect liver from I/R injury by upregulating the content of NO and NOS, and also HIF-1alpha protein expression. These protective effects could be abolished by L-NAME. These findings suggested Rb1 may have the therapeutic potential through ROS-NO-HIF pathway for management of liver warm I/R injury.     

Effect of total flavonoids of Radix Ilicis pubescentis on cerebral ischemia reperfusion model

This paper aims to observe the effects of total flavonoids of Radix Ilicis pubescentis on mouse model of cerebral ischemia reperfusion. Mice were orally given different doses of total flavonoids of Radix Ilicis pubescentis 10 d, and were administered once daily. On the tenth day after the administration of 1 h in mice after anesthesia, we used needle to hook the bilateral common carotid artery (CCA) for 10 min, with 10 min ischemia reperfusion, 10 min ischemia. Then we restored their blood supply, copy the model of cerebral ischemia reperfusion; We then had all mice reperfused for 24 h, and then took their orbital blood samples and measured blood rheology. We quickly removed the brain, with half of the brain having sagittal incision. Then we fixed the brains and sectioned them to observe the pathological changes of brain cells in the hippocampus and cortex. We also measured the other half sample which was made of brain homogenate of NO, NOS, Na⁺-K⁺-, ATP enzyme Mg²⁺-ATPase and Ca²⁺-ATPase. Acupuncture needle hook occlusion of bilateral common carotid arteries can successfully establish the model of cerebral ischemia reperfusion. After comparing with the model mice, we concluded that Ilex pubescens flavonoids not only reduce damage to the brain nerve cells in the hippocampus and cortex, but also significantly reduce the content of NO in brain homogenate, the activity of nitric oxide synthase (NOS) and increases ATP enzyme activity (P < 0.05, P < 0.01). In this way, cerebral ischemia reperfusion injury is improved. Different dosages of Ilex pubescens flavonoids on mouse cerebral ischemia reperfusion model have good effects.     

Inhibition of semicarbazide-sensitive amine oxidase attenuates myocardial ischemia-reperfusion injury in an in vivo rat model

AimsThis study tested the hypothesis that the inhibition of semicarbazide-sensitive amine oxidase (SSAO) after ischemia could attenuate myocardial ischemia–reperfusion (I/R) injury.Main methodsAnesthetized male Sprague–Dawley rats underwent myocardial I/R injury. Saline, semicarbazide (SCZ, 30 mg/kg), hydralazine (HYD, 10 mg/kg), or LJP 1207 (30 mg/kg) was administered intraperitoneally 3 min before reperfusion. After 30 min of ischemia and 180 min of reperfusion, the myocardial infarct size was determined using nitroblue tetrazolium staining. Myocardial myeloperoxidase activity was determined through biochemical assay. HE staining was used for histopathological evaluation. Myocardial SSAO activity was assayed with high performance liquid chromatography analysis. Additionally, the endothelial expression of P-selectin was evaluated using immunohistochemistry after 30 min of ischemia and 20 min of reperfusion.Key findingsMyocardial SSAO activity was increased in myocardial I/R injury. Administration of SCZ, HYD, or LJP 1207 reduced the myocardial infarct size and decreased leukocyte infiltration and endothelial P-selectin expression in myocardial I/R injury in vivo.SignificanceThese data suggest that myocardial I/R injury up-regulates myocardial SSAO activity, and the inhibition of SSAO prior to reperfusion is able to attenuate acute myocardial I/R injury.     

Indirubin-3′-oxime prevents hepatic I/R damage by inhibiting GSK-3β and mitochondrial permeability transition

Indirubin-3′-oxime is an indirubin analogue that shows favorable inhibitory activity targeting glycogen synthase kinase 3β (GSK-3β). In this study, we evaluated if acute treatment with indirubin-3′-oxime (Ind) prevents hepatic ischemia/reperfusion (I/R) damage. Wistar rats were subjected to 150 min of 70% warm ischemia and 16 h of reperfusion. In the treated group 1 μM indirubin-3′-oxime was administered in the hepatic artery 30 min before ischemia. Acute treatment with Ind decreased serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels, comparatively to I/R livers. Bax translocation to the mitochondria and cytochrome c release were higher in I/R livers. Ind treatment significantly attenuated Bax translocation and preserved mitochondrial cytochrome c content. Ind also protected mitochondria from calcium-induced mitochondrial permeability transition (MPT), as well as the decrease in state 3 mitochondrial respiration, the delay in the repolarization after a phosphorylative cycle and the decrease in ATP content caused by I/R. By addressing GSK-3β activity and phosphorylated GSK-3β at Ser⁹ content in liver homogenates and isolated mitochondria, data suggests that inhibition of GSK-3β by indirubin-3′-oxime prevents the increase in mitochondrial phosphorylated GSK-3β at Ser⁹ induced by I/R, thus correlating with MPT inhibition and preservation of cytochrome c content. Pre-treatment with indirubin-3′-oxime in conditions of hepatic I/R, protects the liver by maintaining mitochondrial function and hepatic energetic balance.     

