Melatonin ameliorates cerulein‐induced pancreatitis by the modulation of nuclear erythroid 2‐related factor 2 and nuclear factor‐kappaB in rats

Abstract: Melatonin exhibits a wide variety of biological effects, including antioxidant and anti‐inflammatory functions. Its antioxidant role impedes the etiopathogenesis of pancreatitis, but little is known about the signaling pathway of melatonin in the induction of antioxidant enzymes in acute pancreatitis (AP). The aim of this study was to determine whether melatonin could prevent cerulein‐induced AP through nuclear factor erythroid 2‐related factor 2 (Nrf2) and curtail inflammation by inhibition of NF‐κB. AP was induced by two intraperitoneal (i.p.) injections of cerulein at 2 h intervals (50 μg/kg) in Sprague‐Dawley rats. Melatonin (10 or 50 mg/kg/daily, i.p.) was administered 24 h before each injection of cerulein. The rats were killed 12 h after the last injection. Acinar cell degeneration, pancreatic edema, and inflammatory infiltration were significantly different in cerulein‐ and melatonin‐treated rats. Melatonin significantly reduced amylase, lipase, MPO, and MDA levels, and increased antioxidant enzyme activities including SOD and GPx, which were decreased in AP (P < 0.05). Melatonin increased the expression of NQO1, HO‐1, and SOD2 when compared with the cerulein‐induced AP group (P < 0.05). In addition, melatonin increased Nrf2 expression, and reduced expressions of tumor necrosis factor‐alpha, IL‐1β, IL‐6, IL‐8, and iNOS. The elevated nuclear binding of NF‐κB in the cerulein‐induced pancreatitis group was inhibited by melatonin. These results show that melatonin increases antioxidant enzymes and Nrf2 expression, and limits inflammatory mediators in cerulein‐induced AP. It is proposed that melatonin may play an important role in oxidative stress via the Nrf2 pathway in parallel with reduction of inflammation by NF‐κB inhibition.     

Melatonin stabilizes rupture‐prone vulnerable plaques via regulating macrophage polarization in a nuclear circadian receptor RORα‐dependent manner

Rupture of vulnerable plaques is the main trigger of acute cardio‐cerebral vascular events, but mechanisms responsible for transforming a stable atherosclerotic into a vulnerable plaque remain largely unknown. Melatonin, an indoleamine hormone secreted by the pineal gland, plays pleiotropic roles in the cardiovascular system; however, the effect of melatonin on vulnerable plaque rupture and its underlying mechanisms remains unknown. Here, we generated a rupture‐prone vulnerable carotid plaque model induced by endogenous renovascular hypertension combined with low shear stress in hypercholesterolemic ApoE^?/? mice. Melatonin (10 mg/kg/d by oral administration for 9 weeks) significantly prevented vulnerable plaque rupture, with lower incidence of intraplaque hemorrhage (42.9% vs. 9.5%, P = 0.014) and of spontaneous plaque rupture with intraluminal thrombus formation (38.1% vs. 9.5%, P = 0.029). Mechanistic studies indicated that melatonin ameliorated intraplaque inflammation by suppressing the differentiation of intraplaque macrophages toward the proinflammatory M1 phenotype, and circadian nuclear receptor retinoid acid receptor‐related orphan receptor‐α (RORα) mediated melatonin‐exerted vasoprotection against vulnerable plaque instability and intraplaque macrophage polarization. Further analysis in human monocyte‐derived macrophages confirmed the role of melatonin in regulating macrophage polarization by regulating the AMPKα‐STATs pathway in a RORα‐dependent manner. In summary, our data provided the first evidence that melatonin‐RORα axis acts as a novel endogenous protective signaling pathway in the vasculature, regulates intraplaque inflammation, and stabilizes rupture‐prone vulnerable plaques.     

Macrophage functions in lean and obese adipose tissue

Interactions between macrophages and adipocytes influence both metabolism and inflammation. Obesity-induced changes to macrophages and adipocytes lead to chronic inflammation and insulin resistance. This paper reviews the various functions of macrophages in lean and obese adipose tissue and how obesity alters adipose tissue macrophage phenotypes. Metabolic disease and insulin resistance shift the balance between numerous pro- and anti-inflammatory regulators of macrophages and create a feed-forward loop of increasing inflammatory macrophage activation and worsening adipocyte dysfunction. This ultimately leads to adipose tissue fibrosis and diabetes. The molecular mechanisms underlying these processes have therapeutic implications for obesity, metabolic syndrome, and diabetes.     

