Cytoprotective effects of melatonin against necrosis and apoptosis induced by ischemia/reperfusion injury in rat liver

Abstract:  Melatonin protects against organ ischemia; this effect has mainly been attributed to the antioxidant properties of the indoleamine. This study examined the cytoprotective properties of melatonin against injury to the liver caused by ischemia/reperfusion (I/R). Rats were subjected to 60 min of ischemia followed by 5 hr of reperfusion. Melatonin (10 mg/kg) or the vehicle was administered intraperitoneally 15 min before ischemia and immediately before reperfusion. The serum aminotransferase activity and lipid peroxidation levels were increased markedly by hepatic I/R, which were suppressed significantly by melatonin. In contrast, the glutathione content, which is an index of the cellular redox state, and mitochondrial glutamate dehydrogenase activity, which is a maker of the mitochondrial membrane integrity, were lower in the I/R rats. These decreases were attenuated by melatonin. The rate of mitochondrial swelling, which reflects the extent of the mitochondrial permeability transition, was higher after 5 hr of reperfusion but was attenuated by melatonin. Melatonin limited the release of cytochrome c into the cytosol and the activation of caspase-3 observed in the I/R rats. The melatonin-treated rats showed markedly fewer apoptotic (TUNEL positive) cells and DNA fragmentation than did the I/R rats. These results suggest that melatonin ameliorates I/R-induced hepatocytes damage by inhibiting the level of oxidative stress and the apoptotic pathway. Consequently, melatonin may provide a new pharmacological intervention strategy for hepatic I/R injuries.     

Melatonin prevents hepatic injury-induced decrease in Akt downstream targets phosphorylations

Melatonin is a potent scavenger of reactive oxygen species and a strong antioxidant. Melatonin exerts protective effects against damage by the enhancing the Akt signal pathway, thus regulating apoptotic cell death. Akt phosphorylates pro-apoptotic proteins such as Bad and FoxO1 and inhibits the pro-apoptotic functions of these proteins. This study investigated the protective effects of melatonin through Akt and its downstream targets, Bad and FoxO1, in hepatic ischemia-reperfusion (I/R) damage. Adult mice were subjected to 1 h of hepatic ischemia and 3 h of reperfusion. Hepatic ischemia was induced by occlusions of the hepatic artery, portal vein, and bile duct. Melatonin (10 mg/kg, i.p.) or vehicle was administrated 15 min prior to ischemia and just before reperfusion. Serum aspartate aminotransferase and alanine aminotransferase levels were higher in I/R group than in sham-operated group. Melatonin attenuated increases in these levels. Moreover, melatonin attenuates injury-induced increases in positive TUNEL staining in hepatic tissues. Hepatic I/R injury induced reductions in the Akt up-stream target, PDK1 phosphorylation. The levels of phospho-Akt, phospho-Bad, and phospho-FoxO1 were decreased in vehicle-treated animals. However, melatonin prevented hepatic I/R injury-induced decreases in these proteins levels. Moreover, the interaction levels between phospho-Bad and 14-3-3 and between phospho-FoxO1 and 14-3-3 are reduced in vehicle-treated animals, and melatonin attenuated decreases in the binding levels of these proteins. 14-3-3 exerts an anti-apoptotic function by sequestration of Bad and FoxO1. These findings suggest that melatonin exerts protective effects in case of hepatic I/R damage by maintaining the binding of phospho-Bad and 14-3-3 and the binding of phospho-FoxO1 and 14-3-3, thus preventing activation of apoptotic cell death.     

Melatonin protects liver against ischemia and reperfusion injury through inhibition of toll-like receptor signaling pathway

