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.     

Melatonin receptor‐mediated protection against myocardial ischemia/reperfusion injury: role of SIRT1

Melatonin confers cardioprotective effect against myocardial ischemia/reperfusion (MI/R) injury by reducing oxidative stress. Activation of silent information regulator 1 (SIRT1) signaling also reduces MI/R injury. We hypothesize that melatonin may protect against MI/R injury by activating SIRT1 signaling. This study investigated the protective effect of melatonin treatment on MI/R heart and elucidated its potential mechanisms. Rats were exposed to melatonin treatment in the presence or the absence of the melatonin receptor antagonist luzindole or SIRT1 inhibitor EX527 and then subjected to MI/R operation. Melatonin conferred a cardioprotective effect by improving postischemic cardiac function, decreasing infarct size, reducing apoptotic index, diminishing serum creatine kinase and lactate dehydrogenase release, upregulating SIRT1, Bcl‐2 expression and downregulating Bax, caspase‐3 and cleaved caspase‐3 expression. Melatonin treatment also resulted in reduced myocardium superoxide generation, gp91^phox expression, malondialdehyde level, and increased myocardium superoxide dismutase (SOD) level, which indicate that the MI/R‐induced oxidative stress was significantly attenuated. However, these protective effects were blocked by EX527 or luzindole, indicating that SIRT1 signaling and melatonin receptor may be specifically involved in these effects. In summary, our results demonstrate that melatonin treatment attenuates MI/R injury by reducing oxidative stress damage via activation of SIRT1 signaling in a receptor‐dependent manner.     

Activation of melatonin receptor 2 but not melatonin receptor 1 mediates melatonin‐conferred cardioprotection against myocardial ischemia/reperfusion injury

Accumulated pieces of evidence have proved the beneficial effects of melatonin on myocardial ischemia/reperfusion (MI/R) injury, and these effects were largely dependent on melatonin membrane receptor activation. In humans and other mammals, there are two types of melatonin receptors, including the melatonin receptor 1 (MT1, melatonin receptor 1a or MTNR1A) and melatonin receptor 1 (MT2, melatonin receptor 1b or MTNR1B) receptor subtypes. However, which receptor mediates melatonin‐conferred cardioprotection remains unclear. In this study, we employed both loss‐of‐function and gain‐of‐function approaches to reveal the answer. Mice (wild‐type; MT1 or MT2 silencing by in vivo minicircle vector; and those overexpressing MT1 or MT2 by in vivo AAV9 vector) were exposed to MI/R injury. Both MT1 and MT2 were present in wild‐type myocardium. MT2, but not MT1, was essentially upregulated after MI/R Melatonin administration significantly reduced myocardial injury and improved cardiac function after MI/R Mechanistically, melatonin treatment suppressed MI/R‐initiated myocardial oxidative stress and nitrative stress, alleviated endoplasmic reticulum stress and mitochondrial injury, and inhibited myocardial apoptosis. These beneficial actions of melatonin were absent in MT2‐silenced heart, but not the MT1 subtype. Furthermore, AAV9‐mediated cardiomyocyte‐specific overexpression of MT2, but not MT1, mitigated MI/R injury and improved cardiac dysfunction, which was accompanied by significant amelioration of oxidative stress, endoplasmic reticulum stress, and mitochondrial dysfunction. Mechanistically, MT2 protected primary cardiomyocytes against hypoxia/reoxygenation injury via MT2/Notch1/Hes1/RORα signaling. Our study presents the first direct evidence that the MT2 subtype, but not MT1, is a novel endogenous cardiac protective receptor against MI/R injury. Medications specifically targeting MT2 may hold promise in fighting ischemic heart disease.     