Role of high-fat diet in regulation of gene expression of drug metabolizing enzymes and transporters

AimsLate phase ischemic preconditioning (LPC) protects the heart against ischemia–reperfusion (I/R) injury. However, its effect on myocardial tissue oxygenation and related mechanism(s) is unknown. The aim of the current study is to determine whether LPC attenuates post-ischemic myocardial tissue hyperoxygenation through preserving mitochondrial oxygen metabolism.Main methodsC57BL/6 mice were subjected to 30 min coronary ligation followed by 60 min or 24 h reperfusion with or without LPC (3 cycles of 5 min I/5 min R): Sham, LPC, I/R, and LPC + I/R group. Myocardial tissue Po₂ and redox status were measured with electron paramagnetic resonance (EPR) spectroscopy.Key findingsUpon reperfusion, tissue Po₂ rose significantly above the pre-ischemic level in the I/R mice (23.1 ± 2.2 vs. 12.6 ± 1.3 mm Hg, p < 0.01). This hyperoxygenation was attenuated by LPC in the LPC + I/R mice (11.9 ± 2.0 mm Hg, p < 0.01). Activities of NADH dehydrogenase (NADH-DH), succinate-cytochrome c reductase (SCR) and cytochrome c oxidase (CcO) were preserved or increased in the LPC group, significantly reduced in the I/R group, and conserved in the LPC + I/R group. Manganese superoxide dismutase (Mn-SOD) protein expression was increased by LPC in the LPC and LPC + I/R mice compared to that in the Sham control (1.24 ± 0.01 and 1.23 ± 0.01, p < 0.05). Tissue redox status was shifted to the oxidizing state with I/R (0.0268 ± 0.0016/min) and was corrected by LPC in the LPC + I/R mice (0.0379 ± 0.0023/min). Finally, LPC reduced the infarct size in the LPC + I/R mice (10.5 ± 0.4% vs. 33.3 ± 0.6%, p < 0.05).SignificanceThus, LPC preserved mitochondrial oxygen metabolism, attenuated post-ischemic myocardial tissue hyperoxygenation, and reduced I/R injury.     

Functional polymorphisms in the IL6 gene promoter and the risk of urinary bladder cancer in India

BackgroundInterleukin-6 is a multifunctional cytokine, which plays a key role in tumor proliferation and differentiation. Variations in its gene (IL6) sequence may affect the risk of developing various cancers, including urinary bladder cancer. The present study was done to find the association of functional polymorphisms in the IL6 promoter with urinary bladder cancer.Materials and methodsSingle nucleotide polymorphisms were genotyped in histologically confirmed 232 cases of urinary bladder cancer and 250 healthy controls. The controls subjects were matched to the cases by age, sex, and ethnicity. Genotyping of the polymorphisms (−174G>C; −572G>C, −596A>G) was undertaken by direct DNA sequencing. The level of association between the genotypes and urinary bladder cancer risk was estimated by odds ratios and 95% confidence intervals generated by applying the chi-square test. Linkage disequilibrium (LD) between SNPs and haplotype analysis were performed using Haploview software.ResultSignificantly higher number of smokers (p = 0.047), tobacco chewers (p = <0.001) and those with non-vegetarian food habits (p = 0.016) were seen in the case group. The distribution of genotypes at −174G>C locus differed significantly between cases and controls and the variant genotypes GC + CC were significantly rarer in the cases (p = 0.00073; OR = 0.52 95% CI 0.35–0.75). Variant genotypes (GC + CC) were more common in grade I than grade III tumors (p = 0.032), further suggesting a protective effect. No LD was found between the SNPs; however, the frequency of haplotype AGC was significantly lesser in the cases than controls (p = 0.0103), suggesting a protective effect. Genotype distribution at the other two loci (−572G>C and −596A>G) did not show association with bladder cancer.ConclusionsIL6 (−174G>C) substitution confers significant protection against the risk of urinary bladder cancer in the study population, while other substitutions in this gene (−572G>C and −596A>G) do not affect the risk. In general, there is a lack of studies on the cytokine gene polymorphisms in urinary bladder cancer.     