Abstract: This study investigated the immunomodulating effect of melatonin on toll‐like receptor (TLR)‐stimulated signal transduction. Rats were subjected to 60 min of ischemia followed by 1 or 5 hr of reperfusion. Melatonin (10 mg/kg) or the vehicle was administered intraperitoneally 15 min prior to ischemia and immediately before reperfusion. Melatonin treatment significantly reduced the level of serum alanine aminotransferase activity. Increased levels of TLR3 and TLR4 protein expression induced by ischemia/reperfusion (I/R) were attenuated by melatonin. Serum level of high‐mobility group box 1 (HMGB1), a potent alarmin of the TLR system, increased significantly in the I/R group, and melatonin inhibited this release. Melatonin suppressed the increase in myeloid differentiation factor 88 (MyD88) protein expression, extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) phosphorylation and nuclear translocation of nuclear factor κB (NF‐κB) and phosphorylated c‐Jun, a component of activator protein 1. The increased level of toll‐receptor‐associated activator of interferon (TRIF) expression, phosphorylation of interferon (IFN) regulatory factor 3 (IRF3) and serum IFN‐β was attenuated by melatonin. Melatonin attenuated the levels of tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐6 and inducible nitric oxide synthase (iNOS) protein and mRNA expression, while the level of heme oxygenase‐1 (HO‐1) was augmented. Our results suggest that melatonin ameliorates I/R‐induced liver damage by modulation of TLR‐mediated inflammatory responses.     

Beneficial effects of melatonin on reperfusion injury in rat sciatic nerve

Studies have shown that ischemia-reperfusion (I/R) produces free radicals leading to lipid peroxidation and to damage of the nervous tissue. Melatonin, a main secretory product of the pineal gland, has free radical scavenging and antioxidant properties and has been shown to diminish I/R injury in many tissues. There are a limited number of studies related to the effects of melatonin on I/R injury in the peripheral nervous system. Therefore, in the present study, the protective effect of melatonin was investigated in rats subjected to 2 hr of sciatic nerve ischemia followed by 3 hr of reperfusion. Following reperfusion, nerve tissue samples were collected for quantitative assessment of malondialdehyde (MDA), an oxidative stress marker, and superoxide dismutase (SOD), a principal antioxidant enzyme. Samples were further evaluated at electron microscopic level to examine the neuropathological changes. I/R elevated the concentration of MDA significantly while there was a reduction at SOD levels. Melatonin treatment reversed the I/R-induced increase and decrease in MDA and SOD levels, respectively. Furthermore, melatonin salvaged the nerve fibers from ischemic degeneration. Histopathologic findings in the samples of melatonin-treated animals indicated less edema and less damage to the myelin sheaths and axons than those observed in the control samples. Our results suggest that administration of melatonin protects the sciatic nerve from I/R injury, which may be attributed to its antioxidant property.     

Melatonin reduces torsion-detorsion injury in rat ovary: biochemical and histopathologic evaluation

This experimental study was designed to determine the effects of melatonin on the levels of malondialdehyde (MDA), reduced glutathione (GSH), xanthine oxidase (XO) after adnexial torsion/detorsion (ischemia/reperfusion, I/R) of the ovaries of in rats. Forty adult albino rats were divided into five groups: sham operation, torsion, I/R plus saline, I/R plus melatonin and torsion plus melatonin. Rats in the sham-operated group underwent a surgical procedure similar to the other groups but the adnexa was not occluded. Rats in the torsion group were killed after adnexal torsion for 3 hr. Melatonin and saline were injected intraperitoneally (10 mg/kg) 30 min before detorsion to the I/R plus melatonin group and I/R plus saline group respectively. After 3 hr of ovarian detorsion, the rats were killed and ovaries were removed. Melatonin was injected intraperitoneally (10 mg/kg) 30 min before torsion to the torsion plus melatonin group. After 3 hr of ovarian torsion, the rats were killed and ovaries were harvested. The tissue levels of MDA, GSH and XO were measured. MDA and XO levels in the I/R plus saline group increased significantly when compared with torsion and sham-operated groups (P < 0.001). MDA and XO levels in the I/R plus melatonin group were lower than I/R plus saline and differences between the two groups were statistically significant (P < 0.001). GSH levels in the I/R plus saline group decreased significantly when compared with ischemia and sham-operated groups (P < 0.001). GSH levels in the I/R plus melatonin treated rats were significantly higher than I/R plus saline and ischemia groups (P < 0.001). The tissue levels of XO, MDA and GSH were similar between ischemia and ischemia plus melatonin groups. Morphologically, polymorphonuclear neutrophil infiltration and vascular dilatation were obvious in the I/R-damaged ovaries, and the changes also partially reversed by melatonin. This study demonstrates that melatonin protects the ovaries against oxidative damage associated with reperfusion following an ischemic insult.     