Melatonin attenuates diabetic cardiomyopathy and reduces myocardial vulnerability to ischemia‐reperfusion injury by improving mitochondrial quality control: Role of SIRT6

Targeting mitochondrial quality control with melatonin has been found promising for attenuating diabetic cardiomyopathy (DCM), although the underlying mechanisms remain largely undefined. Activation of SIRT6 and melatonin membrane receptors exerts cardioprotective effects while little is known about their roles during DCM. Using high‐fat diet‐streptozotocin‐induced diabetic rat model, we found that prolonged diabetes significantly decreased nocturnal circulatory melatonin and heart melatonin levels, reduced the expressions of cardiac melatonin membrane receptors, and decreased myocardial SIRT6 and AMPK‐PGC‐1α‐AKT signaling. 16 weeks of melatonin treatment inhibited the progression of DCM and the following myocardial ischemia‐reperfusion (MI/R) injury by reducing mitochondrial fission, enhancing mitochondrial biogenesis and mitophagy via re‐activating SIRT6 and AMPK‐PGC‐1α‐AKT signaling. After the induction of diabetes, adeno‐associated virus carrying SIRT6‐specific small hairpin RNA or luzindole was delivered to the animals. We showed that SIRT6 knockdown or antagonizing melatonin receptors abolished the protective effects of melatonin against mitochondrial dysfunction as evidenced by aggravated mitochondrial fission and reduced mitochondrial biogenesis and mitophagy. Additionally, SIRT6 shRNA or luzindole inhibited melatonin‐induced AMPK‐PGC‐1α‐AKT activation as well as its cardioprotective actions. Collectively, we demonstrated that long‐term melatonin treatment attenuated the progression of DCM and reduced myocardial vulnerability to MI/R injury through preserving mitochondrial quality control. Melatonin membrane receptor‐mediated SIRT6‐AMPK‐PGC‐1α‐AKT axis played a key role in this process. Targeting SIRT6 with melatonin treatment may be a promising strategy for attenuating DCM and reducing myocardial vulnerability to ischemia‐reperfusion injury in diabetic patients.     

Membrane receptor‐dependent Notch1/Hes1 activation by melatonin protects against myocardial ischemia–reperfusion injury: in vivo and in vitro studies

Melatonin confers profound protective effect against myocardial ischemia–reperfusion injury (MI/RI). Activation of Notch1/Hairy and enhancer of split 1 (Hes1) signaling also ameliorates MI/RI. We hypothesize that melatonin attenuates MI/RI‐induced oxidative damage by activating Notch1/Hes1 signaling pathway with phosphatase and tensin homolog deleted on chromosome 10 (Pten)/Akt acting as the downstream signaling pathway in a melatonin membrane receptor‐dependent manner. Male Sprague Dawley rats were treated with melatonin (10 mg/kg/day) for 4 wk and then subjected to MI/R surgery. Melatonin significantly improved cardiac function and decreased myocardial apoptosis and oxidative damage. Furthermore, in cultured H9C2 cardiomyocytes, melatonin (100 μmol/L) attenuated simulated ischemia–reperfusion (SIR)‐induced myocardial apoptosis and oxidative damage. Both in vivo and in vitro study demonstrated that melatonin treatment increased Notch1, Notch1 intracellular domain (NICD), Hes1, Bcl‐2 expressions, and p‐Akt/Akt ratio and decreased Pten, Bax, and caspase‐3 expressions. However, these protective effects conferred by melatonin were blocked by DAPT (the specific inhibitor of Notch1 signaling), luzindole (the antagonist of melatonin membrane receptors), Notch1 siRNA, or Hes1 siRNA administration. In summary, our study demonstrates that melatonin treatment protects against MI/RI by modulating Notch1/Hes1 signaling in a receptor‐dependent manner and Pten/Akt signaling pathways are key downstream mediators.     

