Melatonin reduces endoplasmic reticulum stress and autophagy in liver of leptin‐deficient mice

The sedentary lifestyle of modern society along with the high intake of energetic food has made obesity a current worldwide health problem. Despite great efforts to study the obesity and its related diseases, the mechanisms underlying the development of these diseases are not well understood. Therefore, identifying novel strategies to slow the progression of these diseases is urgently needed. Experimental observations indicate that melatonin has an important role in energy metabolism and cell signalling; thus, the use of this molecule may counteract the pathologies of obesity. In this study, wild‐type and obese (ob/ob) mice received daily intraperitoneal injections of melatonin at a dose of 500 μg/kg body weight for 4 weeks, and the livers of these mice were used to evaluate the oxidative stress status, proteolytic (autophagy and proteasome) activity, unfolded protein response, inflammation and insulin signalling. Our results show, for the first time, that melatonin could significantly reduce endoplasmic reticulum stress in leptin‐deficient obese animals and ameliorate several symptoms that characterize this disease. Our study supports the potential of melatonin as a therapeutic treatment for the most common type of obesity and its liver‐associated disorders.     

Melatonin and endoplasmic reticulum stress: relation to autophagy and apoptosis

Endoplasmic reticulum (ER) is a dynamic organelle that participates in a number of cellular functions by controlling lipid metabolism, calcium stores, and proteostasis. Under stressful situations, the ER environment is compromised, and protein maturation is impaired; this causes misfolded proteins to accumulate and a characteristic stress response named unfolded protein response (UPR). UPR protects cells from stress and contributes to cellular homeostasis re‐establishment; however, during prolonged ER stress, UPR activation promotes cell death. ER stressors can modulate autophagy which in turn, depending of the situation, induces cell survival or death. Interactions of different autophagy‐ and apoptosis‐related proteins and also common signaling pathways have been found, suggesting an interplay between these cellular processes, although their dynamic features are still unknown. A number of pathologies including metabolic, neurodegenerative and cardiovascular diseases, cancer, inflammation, and viral infections are associated with ER stress, leading to a growing interest in targeting components of the UPR as a therapeutic strategy. Melatonin has a variety of antioxidant, anti‐inflammatory, and antitumor effects. As such, it modulates apoptosis and autophagy in cancer cells, neurodegeneration and the development of liver diseases as well as other pathologies. Here, we review the effects of melatonin on the main ER stress mechanisms, focusing on its ability to regulate the autophagic and apoptotic processes. As the number of studies that have analyzed ER stress modulation by this indole remains limited, further research is necessary for a better understanding of the crosstalk between ER stress, autophagy, and apoptosis and to clearly delineate the mechanisms by which melatonin modulates these responses.     

Melatonin prevents the decreased activity of antioxidant enzymes and activates nuclear erythroid 2‐related factor 2 signaling in an animal model of fulminant hepatic failure of viral origin

Abstract: This work was undertaken to investigate whether treatment with melatonin prevents oxidative stress and changes in the expression and activity of factor erythroid 2‐related factor 2 (Nrf2)‐mediated antioxidant enzymes in an animal model of fulminant hepatic failure of viral origin. Rabbits were experimentally infected with 2 × 10⁴ hemagglutination units of a rabbit hemorrhagic disease virus (RHDV) isolate and received melatonin at two concentrations of 10 mg/kg and 20 mg/kg at 0, 12 and 24 hr postinfection. Blood transaminases, blood lactate dehydrogenase, liver concentration of thiobarbituric reactive acid substances and the liver oxidized to reduced glutathione ratio significantly increased at 36 hr postinfection in infected animals. Significant decreases were found in the mRNA levels and in the liver activities of Mn‐superoxide dismutase, glutathione peroxidase and glutathione‐S‐transferase in infected rabbits. These effects were prevented by melatonin administration in a concentration‐dependent manner. Melatonin treatment was not accompanied by changes in protein levels of Kelch‐like ECH‐associating protein 1 (Keap1) but resulted in an increased protein expression of Nrf2 in the cytoplasm and the nucleus, which was confirmed by the results of Nrf2 immunostaining. Nuclear extracts from livers of melatonin‐treated rats displayed an enhanced antioxidant responsive element (ARE)‐binding activity of Nrf2. Our results suggest a potential hepatoprotective role of melatonin in fulminant hepatic failure, partially mediated through the abrogation of oxidative stress and the prevention of the decreased activity of antioxidant enzymes via the Nrf2 pathways.     