Melatonin inhibits type 1 interferon signaling of toll-like receptor 4 via heme oxygenase-1 induction in hepatic ischemia/reperfusion

The cytoprotective mechanisms of melatonin in hepatic ischemia/reperfusion (I/R) injury associated with heme oxygenase-1 (HO-1) induction and type 1 interferon (IFN) signaling pathway downstream of toll-like receptor 4 (TLR4) were investigated. Rats were subjected to 60min of ischemia followed by 5-hr reperfusion. Melatonin (10mg/kg) or vehicle (5% ethanol in saline) was administered intraperitoneally 15min prior to ischemia and immediately before reperfusion. Rats were pretreated with zinc protoporphyrin (ZnPP, 10mg/kg, i.p.), a HO-1 inhibitor, at 16 and 3hr prior to ischemia. Melatonin attenuated the I/R-induced increase in serum alanine aminotransferase activity, and ZnPP reversed this attenuation. Melatonin augmented the levels of HO activity and HO-1 protein and mRNA expression, and this enhancement was reversed by ZnPP. Melatonin enhanced the level of NF-E2-related factor-2 (Nrf2) nuclear translocation, and ZnPP reversed this increase. Overexpression of TLR4 and its adaptor proteins, toll-receptor-associated activator of interferon (TRIF), and myeloid differentiation factor 88 (MyD88), induced by I/R, was attenuated by melatonin; ZnPP reversed the effect of melatonin on TLR4 and TRIF expression. Melatonin suppressed the increased interaction between TLR4/TRIF and TLR4/MyD88, which was reversed by ZnPP. Melatonin attenuated the increased levels of JAK2 and STAT1 activation as well as IFN-β, and ZnPP reversed these inhibitory effects of melatonin. Melatonin inhibited the level of chemokine (C-X-C motif) ligand 10 (CXCL-10), and ZnPP reversed this inhibition. Our findings suggest that melatonin protects the liver against I/R injury by HO-1 overexpression, which suppresses the type 1 IFN signaling pathway downstream of TLR4.     

Microcirculatory effects of melatonin in rat skeletal muscle after prolonged ischemia

The purpose of this study was to determine microcirculatory effects and response of nitric oxide synthase (NOS) to melatonin in skeletal muscle after prolonged ischemia. A vascular pedicle isolated rat cremaster muscle model was used. Each muscle underwent 4 hr of zero-flow warm ischemia followed by 2 hr of reperfusion. Melatonin (10 mg/kg) or saline as a vehicle was given by intraperitoneal injection at 30 min prior to reperfusion and the same dose was given immediately after reperfusion. After reperfusion, microcirculation measurements including arteriole diameter, capillary perfusion and endothelial-dependent and -independent vasodilatation were performed. The cremaster muscle was then harvested to measure endothelial NOS (eNOS) and inducible NOS (iNOS) gene expression and enzyme activity. Three groups of rats were used: sham-ischemia/reperfusion (I/R), vehicle + I/R and melatonin + I/R. As compared with vehicle + I/R group, administration of melatonin significantly enhanced arteriole diameter, improved capillary perfusion, and attenuated endothelial dysfunction in the microcirculation of skeletal muscle after 4 hr warm ischemia. Prolonged warm ischemia followed by reperfusion significantly depressed eNOS gene expression and constitutive NOS activity and enhanced iNOS gene expression. Administration of melatonin did not significantly alter NOS gene expression or activity in skeletal muscle after prolonged ischemia and reperfusion. Melatonin provided a significant microvascular protection from reperfusion injury in skeletal muscle. This protection is probably attributable to the free radical scavenging effect of melatonin, but not to its anti-inflammatory effect.     

Proteomic identification of proteins differentially expressed by melatonin in hepatic ischemia‐reperfusion injury