Melatonin protects mouse testes from palmitic acid-induced lipotoxicity by attenuating oxidative stress and DNA damage in a SIRT1-dependent manner

Palmitic acid (PA), the main component of dietary saturated fat, has been known to increase in patients with obesity, and PA-induced lipotoxicity may contribute to obesity-related male infertility. Melatonin has beneficial effects on reproductive processes; however, the effect and the underlying molecular mechanism of melatonin's involvement in PA-induced cytotoxicity in the testes are poorly understood. Our findings showed that lipotoxicity was observed in mouse testes after long-term PA treatment and that melatonin therapy restored spermatogenesis and fertility in these males. Moreover, melatonin therapy suppressed PA-induced apoptosis by modulating apoptosis-associated proteins such as Bcl2, Bax, C-Caspase3, C-Caspase12, and CHOP in type B spermatogonial stem cells. Changes in the expression of endoplasmic reticulum (ER) stress markers (p-IRE1, p-PERK, ATF4) and intracellular Ca²⁺ levels showed that melatonin relieved PA-induced ER stress. Mechanistically, melatonin stimulated the expression and nuclear translocation of SIRT1 through its receptors and prevented PA-induced ROS production and mitochondrial dysfunction via SIRT1 signaling pathway. Furthermore, melatonin promoted SIRT1-mediated p53 deacetylation, thereby relieving G2/M arrest in response to PA-stimulated DNA damage. Collectively, these findings indicate that melatonin protects the testes from PA-induced lipotoxicity through the activation of SIRT1, which alleviates oxidative stress, ER stress, mitochondrial dysfunction, and DNA damage.     

Melatonin facilitates adipose‐derived mesenchymal stem cells to repair the murine infarcted heart via the SIRT1 signaling pathway

Mesenchymal stem cells (MSCs)‐based therapy provides a promising therapy for the ischemic heart disease (IHD). However, engrafted MSCs are subjected to acute cell death in the ischemic microenvironment, characterized by excessive inflammation and oxidative stress in the host's infarcted myocardium. Melatonin, an indole, which is produced by many organs including pineal gland, has been shown to protect bone marrow MSCs against apoptosis although the mechanism of action remains elusive. Using a murine model of myocardial infarction (MI), this study was designed to evaluate the impact of melatonin on adipose‐derived mesenchymal stem cells (AD‐MSCs)‐based therapy for MI and the underlying mechanism involved with a focus on silent information regulator 1(SIRT1) signaling. Our results demonstrated that melatonin promoted functional survival of AD‐MSCs in infarcted heart and provoked a synergetic effect with AD‐MSCs to restore heart function. This in vivo effect of melatonin was associated with alleviated inflammation, apoptosis, and oxidative stress in infarcted heart. In vitro studies revealed that melatonin exert cytoprotective effects on AD‐MSCs against hypoxia/serum deprivation (H/SD) injury via attenuating inflammation, apoptosis, and oxidative stress. Mechanistically, melatonin enhanced SIRT1 signaling, which was accompanied with the increased expression of anti‐apoptotic protein Bcl2, and decreased the expression of Ac‐FoxO1, Ac‐p53, Ac‐NF‐ΚB, and Bax. Taken together, our findings indicated that melatonin facilitated AD‐MSCs‐based therapy in MI, possibly through promoting survival of AD‐MSCs via SIRT1 signaling. Our data support the promise of melatonin as a novel strategy to improve MSC‐based therapy for IHD, possibly through SIRT1 signaling evocation.     

Melatonin improves insulin resistance and hepatic steatosis through attenuation of alpha‐2‐HS‐glycoprotein