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.     

Melatonin reverses tunicamycin‐induced endoplasmic reticulum stress in human hepatocellular carcinoma cells and improves cytotoxic response to doxorubicin by increasing CHOP and decreasing Survivin

Chemoresistance in hepatocellular carcinoma (HCC) is associated with multiple cellular responses to environmental stresses, such as nutrient deprivation and hypoxia. Nevertheless, whether ER stress resulting from nutrient deprivation and tumor hypoxia contributes to drug resistance remains unclear. Melatonin increased the efficacy of chemotherapeutic drugs in hepatocellular carcinoma in our previous studies. However, the effects of melatonin on endoplasmic reticulum (ER) stress‐induced resistance to chemotherapeutic agents in HCC have not been tested. The effect of the endoplasmic reticulum (ER) stress response during resistance of human hepatocellular carcinoma cells against doxorubicin was investigated in this study. Pretreatment of HepG2 and SMMC‐7721 cells (two human hepatocellular carcinoma cell lines) with tunicamycin, an ER stress inducer, drastically decreased the rate of apoptosis generated by doxorubicin. Interestingly, co‐pretreatment with tunicamycin and melatonin significantly increased apoptosis induced by doxorubicin. Simultaneously, the expression of phosphorylated AKT (p‐AKT) was elevated in HepG2 and SMMC‐7721 cells given tunicamycin but reduced in the presence of melatonin. Furthermore, consistent with inhibition of AKT activation by using the PI3K inhibitor LY294002, melatonin elevated the levels of CHOP (C/EBP‐homologous protein) and reduced the levels of Survivin (a member of the inhibitor of apoptosis protein family)suggesting that inhibition of the PI3K/AKT pathway by melatonin‐reversed ER stress‐induced resistance to doxorubicin in human hepatocellular carcinoma cells. These results demonstrate that melatonin attenuates ER stress‐induced resistance to doxorubicin in human hepatocellular carcinoma cells by down‐regulating the PI3K/AKT pathway, increasing the levels of CHOP and decreasing the levels of Survivin.     

Melatonin reduces endoplasmic reticulum stress and corneal dystrophy‐associated TGFBIp through activation of endoplasmic reticulum‐associated protein degradation

Endoplasmic reticulum (ER) stress is emerging as a factor for the pathogenesis of granular corneal dystrophy type 2 (GCD2). This study was designed to investigate the molecular mechanisms underlying the protective effects of melatonin on ER stress in GCD2. Our results showed that GCD2 corneal fibroblasts were more susceptible to ER stress‐induced death than were wild‐type cells. Melatonin significantly inhibited GCD2 corneal cell death, caspase‐3 activation, and poly (ADP‐ribose) polymerase 1 cleavage caused by the ER stress inducer, tunicamycin. Under ER stress, melatonin significantly suppressed the induction of immunoglobulin heavy‐chain‐binding protein (BiP) and activation of inositol‐requiring enzyme 1α (IRE1α), and their downstream target, alternative splicing of X‐box binding protein 1(XBP1). Notably, the reduction in BiP and IRE1α by melatonin was suppressed by the ubiquitin‐proteasome inhibitor, MG132, but not by the autophagy inhibitor, bafilomycin A1, indicating involvement of the ER‐associated protein degradation (ERAD) system. Melatonin treatment reduced the levels of transforming growth factor‐β‐induced protein (TGFBIp) significantly, and this reduction was suppressed by MG132. We also found reduced mRNA expression of the ERAD system components HRD1 and SEL1L, and a reduced level of SEL1L protein in GCD2 cells. Interestingly, melatonin treatments enhanced SEL1L levels and suppressed the inhibition of SEL1L N‐glycosylation caused by tunicamycin. In conclusion, this study provides new insights into the mechanisms by which melatonin confers its protective actions during ER stress. The results also indicate that melatonin might have potential as a therapeutic agent for ER stress‐related diseases including GCD2.     