Abstract: Hepatic ischemia‐reperfusion (I‐R) injury induces hepatic dysfunction or failure. Melatonin is a potent free radical scavenger and a strong antioxidant. Although many studies have demonstrated the protective effect of melatonin in hepatic injury, the molecular mechanisms of this protection are unclear. We identified specific proteins that are differentially expressed by melatonin treatment in hepatic I‐R injury. Adult mice were subjected to 1 hr of ischemia and 3 hr of reperfusion. Animals were treated with vehicle or melatonin (10 mg/kg, i.p.) 15 min prior to ischemia and just before reperfusion. Serum aspartate aminotransferase and alanine aminotransferase levels were higher in I‐R group than in sham‐operated group, and these increases were reduced by melatonin treatment. Proteins that were differentially expressed following melatonin treatment during hepatic I‐R injury were detected using two‐dimensional gel electrophoresis. Hepatic I‐R injury induced down‐regulation of glyoxalase I, glutaredoxin‐3, spermidine synthase, proteasome subunit beta type‐4, and dynamin like protein‐1 (DLP‐1). However, melatonin prevented the reductions in these proteins induced by I‐R injury. Among the identified proteins, we focused on DLP‐1, which is essential for the maintenance of mitochondrial and endoplasmic reticulum morphology. Western blot analysis confirmed that melatonin prevents the hepatic I‐R injury‐induced decrease in DLP‐1. These results suggest that melatonin protects hepatic cells against hepatic I‐R injury and that its protective effects involve the regulation of specific proteins.     

Dihydrolipoyl histidinate zinc complex, a new antioxidant, attenuates hepatic ischemia-reperfusion injury in rats

Background and Aims: Ischemia/reperfusion (I/R) injury is characterized by significant oxidative stress, which induces characteristic changes in the antioxidant system and organ injury leading to significant morbidity and mortality. The aim of this study was to evaluate the protective effect of dihydrolipoyl histidinate zinc complex (DHLHZn) on oxidative damage after severe hepatic I/R injury. Methods: Thirty male Wistar rats were subjected to 45 min of hepatic ischemia by clamping of the hepatic artery and portal vein, followed by a 6‐h reperfusion period. DHLHZn (10 mg/kg) (I/R + DHLHZn group) or saline (I/R group) was administered intraperitoneally twice, 30 min before ischemia and at the beginning of the reperfusion. Sham‐operated animals (sham group) received equal amounts of saline. The rats were killed at the end of the reperfusion period. Serum levels of aspartate aminotransferase and alanine aminotransferase were determined, and histological examination and oxidative stress were evaluated in liver tissues. In addition, antimycin A‐stimulated RAW264.7 cells (murine macrophage‐like cells) were treated with DHLHZn to estimate its antioxidant effect. Results: Serum aspartate aminotransferase and alanine aminotransferase levels were increased in the I/R group, but these increases were significantly inhibited in the I/R + DHLHZn group. Similarly, liver tissue damage observed in the I/R group was attenuated in the I/R + DHLHZn group. Cells treated in vitro with both DHLHZn and antimycin A showed reduced reactive oxygen species activity compared to cells treated with antimycin A alone. Conclusion: The new antioxidant DHLHZn may have potential for therapeutic application in liver I/R injury, although this is a limited animal study.     

Melatonin treatment protects against ischemia/reperfusion-induced functional and biochemical changes in rat urinary bladder

Reactive oxygen metabolites play important roles in ischemia/reperfusion (I/R) injury in several systems. The aim of this study was to investigate the role of melatonin against I/R injury of the rat urinary bladder. The abdominal aorta was clamped to induce ischemia for 30 min, then the animals were subjected to 60 min of reperfusion. Melatonin (10 mg/kg, i.p.) or the vehicle (control 1% alcohol i.p.) was administered before I/R. After decapitation, the bladder was removed and the tissue was either used for functional studies or stored for measurement of products of lipid peroxidation (LP), glutathione (GSH) levels and myeloperoxidase activity (MPO). Bladder strips were suspended in oxygenated Tyrode's buffer at 37 degrees C and isometric contractions to carbachol (CCh; 10(-8)-10(-4) m) were recorded. In the I/R group, the contractile responses of the bladder strips were lower than those of the control group (P < 0.01-0.001) and were reversed by treatment with melatonin (P < 0.05-0.001). LP which was higher in I/R group compared with control (27.68 +/- 1.69 and 10.59 +/- 1.27 nmol/g, respectively; P < 0.001) was partially reversed by melatonin (19.01 +/- 1.85 nmol/g; P < 0.01). Similarly, GSH showed a decrease in the I/R group compared with controls (0.27 +/- 0.03 and 0.43 +/- 0.04 micromol/g, respectively; P < 0.05) and melatonin prevented this effect completely (0.45 +/- 0.04 micromol/g; P < 0.05). MPO activity in the I/R group (4.19 +/- 0.08 U/g) was significantly higher than that of the control group (1.41 +/- 0.08 U/g; P < 0.001) and melatonin treatment reduced MPO levels compared with I/R alone (3.16 +/- 0.07; P < 0.001). Melatonin almost completely reversed the low contractile responses of rat urinary bladder strips to CCh and prevented oxidative tissue damage following I/R.     