Melatonin plays an important role in regulating circadian rhythms. It also acts as a potent antioxidant and regulates glucose and lipid metabolism, although the exact action mechanism is not clear. The α2‐HS‐glycoprotein gene (AHSG) and its protein, fetuin‐A (FETUA), are one of the hepatokines and are known to be associated with insulin resistance and type 2 diabetes. The aim of this study was to determine whether melatonin improves hepatic insulin resistance and hepatic steatosis in a FETUA‐dependent manner. In HepG2 cells treated with 300 μmol/L of palmitic acid, phosphorylated AKT expression decreased, and FETUA expression increased, but this effect was inhibited by treatment with 10 μmol/L of melatonin. However, melatonin did not improve insulin resistance in FETUA‐overexpressing cells, indicating that improvement in insulin resistance by melatonin was dependent on downregulation of FETUA. Moreover, melatonin decreased palmitic acid‐induced ER stress markers, CHOP, Bip, ATF‐6, XBP‐1, ATF‐4, and PERK. In addition, in the high‐fat diet (HFD) mice, oral treatment with 100 mg/kg/day melatonin for 10 weeks reduced body weight gain to one‐third of that of the HFD group and hepatic steatosis. Insulin sensitivity and glucose intolerance improved with the upregulation of muscle p‐AKT protein expression. FETUA expression and ER stress markers in the liver and serum of HFD mice were decreased by melatonin treatment. In conclusion, melatonin can improve hepatic insulin resistance and hepatic steatosis through reduction in ER stress and the resultant AHSG expression.     

Melatonin reduces endoplasmic reticulum stress and preserves sirtuin 1 expression in neuronal cells of newborn rats after hypoxia–ischemia

Conditions that interfere with the endoplasmic reticulum (ER) functions cause accumulation of unfolded proteins in the ER lumen, referred to as ER stress, and activate a homeostatic signaling network known as unfolded protein response (UPR). We have previously shown that in neonatal rats subjected to hypoxia–ischemia (HI), melatonin administration significantly reduces brain damage. This study assessed whether attenuation of ER stress is involved in the neuroprotective effect of melatonin after neonatal HI. We found that the UPR was strongly activated after HI. Melatonin significantly reduced the neuron splicing of XBP‐1 mRNA, the increased phosphorylation of eIF2α, and elevated expression of chaperone proteins GRP78 and Hsp70 observed after HI in the brain. CHOP, which plays a convergent role in the UPR, was reduced as well. Melatonin also completely prevented the depletion of SIRT‐1 induced by HI, and this effect was observed in the same neurons that over‐express CHOP. These results demonstrate that melatonin reduces ER stress induced by neonatal HI and preserves SIRT‐1 expression, suggesting that SIRT‐1, due to its action in the modulation of a wide variety of signaling pathways involved in neuroprotection, may play a key role in the reduction of ER stress and neuroprotection observed after melatonin.     

Pre‐ischemia melatonin treatment alleviated acute neuronal injury after ischemic stroke by inhibiting endoplasmic reticulum stress‐dependent autophagy via PERK and IRE1 signalings

Melatonin has demonstrated a potential protective effect in central nervous system. Thus, it is interesting to determine whether pre‐ischemia melatonin administration could protect against cerebral ischemia/reperfusion (IR)‐related injury and the underlying molecular mechanisms. In this study, we revealed that IR injury significantly activated endoplasmic reticulum (ER) stress and autophagy in a middle cerebral artery occlusion mouse model. Pre‐ischemia melatonin treatment was able to attenuate IR‐induced ER stress and autophagy. In addition, with tandem RFP‐GFP‐LC3 adeno‐associated virus, we demonstrated pre‐ischemic melatonin significantly alleviated IR‐induced autophagic flux. Furthermore, we showed that IR induced neuronal apoptosis through ER stress related signalings. Moreover, IR‐induced autophagy was significantly blocked by ER stress inhibitor (4‐PBA), as well as ER‐related signaling inhibitors (PERK inhibitor, GSK; IRE1 inhibitor, 3,5‐dibromosalicylaldehyde). Finally, we revealed that melatonin significantly alleviated cerebral infarction, brain edema, neuronal apoptosis, and neurological deficiency, which were remarkably abolished by tunicamycin (ER stress activator) and rapamycin (autophagy activator), respectively. In summary, our study provides strong evidence that pre‐ischemia melatonin administration significantly protects against cerebral IR injury through inhibiting ER stress‐dependent autophagy. Our findings shed light on the novel preventive and therapeutic strategy of daily administration of melatonin, especially among the population with high risk of cerebral ischemic stroke.     