Melatonin attenuates apoptotic liver damage in fulminant hepatic failure induced by the rabbit hemorrhagic disease virus

Hepatocyte apoptosis plays an important role in the development of fulminant hepatic failure (FHF). The objective of this study was to investigate the antiapoptotic effect of melatonin in an animal model of FHF of viral origin induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2 × 10(4) hemagglutination units of a RHDV isolate and received melatonin at two concentrations of 10 and 20 mg/kg at 0, 12, and 24 hr postinfection. RHDV infection induced liver apoptosis, with increased caspase-3 immunoexpression and activity and poly(ADP-ribose)polymerase-1 (PARP-1) proteolysis. These effects were attenuated by melatonin in a concentration-dependent manner. Antiapoptotic effects of melatonin were related to a reduced expression of Bax and cytosolic cytochrome c release, increased expression of Bcl-2 and Bcl-xL, and inhibition of caspase-9 activity. Increased thiobarbituric reactive acid substances concentration and oxidized-to-reduced glutathione ratio were significantly prevented by melatonin administration. Melatonin treatment also resulted in a reduction in caspase-8 activity, tumor necrosis factor receptor-1 (TNF-R1) expression, and phosphorylated Janus kinase (JNK) expression, and increased expression of cellular FLICE-inhibitory protein (c-FLIP). Our findings show that inhibition of apoptotic mechanisms contributes to the beneficial effects of melatonin in rabbits with experimental infection by RHDV and supports a potential hepatoprotective role of melatonin in FHF.     

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.     

Ghrelin通过抑制ERS减轻ISO所致的心肌损伤和心肌细胞凋亡

目的探讨ghrelin（G）减轻异丙基肾上腺素（isoproterenol,ISO）所致心肌损伤和凋亡是否与其抑制心肌内质网应激（endoplasmic reticulum stress,ERS）有关。方法：将29只雄性Sprague-Dawley（SD）大鼠随机分为对照（Con）组、ISO组和ISO＋G组。Con组皮下注射生理盐水,ISO组皮下注射ISO,ISO＋G组皮下注射ISO前2 d皮下注射ghrelin。采用生理记录仪测定各组大鼠心功能。以HE染色及血浆乳酸脱氢酶（LDH）及肌酸激酶MB（CK-MB）活性的测定评估心肌损伤的程度。用TUNEL法测定心肌细胞的凋亡。用Western blot检测各组心肌组织中ERS标志性分子C／EBP同源蛋白（C／EBP-homologous protein,CHOP）和easpase．12的表达。结果：外源性ghrelin可显著缓解ISO所致大鼠的心功能不全,减轻ISO造成的心肌损伤,减少心肌细胞凋亡,并降低CHOP和剪切的caspase-12的表达。结论：ghrelin通过抑制ERS可减轻ISO所致心肌损伤和心肌细胞凋亡。     

Melatonin alleviates lipopolysaccharide-induced placental cellular stress response in mice

Abstract: Melatonin protects mice from lipopolysaccharide (LPS)‐induced fetal death and intra‐uterine growth retardation. Nevertheless, its molecular mechanism remains obscure. In the present study, we investigated the effects of melatonin on LPS‐induced cellular stress in placenta. Pregnant mice were given with melatonin [5.0 mg/kg, intraperitoneal (i.p.)] 30 min before and 150 min after LPS (300 μg/kg, i.p.) on gestational day 15. Oxidative stress, endoplasmic reticulum (ER) stress, hypoxic stress, and heat stress in placenta were analyzed at 4 hr after LPS. As expected, maternal LPS administration resulted in placental glutathione (GSH) depletion and up‐regulated the expression of placental antioxidative enzymes. In addition, LPS significantly increased the level of inducible nitric oxide synthase (iNOS) and enhanced the intensity of placental 3‐nitrotyrosine residues. An ER stress, as determined by a decreased GRP78 expression, an obvious eIF2α and JNK phosphorylation, and an increased CHOP expression, were observed in placenta of pregnant mice injected with LPS. In addition, LPS significantly increased mRNA level of placental HIF‐1α, VEGF, and ET‐1, the markers of hypoxic stress. Heme oxygenase (HO)‐1, a marker of heat stress, was also up‐regulated in placenta of LPS‐treated pregnant mice. Interestingly, LPS‐induced placental oxidative stress, hypoxic stress, and ER stress were significantly alleviated when pregnant mice were given with melatonin, whereas melatonin had little effect on LPS‐evoked placental HO‐1 expression. In conclusion, maternally administered melatonin alleviates LPS‐induced cellular stress in the placenta. Melatonin may be useful as pharmacological agents to protect the fetuses against LPS‐induced intra‐uterine fetal death and intra‐uterine growth restriction.     