The protective effect of melatonin on renal ischemia-reperfusion injury in the rat

Oxygen free radicals are considered to be important components involved in the pathophysiological tissue alterations observed during ischemia-reperfusion (I/R). In this study, we investigated the putative protective effects of melatonin treatment on renal I/R injury. Wistar albino rats were unilaterally nephrectomized and subjected to 45 min of renal pedicle occlusion followed by 1, 3, 6, 24, 48 hr or 1 wk of reperfusion. Melatonin (10 mg/kg, s.c.) or vehicle was administered twice, 15 min prior to ischemia and immediately before the reperfusion period. At the end of the reperfusion periods, rats were decapitated. Kidney samples were taken for histological examination or the determination of renal malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity and protein oxidation (PO). Serum creatinine and blood urea nitrogen (BUN) concentrations were measured for the evaluation of renal function. The results revealed that I/R induced nephrotoxicity, as evidenced by increases in BUN and creatinine levels at each time point, was reversed by melatonin treatment. The decrease in GSH and increases in MDA, MPO and PO induced by I/R indicated that renal injury involves free radical formation. As melatonin administration reversed these oxidant responses, improved renal function and microscopic damage, it seems likely that melatonin protects kidney tissue against oxidative damage.     

Melatonin ameliorates myocardial ischemia reperfusion injury through SIRT3‐dependent regulation of oxidative stress and apoptosis

Sirtuins are a family of highly evolutionarily conserved nicotinamide adenine nucleotide‐dependent histone deacetylases. Sirtuin‐3 (SIRT3) is a member of the sirtuin family that is localized primarily to the mitochondria and protects against oxidative stress‐related diseases, including myocardial ischemia/reperfusion (MI/R) injury. Melatonin has a favorable effect in ameliorating MI/R injury. We hypothesized that melatonin protects against MI/R injury by activating the SIRT3 signaling pathway. In this study, mice were pretreated with or without a selective SIRT3 inhibitor and then subjected to MI/R operation. Melatonin was administered intraperitoneally (20 mg/kg) 10 minutes before reperfusion. Melatonin treatment improved postischemic cardiac contractile function, decreased infarct size, diminished lactate dehydrogenase release, reduced the apoptotic index, and ameliorated oxidative damage. Notably, MI/R induced a significant decrease in myocardial SIRT3 expression and activity, whereas the melatonin treatment upregulated SIRT3 expression and activity, and thus decreased the acetylation of superoxide dismutase 2 (SOD2). In addition, melatonin increased Bcl‐2 expression and decreased Bax, Caspase‐3, and cleaved Caspase‐3 levels in response to MI/R. However, the cardioprotective effects of melatonin were largely abolished by the selective SIRT3 inhibitor 3‐(1H‐1,2,3‐triazol‐4‐yl)pyridine (3‐TYP), suggesting that SIRT3 plays an essential role in mediating the cardioprotective effects of melatonin. In vitro studies confirmed that melatonin also protected H9c2 cells against simulated ischemia/reperfusion injury (SIR) by attenuating oxidative stress and apoptosis, while SIRT3‐targeted siRNA diminished these effects. Taken together, our results demonstrate for the first time that melatonin treatment ameliorates MI/R injury by reducing oxidative stress and apoptosis via activating the SIRT3 signaling pathway.     

Hepatoprotective actions of melatonin: possible mediation by melatonin receptors

Melatonin,the hormone of darkness and messenger of the photoperiod,is also well known to exhibit strong direct and indirect antioxidant properties. Melatonin has previously been demonstrated to be a powerful organ protective substance in numerous models of injury; these beneficial effects have been attributed to the hormone’s intense radical scavenging capacity. The present report reviews the hepatoprotective potential of the pineal hormone in various models of oxidative stress in vivo,and summarizes the extensive literature showing that melatonin may be a suitable experimental substance to reduce liver damage after sepsis,hemorrhagic shock,ischemia/reperfusion,and in numerous models of toxic liver injury. Melatonin’s influence on hepatic antioxidant enzymes and other potentially relevant pathways,such as nitric oxide signaling,hepatic cytokine and heat shock protein expression,are evaluated. Based on recent literature demonstrating the functional relevance of melatonin receptor activation for hepatic organ protection,this article finally suggests that melatonin receptors could mediate the hepatoprotective actions of melatonin therapy.     