褪黑素减轻心肌缺血/再灌注损伤的作用及机制

目的:探讨褪黑素（melatonin,Mel）在大鼠心肌缺血/再灌注（MI/R）损伤中的拮抗作用及其机制。方法:80只体质量200~250 g雄性SD大鼠随机分为4组:假手术（Sham）组、溶剂对照（MI/R+V）组、Mel治疗（MI/R+Mel）组、Mel+EX527（MI/R+Mel+EX）组。常规结扎左冠状动脉前降支行心肌缺血/再灌注手术,缺血30 min,再灌72 h后超声心动图法检测各组大鼠心功能,再灌6 h后ELISA法检测血清酶学指标,TUNEL法检测心肌细胞凋亡率,Evans blue-TTC双染法测定梗死面积,Western blot法检测沉默信息转录调控因子1（SIRT1）、乙酰化叉头转录因子1（Ac-Foxo1）及凋亡相关蛋白表达水平。结果:Mel治疗可显著改善MI/R损伤后心功能,降低血清肌酸激酶（CK）及乳酸脱氢酶（LDH）水平,降低凋亡率及梗死面积,上调SIRT1表达,下调Ac-Foxo1水平,降低凋亡相关蛋白表达。而使用EX527阻断SIRT1信号后逆转Mel的上述作用（均P<0.01）。结论:Mel可发挥抗凋亡作用减轻MI/R损伤并改善心功能,其作用机制可能与其激活SIRT1信号通路并降低Ac-Foxo1水平有关。     

Melatonin rescues cardiac thioredoxin system during ischemia‐reperfusion injury in acute hyperglycemic state by restoring Notch1/Hes1/Akt signaling in a membrane receptor‐dependent manner

Stress hyperglycemia is commonly observed in patients suffering from ischemic heart disease. It not only worsens cardiovascular prognosis but also attenuates the efficacies of various cardioprotective agents. This study aimed to investigate the protective effect of melatonin against myocardial ischemia‐reperfusion (MI/R) injury in acute hyperglycemic state with a focus on Notch1/Hes1/Akt signaling and intracellular thioredoxin (Trx) system. Sprague Dawley rats were subjected to MI/R surgery and high‐glucose (HG, 500 g/L) infusion (4 mL/kg/h) to induce temporary hyperglycemia. Rats were treated with or without melatonin (10 mg/kg/d) during the operation. Furthermore, HG (33 mmol/L)‐incubated H9c2 cardiomyoblasts were treated in the presence or absence of luzindole (a competitive melatonin receptor antagonist), DAPT (a γ‐secretase inhibitor), LY294002 (a PI3‐kinase/Akt inhibitor), or thioredoxin‐interacting protein (Txnip) adenoviral vectors. We found that acute hyperglycemia aggravated MI/R injury by suppressing Notch1/Hes1/Akt signaling and intracellular Trx activity. Melatonin treatment effectively ameliorated MI/R injury by reducing infarct size, myocardial apoptosis, and oxidative stress. Moreover, melatonin also markedly enhanced Notch1/Hes1/Akt signaling and rescued intracellular Trx system by upregulating Notch1, N1ICD, Hes1, and p‐Akt expressions, increasing Trx activity, and downregulating Txnip expression. However, these effects were blunted by luzindole, DAPT, or LY294002. Additionally, Txnip overexpression not only decreased Trx activity, but also attenuated the cytoprotective effect of melatonin. We conclude that impaired Notch1 signaling aggravates MI/R injury in acute hyperglycemic state. Melatonin rescues Trx system by reducing Txnip expression via Notch1/Hes1/Akt signaling in a membrane receptor‐dependent manner. Its role as a prophylactic/therapeutic drug deserves further clinical study.     