Melatonin reduces inflammatory response in human intestinal epithelial cells stimulated by interleukin‐1β

Melatonin is the main secretory product of the pineal gland, and it is involved in the regulation of periodic events. A melatonin production independent of the photoperiod is typical of the gut. However, the local physiological role of melatonin at the intestinal tract is poorly characterized. In this study, we evaluated the anti‐inflammatory activities of melatonin in an in vitro model of inflamed intestinal epithelium. To this purpose, we assessed different parameters usually associated with intestinal inflammation using IL‐1β‐stimulated Caco‐2 cells. Differentiated monolayers of Caco‐2 cells were preincubated with melatonin (1 nmol/L‐50 μmol/L) and then exposed to IL‐1β. After each treatment, different inflammatory mediators, DNA‐breakage, and global DNA methylation status were assayed. To evaluate the involvement of melatonin membrane receptors, we also exposed differentiated monolayers to melatonin in the presence of luzindole, a MT1 and MT2 antagonist. Our results showed that melatonin, at concentrations similar to those obtained in the lumen gut after ingestion of dietary supplements for the treatment of sleep disorders, was able to attenuate the inflammatory response induced by IL‐1β. Anti‐inflammatory effects were expressed as both a decrease of the levels of inflammatory mediators, including IL‐6, IL‐8, COX‐2, and NO, and a reduced increase in paracellular permeability. Moreover, the protection was associated with a reduced NF‐κB activation and a prevention of DNA demethylation. Conversely, luzindole did not reverse the melatonin inhibition of stimulated‐IL‐6 release. In conclusion, our findings suggest that melatonin, through a local action, can modulate inflammatory processes at the intestinal level, offering new opportunities for a multimodal management of IBD.     

Melatonin attenuates (‐)‐epigallocatehin‐3‐gallate‐triggered hepatotoxicity without compromising its downregulation of hepatic gluconeogenic and lipogenic genes in mice

(‐)‐Epigallocatehin‐3‐gallate (EGCG), a major constituent of green tea, can ameliorate metabolic syndrome at least in part through reducing gluconeogenesis and lipogenesis. Green tea extracts, of which EGCG is a key constituent, have been used for weight loss in humans. A potential adverse effect of high‐dose EGCG or green tea extracts is hepatotoxicity. Melatonin, an endogenous antioxidant with a high safety profile, is effective in preventing various types of tissue damage. The current study investigated the influence of melatonin on EGCG‐triggered hepatotoxicity and EGCG‐downregulated hepatic genes responsible for gluconeogenesis and lipogenesis in mice. We found that (i) melatonin extended survival time of mice intoxicated with lethal doses of EGCG; (ii) melatonin ameliorated acute liver damage and associated hepatic Nrf2 suppression caused by a nonlethal toxic dose of EGCG; (iii) melatonin reduced subacute liver injury and hepatic Nrf2 activation caused by lower toxic doses of EGCG; and (iv) melatonin did not compromise the action of pharmacological doses of EGCG in downregulating a battery of hepatic genes responsible for gluconeogenesis and lipogenesis, including G6Pc, PEPCK, FOXO1α, SCD1, Fasn, leptin, ACCα, ACCβ, GAPT, and Srebp‐1. Taken together, these results suggest that the combination of EGCG and melatonin is an effective approach for preventing potential adverse effects of EGCG as a dietary supplement for metabolic syndrome alleviation and body weight reduction.     

Melatonin suppresses activation of hepatic stellate cells through RORα‐mediated inhibition of 5‐lipoxygenase