Abdominal compartment syndrome in patients with severe acute pancreatitis in early stage

AIM： To study retrospectively the influence of intra-abdominal hypertension （IAH） and abdominal compartment syndrome （ACS） in patients with early acute pancreatitis （AP） （during the first week after admission） on physiological functions, and the association of the presence of IAH/ACS and outcome.
METHODS： Patients （n = 74） with AP recruited in this study were divided into two groups according to intra-abdominal pressure （IAP） determined by indirect measurement using the transvesical route via Foley bladder catheter during the first week after admission. Patients （n = 44） with IAP ≥ 12 mmHg were assigned in IAH group, and the remaining patients （n = 30） with IAP 〈 12 mmHg in normal IAP group. For analysis of the influence of IAH/ACS on organ function and outcome, the physiological parameters and the occurrence of organ dysfunction during intensive care unit （ICU） stay were recorded, as were the incidences of pancreatic infection and in-hospital mortality.
RESULTS： IAH within the first week after admission was found in 44 patients （59.46%）. Although the APACHE Ⅱ scores on admission and the Ranson scores within 48 h after hospitalization were elevated in IAH patients in early stage, they did not show the statistically significant differences from patients with normal IAP within a week after admission （16.18 ± 3.90 vs 15.70 ± 4.25, P = 0.616; 3.70 ± 0.93 vs 3.47 ± 0.94, P = 0.285, respectively）. ACS in early AP was recorded in 20 patients （27.03%）. During any 24-h period of the first week after admission, the recorded mean IAP correlated significantly with the Marshall score calculated at the same time interval in IAH group （r = 0.635, P 〈 0.001）. Although ACS patients had obvious amelioration in physiological variables within 24 h after decompression, the incidences of pancreatitic infection, septic shock, multiple organ dysfunction syndrome （MODS） and death in the patients with ACS were significantly higher than that in other patients wit     

Early recognition of abdominal compartment syndrome in patients with acute pancreatitis

AIM： To assess the value of widely used clinical scores in the early identification of acute pancreatitis （AP） patients who are likely to suffer from intra-abdominal hypertension （IAH） and abdominal compartment syndrome （ACS）.
METHODS： Patients （η = 44） with AP recruited in this study were divided into two groups （ACS and non-ACS） according to intra-abdominal pressure （IAP） determined by indirect measurement using the transvesical route via Foley bladder catheter. On admission and at regular intervals, the severity of the AP and presence of organ dysfunction were assessed utilizing different multifactorial prognostic systems： Glasgow-Imrie score, Acute Physiology and Chronic Health Evaluation Ⅱ （APACHE-Ⅱ） score, and Multiorgan Dysfunction Score （MODS）. The diagnostic performance of scores predicting ACS development, cut-off values and specificity and sensitivity were established using receiver operating characteristic （ROC） curve analysis.
RESULTS： The incidence of ACS in our study population was 19.35%. IAP at admission in the ACS group was 22.0 （18.5-25.0） mmHg and 9.25 （3.0-12.4） mmHg in the non-ACS group （P 〈 0.01）. Univariate statistical analysis revealed that patients in the ACS group had significantly higher multifactorial clinical scores （APACHE Ⅱ, Glasgow-Imrie and MODS） on admission and higher maximal scores during hospitalization （P 〈 0.01）. ROC curve analysis revealed that APACHE Ⅱ, Glasgow-Imrie, and MODS are valuable tools for early prediction of ACS with high sensitivity and specificity, and that cut-off values are similar to those used for stratification of patients with severe acute pancreatitis （SAP）.
CONCLUSION： IAH and ACS are rare findings in patients with mild AR Based on the results of our study we recommend measuring the IAP in cases when patients present with SAP （APACHE Ⅱ 〉 7; MODS 〉 2 or Glasgow-Imrie score 〉 3）.     