Protective effect of heme oxygenase-1 induction against hepatic injury in alcoholic steatotic liver exposed to cold ischemia/reperfusion

AimsThe purpose of this study was to investigate the cytoprotective role of heme oxygenase-1 (HO-1) induction in hepatic injury in alcoholic steatotic liver exposed to cold ischemia/reperfusion (I/R).Main methodsAnimals were fed an ethanol liquid diet or isocaloric control diet for 5 weeks. Isolated perfused rat livers were preserved in Histidine–Tryptophan–Ketoglutarate at 4 °C. After 24 h of storage, livers were subjected to 120 min of reperfusion with Krebs–Henseleit bicarbonate buffer at 37 °C. Animals were pretreated with cobalt protoporphyrin (CoPP, 5 mg/kg, i.p.) or zinc protoporphyrin (ZnPP, 25 mg/kg, i.p.), HO-1 inducer and antagonist, respectively.Key findingsIn the model of ischemia/isolated perfusion, endogenous HO-1 was downregulated in the livers fed with ethanol diet (ED I/R). In ED I/R group, portal pressure and lactate dehydrogenase release were significantly increased, while bile output and hyaluronic acid clearance decreased compared to rats fed on control diet (CD I/R). Furthermore, hepatic glutathione content decreased and lipid peroxidation increased in the ED I/R group compared to the CD I/R group. These alterations were attenuated by upregulation of HO-1 with CoPP pretreatment.SignificanceOur results suggest that chronic ethanol consumption aggravates hepatic injury during cold I/R and it is likely due to downregulation of endogenous HO-1. Prior induction of HO-1 expression may provide a new strategy to protect livers against hepatic I/R injury or to increase the donor transplant pool through modulation of marginal alcoholic steatotic livers.     

Toll-like receptor 3 plays a role in myocardial infarction and ischemia/reperfusion injury

Innate immune and inflammatory responses mediated by Toll like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. This study examined the role of TLR3 in myocardial injury induced by two models, namely, myocardial infarction (MI) and I/R. First, we examined the role of TLR3 in MI. TLR3 deficient (TLR3^−/−) and wild type (WT) mice were subjected to MI induced by permanent ligation of the left anterior descending (LAD) coronary artery for 21 days. Cardiac function was measured by echocardiography. Next, we examined whether TLR3 contributes to myocardial I/R injury. TLR3^−/− and WT mice were subjected to myocardial ischemia (45 min) followed by reperfusion for up to 3 days. Cardiac function and myocardial infarct size were examined. We also examined the effect of TLR3 deficiency on I/R-induced myocardial apoptosis and inflammatory cytokine production. TLR3^−/− mice showed significant attenuation of cardiac dysfunction after MI or I/R. Myocardial infarct size and myocardial apoptosis induced by I/R injury were significantly attenuated in TLR3^−/− mice. TLR3 deficiency increases B-cell lymphoma 2 (BCL2) levels and attenuates I/R-increased Fas, Fas ligand or CD95L (FasL), Fas-Associated protein with Death Domain (FADD), Bax and Bak levels in the myocardium. TLR3 deficiency also attenuates I/R-induced myocardial nuclear factor KappaB (NF-κB) binding activity, Tumor necrosis factor alpha (TNF-α) and Interleukin-1 beta (IL-1β) production as well as I/R-induced infiltration of neutrophils and macrophages into the myocardium. TLR3 plays an important role in myocardial injury induced by MI or I/R. The mechanisms involve activation of apoptotic signaling and NF-κB binding activity. Modulation of TLR3 may be an effective approach for ameliorating heart injury in heart attack patients.     

Post-infarct treatment with [Pyr1]-apelin-13 reduces myocardial damage through reduction of oxidative injury and nitric oxide enhancement in the rat model of myocardial infarction

Apelin is a newly discovered peptide that has been recently shown to have cardioprotective effects in the animal model of myocardial infarction (MI) and ischemia/reperfusion (I/R) injuries. The aim of the present study was to investigate the long term cardioprotective effect of [Pyr1]-apelin-13 in the rat model of MI. Male Wistar rats (n = 22) were randomly divided into three groups: (1) sham operated group (2) control MI group and (3) MI treated with apelin (MI-AP group). MI animals were subjected to 30 min of left anterior descending coronary artery (LAD) ligation and 14 days of reperfusion. 24 h after LAD ligation, apelin (10 nmol/kg/day) was administered i.p. for 5 days. Blood sampling was performed at days 1, 3, 5 and 7 after MI for determination of serum changes of lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), malondialdehyde (MDA) and nitric oxide (NO). Myocardial infarct size (IS) and hemodynamic function were also measured at the end of the study at day 14. We found out that post infarct treatment with apelin decreases infarct size, serum levels of LDH, CK-MB and MDA and increases heart rate and serum level of NO in the consecutive days, but there were no significant differences in blood pressure in the MI-AP group in comparison with MI. In conclusion, apelin has long term cardioprotective effects against myocardial infarction through attenuation of cardiac tissue injury and lipid peroxidation and enhancement of NO production.