Porcine Circovirus 2 Deploys PERK Pathway and GRP78 for Its Enhanced Replication in PK-15 Cells

Porcine circovirus type 2 (PCV2) infection induces autophagy and apoptosis. These cellular responses could be connected with endoplasmic reticulum (ER) stress. It remains unknown if PCV2 induces ER stress and if autophagy or apoptosis is primary to PCV2 infection or secondary responses following ER stress. Here, we demonstrate that PCV2 triggered unfolded protein response (UPR) in PK-15 cells by activating the PERK/eIF2α pathway without concomitant activation of IRE1 or ATF6. Since ATF4 and CHOP were induced later than PERK/eIF2α, it is clear that persistent PCV2 infection could lead to selective activation of PERK via the PERK-eIF2α-ATF4-CHOP axis. Therefore, PERK activation could be part of the pro-apoptotic signaling via induced expression of CHOP by PCV2. Since PERK inhibition by GSK2606414 or RNA silencing or suppression of eIF2α dephosphorylation by salubrinal limited viral replication, we suppose that PCV2 deploys UPR to enhance its replication. Over-expression of GRP78 or treatment with tauroursodeoxycholic acid could enhance viral capsid expression and/or viral titers, indicating that these chaperones, endogenous or exogenous, could help correct folding of viral proteins. Our findings provide the first evidence that ER stress plays a role in the pathogenesis of PCV2 infection probably as part of autophagic and apoptotic responses.     

The impaired myocardial ischemic tolerance in adult offspring of diabetic pregnancy is restored by maternal melatonin treatment

Diabetic pregnancy, with ever increasing prevalence, adversely affects embryogenesis and increases vasculometabolic disorder risks in adult offspring. However, it remains poorly understood whether maternal diabetes increases the offspring's susceptibility to heart injuries in adulthood. In this study, we observed that cardiac function and structure were comparable between adult offspring born to diabetic mice and their counterparts born to nondiabetic mice at baseline. However, in response to myocardial ischemia/reperfusion (MIR), diabetic mother offspring exhibited augmented infarct size, cardiac dysfunction, and myocardial apoptosis compared with control, in association with exaggerated activation of mitochondria‐ and endoplasmic reticulum (ER) stress‐mediated apoptosis pathways and oxidative stress. Molecular analysis showed that the impaired myocardial ischemic tolerance in diabetic mother offspring was mainly attributable to blunted cardiac insulin receptor substrate (IRS)‐1/Akt signaling. Furthermore, the effect of maternal melatonin administration on offspring's response to MIR was determined, and the results indicated that melatonin treatment in diabetic dams during pregnancy significantly improved the tolerance to MIR injury in their offspring, via restoring cardiac IRS‐1/Akt signaling. Taken together, these data suggest that maternal diabetes predisposes offspring to augmented MIR injury in adulthood, and maternal melatonin supplementation during diabetic pregnancy may hold promise for improving myocardial ischemic tolerance in the offspring.     

JAK2/STAT3 activation by melatonin attenuates the mitochondrial oxidative damage induced by myocardial ischemia/reperfusion injury

Ischemia/reperfusion injury (IRI) is harmful to the cardiovascular system and causes mitochondrial oxidative stress. Numerous data indicate that the JAK2/STAT3 signaling pathway is specifically involved in preventing myocardial IRI. Melatonin has potent activity against IRI and may regulate JAK2/STAT3 signaling. This study investigated the protective effect of melatonin pretreatment on myocardial IRI and elucidated its potential mechanism. Perfused isolated rat hearts and cultured neonatal rat cardiomyocytes were exposed to melatonin in the absence or presence of the JAK2/STAT3 inhibitor AG490 or JAK2 siRNA and then subjected to IR. Melatonin conferred a cardio‐protective effect, as shown by improved postischemic cardiac function, decreased infarct size, reduced apoptotic index, diminished lactate dehydrogenase release, up‐regulation of the anti‐apoptotic protein Bcl2, and down‐regulation of the pro‐apoptotic protein Bax. AG490 or JAK2 siRNA blocked melatonin‐mediated cardio‐protection by inhibiting JAK2/STAT3 signaling. Melatonin exposure also resulted in a well‐preserved mitochondrial redox potential, significantly elevated mitochondrial superoxide dismutase (SOD) activity, and decreased formation of mitochondrial hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), which indicates that the IR‐induced mitochondrial oxidative damage was significantly attenuated. However, this melatonin‐induced effect on mitochondrial function was reversed by AG490 or JAK2 siRNA treatment. In summary, our results demonstrate that melatonin pretreatment can attenuate IRI by reducing IR‐induced mitochondrial oxidative damage via the activation of the JAK2/STAT3 signaling pathway.     