Effects of physiological and pharmacological concentrations of melatonin on ischemia–reperfusion arrhythmias in rats: can the incidence of sudden cardiac death be reduced?

Cardiac arrhythmias during ischemia-reperfusion (I/R) are believed to be related to free radicals generated in the heart especially during the period of reperfusion. The pineal secretory product, melatonin, is known to be a potent free radical scavenger and its pharmacological concentrations have been shown to reduce the I/R-induced arrhythmias in isolated rat hearts. However, the physiological role of melatonin in the prevention of these arrhythmias is unknown. Rats were pinealectomized (Px) or sham-operated (non-Px) (control) 2 months before the I/R studies. To produce arrhythmias, left main coronary artery was occluded for 7 min, followed by 7 min reperfusion, in anesthetized rats. The incidence of mortality resulted from irreversible ventricular fibrillation (VF) was found significantly higher in the Px rats (63%) than in the control group (25%). Melatonin administration (0.4 mg/kg, either before ischemia or reperfusion) to Px rats significantly reduced the incidence of total (irreversible plus reversible) and irreversible VF and returned them to control values. On the other hand, melatonin administration (0.4 and 4 mg/kg) to non-Px rats failed to attenuate the I/R arrhythmias, significantly. These results suggest that physiological melatonin concentrations are important to reduce the I/R-induced VF and mortality, while pharmacological concentrations of melatonin did not increase its beneficial effect on these arrhythmias. As melatonin levels have been reported to decrease with age, melatonin replacement therapy may attenuate the incidence of sudden cardiac death especially in older patients.     

Melatonin reduces ventricular arrhythmias and preserves capillary perfusion during ischemia-reperfusion events in cardiomyopathic hamsters

Earlier studies showed that melatonin has powerful antioxidative effects on ischemia-reperfusion (I/R) injury in healthy hamsters. In the present study, the possible protective effects of melatonin in 10-month-old cardiomyopathic (CM) hamsters were evaluated in a model of I/R in the cheek pouches observed by intravital microscopy. In CM (BIO 14.6) hamsters diameter, red blood cell (RBC) velocity and flow in arterioles as well as lipid peroxide and nitrite/nitrate concentrations in the systemic blood, perfused capillary length, vascular permeability, and leukocyte adhesion were measured after melatonin injection (6 mg/kg intraperitoneally daily for 3 weeks), and after I/R. The influence of melatonin on the incidence of postischemic-reperfusion-induced ventricular tachycardia (VT) and ventricular fibrillation (VF) were also measured. Changes in the arteriolar response to NG-monomethyl-L-arginine (L-NMMA), a nitric oxide inhibitor, norepinephrine (NE), and angiotensin II (ANG II) were studied before and after melatonin injection (10 mg/kg intravenously). In CM hamsters, melatonin restored normal arteriolar responses to L-NMMA, NE, and ANG II. I/R elevated lipid peroxide and nitrate/nitrite levels, and vascular permeability while arteriolar diameter, RBC velocity, flow and capillary perfusion were reduced. These effects were more marked in CM versus healthy hamsters. During I/R melatonin reduced oxidative and nitrosative stress, vasoconstriction, leukocyte adhesion, and vascular permeability and increased capillary perfusion. Melatonin reduced the incidence of VT while VF during reperfusion disappeared totally. In conclusion, melatonin prevents both microvascular injury and ventricular arrhythmias during postischemic reperfusion by modulating the lipid peroxide overproduction and nitrative stress which are involved in the development of cardiomyopathy.     

Melatonin protects against ischemia/reperfusion injury in skeletal muscle

Abstract:  Melatonin has been shown to diminish ischemia-reperfusion (I/R) injury in many tissues. The main aim of this study was to evaluate the protective antioxidant effect of melatonin in skeletal muscle during I/R injury. Wistar albino rats were randomly divided into three groups. Hindlimb ischemia was achieved by clamping the common femoral artery in two groups but not in control group. Limbs were rendered ischemic for 1.5 hr; at the end of the reperfusion period of 1.5 hr muscle tissue samples were taken for the histological evaluation and biochemical analysis. Melatonin (10 mg/kg) was injected i.p. in the I/R + Mel group at the onset of ischemia whereas the vehicle solution was injected in the I/R group. In I/R + Mel group histological damage was significantly less than in the I/R group ( P  < 0.001). In the I/R + Mel group, the mean malonedialdehyde level was lower than in the I/R group ( P  < 0.01) and was quite near to the levels in the control group ( P  > 0.05). Glutathione levels were found to be reduced in the I/R group compared with the control ( P  < 0.01) and I/R + Mel group ( P  < 0.01). Melatonin has a protective effect against I/R injury in skeletal muscle and may reduce the incidence of compartment syndrome, especially after acute or chronic peripheral arterial occlusions.     