Liver fibrosis is scar tissue resulting from an uncontrolled wound‐healing process in response to chronic liver injury. Liver damage generates an inflammatory reaction that activates hepatic stellate cells (HSC) that transdifferentiate from quiescent cells that control retinol metabolism to proliferative and migratory myofibroblasts that produce excessive amounts of extracellular matrix proteins, in particular collagen 1a1 (COL1A1). Although liver fibrosis is reversible, no effective drug therapy is available to prevent or reverse HSC activation. Melatonin has potent hepatoprotective properties in a variety of acute and chronic liver injury models and suppresses liver fibrosis. However, it remains unclear whether melatonin acts indirectly or directly on HSC to prevent liver fibrosis. Here, we studied the effect of melatonin on culture‐activated rat HSC. Melatonin dose‐dependently suppressed the expression of HSC activation markers Col1a1 and alpha‐smooth muscle actin (αSMA, Acta2), as well as HSC proliferation and loss of lipid droplets. The nuclear melatonin sensor retinoic acid receptor‐related orphan receptor‐alpha (RORα/Nr1f1) was expressed in quiescent and activated HSC, while the membranous melatonin receptors (Mtrn1a and Mtrn1b) were not. The synthetic RORα agonist SR1078 more potently suppressed Col1a1 and αSma expression, HSC proliferation, and lipid droplet loss, while the RORα antagonist SR1001 blocked the antifibrotic features of melatonin. Melatonin and SR1078 inhibited the expression of Alox5, encoding 5‐lipoxygenase (5‐LO). The pharmacological 5‐LO inhibitor AA861 reduced Acta2 and Col1a1 expression in activated HSC. We conclude that melatonin directly suppresses HSC activation via RORα‐mediated inhibition of Alox5 expression, which provides novel drug targets to treat liver fibrosis.     

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 alleviates cadmium‐induced liver injury by inhibiting the TXNIP‐NLRP3 inflammasome

Cadmium (Cd) is a persistent environmental and occupational contaminant that accumulates in the liver and induces oxidative stress and inflammation. Melatonin possesses potent hepatoprotective properties against the development and progression of acute and chronic liver injury. Nevertheless, the molecular mechanism underlying the protective effects of melatonin against Cd‐induced hepatotoxicity remains obscure. In this study, we aimed to investigate the effects of melatonin on Cd‐induced liver inflammation and hepatocyte death. Male C57BL/6 mice were intraperitoneally injected with melatonin (10 mg/kg) once a day for 3 days before exposure to CdCl₂ (2.0 mg/kg). We found that Cd induced hepatocellular damage and inflammatory infiltration as well as increased serum ALT/AST enzymes. In addition, we showed that Cd triggered an inflammatory cell death, which is mediated by the NOD‐like receptor pyrin domain containing 3 (NLRP3) inflammasome. Moreover, melatonin treatment significantly alleviated Cd‐induced liver injury by decreasing serum ALT/AST levels, suppressing pro‐inflammatory cytokine production, inhibiting NLRP3 inflammasome activation, ameliorating oxidative stress, and attenuating hepatocyte death. Most importantly, melatonin markedly abrogated Cd‐induced TXNIP overexpression and decreased the interaction between TXNIP and NLRP3 in vivo and in vitro. However, treatment with siRNA targeting TXNIP blocked the protective effects of melatonin in Cd‐treated primary hepatocytes. Collectively, our results suggest that melatonin confers protection against Cd‐induced liver inflammation and hepatocyte death via inhibition of the TXNIP‐NLRP3 inflammasome pathway.     

Melatonin induces browning of inguinal white adipose tissue in Zucker diabetic fatty rats

Melatonin limits obesity in rodents without affecting food intake and activity, suggesting a thermogenic effect. Identification of brown fat (beige/brite) in white adipose tissue (WAT) prompted us to investigate whether melatonin is a brown‐fat inducer. We used Zücker diabetic fatty (ZDF) rats, a model of obesity‐related type 2 diabetes and a strain in which melatonin reduces obesity and improves their metabolic profiles. At 5 wk of age, ZDF rats and lean littermates (ZL) were subdivided into two groups, each composed of four rats: control and those treated with oral melatonin in the drinking water (10 mg/kg/day) for 6 wk. Melatonin induced browning of inguinal WAT in both ZDF and ZL rats. Hematoxylin–eosin staining showed patches of brown‐like adipocytes in inguinal WAT in ZDF rats and also increased the amounts in ZL animals. Inguinal skin temperature was similar in untreated lean and obese rats. Melatonin increased inguinal temperature by 1.36 ± 0.02°C in ZL and by 0.55 ± 0.04°C in ZDF rats and sensitized the thermogenic effect of acute cold exposure in both groups. Melatonin increased the amounts of thermogenic proteins, uncoupling protein 1 (UCP1) (by ~2‐fold, P < 0.01) and PGC‐1α (by 25%, P < 0.05) in extracts from beige inguinal areas in ZL rats. Melatonin also induced measurable amounts of UCP1 and stimulated by ~2‐fold the levels of PGC‐1α in ZDF animals. Locomotor activity and circulating irisin levels were not affected by melatonin. These results demonstrate that chronic oral melatonin drives WAT into a brown‐fat‐like function in ZDF rats. This may contribute to melatonin′s control of body weight and its metabolic benefits.     