Melatonin modulates neonatal brain inflammation through endoplasmic reticulum stress,autophagy, and miR‐34a/silent information regulator 1 pathway

Maternal infection/inflammation represents one of the most important factors involved in the etiology of brain injury in newborns. We investigated the modulating effect of prenatal melatonin on the neonatal brain inflammation process resulting from maternal intraperitoneal (i.p.) lipopolysaccharide (LPS) injections. LPS (300 μg/kg) was administered to pregnant rats at gestational days 19 and 20. Melatonin (5 mg/kg) was administered i.p. at the same time as LPS. Melatonin counteracted the LPS sensitization to a second ibotenate‐induced excitotoxic insult performed on postnatal day (PND) 4. As melatonin succeeded in reducing microglial activation in neonatal brain at PND1, pathways previously implicated in brain inflammation regulation, such as endoplasmic reticulum (ER) stress, autophagy and silent information regulator 1 (SIRT1), a melatonin target, were assessed at the same time‐point in our experimental groups. Results showed that maternal LPS administrations resulted in an increase in CHOP and Hsp70 protein expression and eIF2α phosphorylation, indicative of activation of the unfolded protein response consequent to ER stress, and a slighter decrease in the autophagy process, determined by reduced lipidated LC3 and increased p62 expression. LPS‐induced inflammation also reduced brain SIRT1 expression and affected the expression of miR‐34a, miR146a, and miR‐126. All these effects were blocked by melatonin. Cleaved‐caspase‐3 apoptosis pathway did not seem to be implicated in the noxious effect of LPS on the PND1 brain. We conclude that melatonin is effective in reducing maternal LPS‐induced neonatal inflammation and related brain injury. Its role as a prophylactic/therapeutic drug deserves to be investigated by clinical studies.     

The nuclear melatonin receptor RORα is a novel endogenous defender against myocardial ischemia/reperfusion injury

Circadian rhythm disruption or decrease in levels of circadian hormones such as melatonin increases ischemic heart disease risk. The nuclear melatonin receptors RORs are pivotally involved in circadian rhythm regulation and melatonin effects mediation. However, the functional roles of RORs in the heart have never been investigated and were therefore the subject of this study on myocardial ischemia/reperfusion (MI/R) injury pathogenesis. RORα and RORγ subtypes were detected in the adult mouse heart, and RORα but not RORγ was downregulated after MI/R. To determine the pathological consequence of MI/R‐induced reduction of RORα, we subjected RORα‐deficient staggerer mice and wild‐type (WT) littermates to MI/R injury, resulting in significantly increased myocardial infarct size, myocardial apoptosis and exacerbated contractile dysfunction in the former. Mechanistically, RORα deficiency promoted MI/R‐induced endoplasmic reticulum stress, mitochondrial impairments, and autophagy dysfunction. Moreover, RORα deficiency augmented MI/R‐induced oxidative/nitrative stress. Given the emerging evidence of RORα as an essential melatonin effects mediator, we further investigated the RORα roles in melatonin‐exerted cardioprotection, in particular against MI/R injury, which was significantly attenuated in RORα‐deficient mice, but negligibly affected by cardiac‐specific silencing of RORγ. Finally, to determine cell type‐specific effects of RORα, we generated mice with cardiomyocyte‐specific RORα overexpression and they were less vulnerable to MI/R injury. In summary, our study provides the first direct evidence that the nuclear melatonin receptor RORα is a novel endogenous protective receptor against MI/R injury and an important mediator of melatonin‐exerted cardioprotection; melatonin‐RORα axis signaling thus appears important in protection against ischemic heart injury.     