Myocardial ischemia-reperfusion in rats: reduction of infarct size by either supplemental physiological or pharmacological doses of melatonin

Myocardial ischemia-reperfusion (I/R) represents a clinically relevant problem associated with thrombolysis, angioplasty and coronary bypass surgery. I/R injury is believed to be a consequence of free radical generation in the heart especially during the period of reperfusion. The pineal secretory product, melatonin, is known to be a potent free radical scavenger and pharmacological concentrations have been shown to reduce the I/R-induced cardiac damage in isolated rat hearts. However, the physiological role of melatonin in the prevention of this damage is unknown. Rats were pinealectomized or sham-operated (control) 2 months before the I/R studies. To produce cardiac damage, the left main coronary artery was occluded for 30 min, followed by 120 min reperfusion, in anesthetized rats. Infarct size, expressed as the percentage of the risk zone, was found significantly higher in pinealectomized rats (49+/-3.4%) than in the control group (34+/-3.6%). Melatonin administration (4 mg/kg, either before ischemia or reperfusion) to pinealectomized rats significantly reduced the infarct size values and returned them to the control values. On the other hand, melatonin administration (4 mg/kg) to sham-operated rats failed to attenuate significantly the I/R-induced infarct size. These results suggest that physiological melatonin concentrations are important in reducing the I/R-induced myocyte damage, while pharmacological concentrations of melatonin did not add to the beneficial effect. As melatonin levels have been reported to decrease with age, melatonin replacement therapy may attenuate I/R-induced myocardial injury, especially in older patients.     

Reduced silent information regulator 1 signaling exacerbates myocardial ischemia–reperfusion injury in type 2 diabetic rats and the protective effect of melatonin

Diabetes mellitus (DM) increases myocardial oxidative stress and endoplasmic reticulum (ER) stress. Melatonin confers cardioprotective effect by suppressing oxidative damage. However, the effect and mechanism of melatonin on myocardial ischemia–reperfusion (MI/R) injury in type 2 diabetic state are still unknown. In this study, we developed high‐fat diet‐fed streptozotocin (HFD‐STZ) rat, a well‐known type 2 diabetic model, to evaluate the effect of melatonin on MI/R injury with a focus on silent information regulator 1 (SIRT1) signaling, oxidative stress, and PERK/eIF2α/ATF4‐mediated ER stress. HFD‐STZ treated rats were exposed to melatonin treatment in the presence or the absence of sirtinol (a SIRT1 inhibitor) and subjected to MI/R surgery. Compared with nondiabetic animals, type 2 diabetic rats exhibited significantly decreased myocardial SIRT1 signaling, increased apoptosis, enhanced oxidative stress, and ER stress. Additionally, further reduced SIRT1 signaling, aggravated oxidative damage, and ER stress were found in diabetic animals subjected to MI/R surgery. Melatonin markedly reduced MI/R injury by improving cardiac functional recovery and decreasing myocardial apoptosis in type 2 diabetic animals. Melatonin treatment up‐regulated SIRT1 expression, reduced oxidative damage, and suppressed PERK/eIF2α/ATF4 signaling. However, these effects were all attenuated by SIRT1 inhibition. Melatonin also protected high glucose/high fat cultured H9C2 cardiomyocytes against simulated ischemia–reperfusion injury‐induced ER stress by activating SIRT1 signaling while SIRT1 siRNA blunted this action. Taken together, our study demonstrates that reduced cardiac SIRT1 signaling in type 2 diabetic state aggravates MI/R injury. Melatonin ameliorates reperfusion‐induced oxidative stress and ER stress via activation of SIRT1 signaling, thus reducing MI/R damage and improving cardiac function.