内质网应激与酒精性心肌病的关系研究进展

近年来,随着酒精性饮品的摄入增加,酒精性心肌病(ACM)的发病率和死亡率也增加。研究发现ACM发病涉及多层次和水平的调控。而且,细胞信号调控的关键场所内质网应激(ERS)在ACM中的作用受到重视。因此,本文对ACM的概述、发病机制以及ERS在酒精所致心肌损伤中的作用进行简要综述。     

内质网应激与心血管疾病的研究进展

内质网(ER)是真核细胞最主要的膜性结构,是细胞内重要生理过程发生的关键细胞器。在多种内外因素的作用下,ER的稳态受到破坏,导致蛋白质加工运输受阻,未折叠蛋白或错误折叠蛋白在ER腔内聚集,形成内质网应激(ERS),并触发未折叠蛋白反应(UPR)。适度的ERS通过UPR信号通路减少蛋白质合成、促进蛋白质降解、增加协助蛋白质折叠的分子伴侣,最终缓解ER压力。但是,如果ERS过强或持续时间过长,超过细胞的自身调节能力时,UPR可启动细胞凋亡,亦可导致疾病。大量研究表明,ERS与多种心血管疾病(CVD)的发生发展密切相关。该综述主要阐述UPR在几种常见CVD中的研究进展和靶向UPR作为CVD的潜在治疗方法。     

内质网应激在心肌缺血再灌注损伤中的作用

内质网应激（ERS）是细胞对内外环境变化的一种适应性反应,有助于细胞和生命个体的存活,但持续存在或过强时则最终诱发细胞凋亡。缺血,再灌注（I／R）时,钙超载及大量自由基的生成等因素诱导过度ERS导致组织损伤。缺血预处理及稳定Ca2＋稳态可激发适当的ERS,增强细胞耐受长时间缺血刺激的能力,延缓或减轻I／R造成的组织损伤。现综述ERS在缺血再灌注损伤（IRI）中的作用,并指出ERS已成为IRI防治的新靶点。     

Accurate prediction of fulminant hepatic failure in severe acute viral hepatitis: multicenter study

Background: We have attempted to predict the development of fulminant hepatic failure at the stage of severe acute hepatitis before the onset of coma. This prediction is valuable because it may be used to block the development of fulminant hepatic failure with appropriate medical treatment. Methods: To establish a discrimination formula, we retrospectively compared 13 clinical and laboratory variables in 36 patients with acute viral hepatitis and prothrombin levels of 40% or less of the control value who later developed fulminant hepatic failure with these variables in 12 patients who recovered spontaneously. A prospective study of 58 patients who developed fulminant hepatic failure and 18 who spontaneously recovered confirmed the validity of this formula. Results: In the retrospective study, we established the following discrimination equation: Z = −0.89 + 1.74 × (causal viruses, 1 point for type A or type B in acute hepatitis B virus [HBV] infection, 2 points for others) + 0.056 × (total bilirubin, mg/dl) −0.014 × (cholinesterase, U/ml). A positive Z value indicates that fulminant hepatic failure will develop. In the prospective study, the specificity, sensitivity, predictive accuracy, and positive and negative predictive values were 0.833, 0.983, 0.947, 0.950, and 0.938, respectively. Conclusions: The present study indicated that fulminant hepatic failure can be predicted, by a simple discrimination equation, at the stage of severe acute hepatitis. Received: September 4, 2001 / Accepted: May 31, 2002 Acknowledgments. Supported by a grant from the Ministry of Health and Welfare of Japan. Reprint requests to: M. Yoshiba