From chronic overnutrition to insulin resistance: The role of fat-storing capacity and inflammation

AimsWe analyze how the inflammatory state in adipose tissue caused by a condition of chronically positive energy balance can lead to insulin resistance first in adipose tissue, then in all insulin-sensitive tissues.Data synthesisChronic nutrient overload causes an increase in adipose depots that, if adipose tissue expandability is low, are characterized by an increased presence of hypertrophic adipocytes. This adipocyte hypertrophy is a possible stress condition for the endoplasmic reticulum (ER) that would lead to a proinflammatory state in adipose tissue. In this condition, ER stress would activate metabolic pathways that trigger insulin resistance, release of macrophage chemoattractant proteins, and in chronic inflammation, the death of the hypertrophic adipocyte. The infiltrated macrophages in turn release inflammatory proteins causing further recruitment of macrophages to adipose tissue and the release of inflammatory cytokines. Following these events, insulin resistance becomes extended to all adipose tissue. Insulin-resistant adipocytes, characterized by low liposynthetic capacity and high lipolytic capacity, cause increased release of free fatty acids (FFA). FFA released by lipolitic adipocytes may also activate Toll-like receptors 4 and then chemokines and cytokines release amplifying insulin resistance, lipolysis and inflammation in all adipose tissue. Moreover, increased circulating FFA levels, reduced circulating adiponectin levels and leptin resistance lead to decreased lipid oxidation in non-adipose tissues, thereby triggering ectopic accumulation of lipids, lipotoxicity and insulin resistance.ConclusionAll the conditions that increase circulating fatty acids and cause lipid overloading (obesity, lipoatrophy, lipodystrophy, catabolic states, etc.) induce a lipotoxic state in non-adipose tissues that gives rise to insulin resistance.     

Melatonin mediates protective effects on inflammatory response induced by interleukin‐1 beta in human mesenchymal stem cells

Joint diseases like osteoarthritis usually are accompanied with inflammatory processes, in which pro‐inflammatory cytokines mediate the generation of intracellular reactive oxygen species (ROS) and compromise survival of subchondral osteoblasts. Melatonin is capable of manipulating bone formation and osteogenic differentiation of mesenchymal stem cells (MSCs). The aim of this work was to investigate the anti‐inflammatory effect of melatonin on MSC proliferation and osteogenic differentiation in the absence or presence of interleukin‐1 beta (IL‐1β), which was used to induce inflammation. Our data showed that melatonin improved cell viability and reduced ROS generation in MSCs in a dose‐dependent manner. When exposed to 10 ng/mL IL‐1β, various concentrations of melatonin resulted in significant reduction of ROS by 34.9% averagely. Luzindole as a melatonin receptor antagonist reversed the anti‐oxidant effect of melatonin in MSCs with co‐exposure to IL‐1β. Real‐time RT‐PCR data suggested that melatonin treatment up‐regulated the expression of CuZnSOD and MnSOD, while down‐regulated the expression of Bax. To investigate the effect of melatonin on osteogenesis, MSCs were cultured in osteogenic differentiation medium supplemented with IL‐1β, melatonin, or luzindole. After exposed to IL‐1β for 21 days, 1 μm melatonin treatment significantly increased the levels of type I collagen, ALP, and osteocalcin, and 100 μm melatonin treatment yielded the highest level of osteopontin. Our study demonstrated that melatonin maintained MSC survival and promoted osteogenic differentiation in inflammatory environment induced by IL‐1β, suggesting melatonin treatment could be a promising method for bone regenerative engineering in future studies.     

M2‐like macrophages serve as a niche for adipocyte progenitors in adipose tissue

Adipose tissue (AT) is composed not only of adipocytes, but also of macrophages, endothelial cells and preadipocytes. Macrophages are an important component of AT, and are involved in tissue homeostasis, tissue repair and fibrosis. AT‐resident macrophages are categorized into two subtypes, the M1‐like and M2‐like macrophages. M2‐like macrophages are reported to play anti‐inflammatory roles, and to be involved in clearing and removal of dying/dead adipocytes, and recruiting adipocyte progenitors (APs). M2‐like macrophages are also reported to be involved in the promotion of fibrosis in a transforming growth factor‐β‐dependent manner. However, the precise roles of M2‐like macrophages in the AT have not yet been clearly delineated. Recently, we generated genetically engineered transgenic mice in which CD206⁺ M2‐like macrophages can be conditionally depleted. Unexpectedly, we found that the depletion of CD206⁺ M2‐like macrophages resulted in the enhanced generation of smaller adipocytes, improved insulin sensitivity and proliferation of APs. We further clarified that the CD206⁺ M2‐like macrophages directly interact with the APs to regulate their growth/differentiation and adipogenesis, thereby controlling adiposity and systemic insulin sensitivity. In the present review, we discuss how CD206⁺ M2‐like macrophages provide a niche for APs and maintain glucose homeostasis.     