白藜芦醇对糖尿病大鼠心脏缺血再灌注损伤的保护作用

目的探讨白藜芦醇(RSV)对糖尿病大鼠心肌缺血再灌注(MI/R)损伤的保护作用及其机制。方法通过腹腔注射链脲佐菌素诱导2型糖尿病大鼠模型。2周后糖尿病大鼠随机分为假手术(Sham)组、MI/R组和白藜芦醇(RSV)组。通过结扎左冠状动脉前降支诱导MI/R损伤模型。测定各组大鼠乳酸脱氢酶(LDH)、肌酸激酶(CK)、心肌肌钙蛋白I(cTnI)、心肌梗死面积、心脏收缩和舒张功能;TUNEL法检测心肌细胞凋亡指数;Western blot检测沉默信息调节因子1(SIRT1)、p53、乙酰化p53(Acetyl-p53)、Bcl-2、Bax以及细胞浆和线粒体细胞色素C(Cyt C)和凋亡诱导因子(AIF)的表达;HE染色检测心肌损伤评分。结果与Sham组相比,MI/R组心肌梗死面积、心肌损伤评分、心肌LDH、CK、cTnI、Acetyl-p53、Bax、细胞浆Cyt C和AIF表达以及心肌细胞凋亡指数均明显增加,而心脏收缩和舒张功能明显降低,Bcl-2和SIRT1表达以及线粒体Cyt C和AIF表达明显减少。与MI/R组相比,RSV组心肌梗死面积、心肌损伤评分、心肌LDH、CK、cTnI、Acetyl-p53、Bax、细胞浆Cyt C和AIF表达以及心肌细胞凋亡指数均明显降低,而心脏收缩和舒张功能明显改善,Bcl-2和SIRT1表达以及线粒体Cyt C和AIF表达明显增加。3组之间p53表达无差异。结论白藜芦醇可通过抗凋亡作用减轻糖尿病大鼠MI/R损伤,其作用机制与SIRT1/p53信号通路相关。     

Melatonin alleviates brain injury in mice subjected to cecal ligation and puncture via attenuating inflammation,apoptosis, and oxidative stress: the role of SIRT1 signaling

Sepsis is a systemic inflammatory response to infection that causes severe neurological complications. Previous studies have suggested that melatonin is protective during sepsis. Additionally, silent information regulator 1 (SIRT1) was reported to be beneficial in sepsis. However, the role of SIRT1 signaling in the protective effect of melatonin against septic encephalopathy remains unclear. This study aimed to investigate the role of SIRT1 in the protective effect of melatonin. EX527, a SIRT1 inhibitor, was used to reveal the role of SIRT1 in melatonin's action. Cecal ligation and puncture or sham operation was performed in male C57BL/6J mice. Melatonin was administrated intraperitoneally (30 mg/kg). The survival rate of mice was recorded for the 7‐day period following the sham or CLP operation. The blood–brain barrier (BBB) integrity, brain water content, levels of inflammatory cytokines (TNF‐α, IL‐1β, and HMGB1), and the level of oxidative stress (superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA)) and apoptosis were assessed. The expression of SIRT1, Ac‐FoxO1, Ac‐p53, Ac‐NF‐κB, Bcl‐2, and Bax was detected by Western blot. The results suggested that melatonin improved survival rate, attenuated brain edema and neuronal apoptosis, and preserved BBB integrity. Melatonin decreased the production of TNF‐α, IL‐1β, and HMGB1. Melatonin increased the activity of SOD and CAT and decreased the MDA production. Additionally, melatonin upregulated the expression of SIRT1 and Bcl‐2 and downregulated the expression of Ac‐FoxO1, Ac‐p53, Ac‐NF‐κB, and Bax. However, the protective effects of melatonin were abolished by EX527. In conclusion, our results demonstrate that melatonin attenuates sepsis‐induced brain injury via SIRT1 signaling activation.