Melatonin attenuates D‐galactose‐induced memory impairment,neuroinflammation and neurodegeneration via RAGE/NF‐KB/JNK signaling pathway in aging mouse model

Melatonin acts as a pleiotropic agent in various age‐related neurodegenerative diseases. In this study, we examined the underlying neuroprotective mechanism of melatonin against D‐galactose‐induced memory and synaptic dysfunction, elevated reactive oxygen species (ROS), neuroinflammation and neurodegeneration. D‐galactose was administered (100 mg/kg intraperitoneally (i.p.)) for 60 days. After 30 days of D‐galactose administration, vehicle (same volume) or melatonin (10 mg/kg, i.p.) was administered for 30 days. Our behavioral (Morris water maze and Y‐maze test) results revealed that chronic melatonin treatment alleviated D‐galactose‐induced memory impairment. Additionally, melatonin treatment reversed D‐galactose‐induced synaptic disorder via increasing the level of memory‐related pre‐and postsynaptic protein markers. We also determined that melatonin enhances memory function in the D‐galactose‐treated mice possibly via reduction of elevated ROS and receptor for advanced glycation end products (RAGE). Furthermore, Western blot and morphological results showed that melatonin treatment significantly reduced D‐galactose‐induced neuroinflammation through inhibition of microgliosis (Iba‐1) and astrocytosis (GFAP), and downregulating other inflammatory mediators such as p‐IKKβ, p‐NF‐_KB65, COX2, NOS2, IL‐1β, and TNFα. Moreover, melatonin lowered the oxidative stress kinase p‐JNK which suppressed various apoptotic markers, that is, cytochrome C, caspase‐9, caspase‐3 and PARP‐1, and prevent neurodegeneration. Hence, melatonin attenuated the D‐galactose‐induced memory impairment, neuroinflammation and neurodegeneration possibly through RAGE/NF‐_KB/JNK pathway. Taken together, our data suggest that melatonin could be a promising, safe and endogenous compatible antioxidant candidate for age‐related neurodegenerative diseases such as Alzheimer's disease (AD).     

Somatostatin is expressed and secreted by human adipose tissue upon infection and inflammation

Somatostatin (SRIF) is a well-known neuroendocrine secretion product. SRIF expression and secretion are induced after inflammation in murine macrophages and in endotoxin-injected sheep and pigs. Because adipocytes have been demonstrated to produce numerous cytokines and peptide hormones, we investigated the expression of SRIF and its receptors (SSTR1-5) in human adipose tissue after inflammatory stimulation in vitro and in tissues from patients with septic disease.Preadipocyte-derived adipocytes, mesenchymal stem cell-derived adipocytes, and mature explanted adipocytes expressed SRIF-mRNA after endotoxin [lipopolysaccharide (LPS)] or IL-1beta treatments. LPS- and IL-1beta-mediated SRIF-mRNA induction was blocked by pretreatment with dexamethasone. Using cocultures and quantitative real-time PCR, we demonstrate adipocyte SRIF induction by secretion factors from activated peripheral blood mononuclear cell-derived macrophages. In contrast to basal adipocytes, SRIF protein was detected in culture supernatants of LPS-treated and of combined TNFalpha/IL-1beta/LPS-treated adipocytes. SRIF protein was visualized by immunohistochemistry in explanted minced adipose tissue after overnight incubation in culture medium supplemented with combined IL-1beta and LPS. In septic patients, expression of SRIF-mRNA and SRIF protein was found in visceral, but not in sc, adipose tissue. Adipocyte mRNA abundance of SSTR 1-5 was differentially regulated by inflammatory treatments.Thus, human visceral adipose tissue secretes SRIF during inflammation and sepsis and expresses several SSTRs. It is tempting to speculate that visceral adipose tissue-derived SRIF plays a modulatory role in the immunological and metabolic response to inflammation.