Melatonin improves influenza virus infection-induced acute exacerbation of COPD by suppressing macrophage M1 polarization and apoptosis
Mengmeng XuJia-Ying KangQiuyan WangXing ZuoYuan-Yuan TanYuanyuan WeiDawei ZhangLing ZhangHuimei WuGuanghe Fei
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Abstract Background Influenza A viruses (IAV) are extremely common respiratory viruses for the acute exacerbation of chronic obstructive pulmonary disease (AECOPD), in which IAV infection may further evoke abnormal macrophage polarization, amplify cytokine storms. Melatonin exerts potential effects of anti-inflammation and anti-IAV infection, while its effects on IAV infection-induced AECOPD are poorly understood. Methods COPD mice models were established through cigarette smoke exposure for consecutive 24 weeks, evaluated by the detection of lung function. AECOPD mice models were established through the intratracheal atomization of influenza A/H3N2 stocks in COPD mice, and were injected intraperitoneally with melatonin (Mel). Then, The polarization of alveolar macrophages (AMs) was assayed by flow cytometry of bronchoalveolar lavage (BAL) cells. In vitro, the effects of melatonin on macrophage polarization were analyzed in IAV-infected Cigarette smoking extract (CSE)-stimulated Raw264.7 macrophages. Moreover, the roles of the melatonin receptors (MTs) in regulating macrophage polarization and apoptosis were determined using MTs antagonist luzindole. Results The present results demonstrated that IAV/H3N2 infection deteriorated lung function (reduced FEV 20,50 /FVC), exacerbated lung damages in COPD mice with higher dual polarization of AMs. Melatonin therapy improved airflow limitation and lung damages of AECOPD mice by decreasing IAV nucleoprotein (IAV-NP) protein levels and the M1 polarization of pulmonary macrophages. Furthermore, in CSE-stimulated Raw264.7 cells, IAV infection further promoted the dual polarization of macrophages accompanied with decreased MT1 expression. Melatonin decreased STAT1 phosphorylation, the levels of M1 markers and IAV-NP via MTs reflected by the addition of luzindole. Recombinant IL-1β attenuated the inhibitory effects of melatonin on IAV infection and STAT1-driven M1 polarization, while its converting enzyme inhibitor VX765 potentiated the inhibitory effects of melatonin on them. Moreover, melatonin inhibited IAV infection-induced apoptosis by suppressing IL-1β/STAT1 signaling via MTs. Conclusions These findings suggested that melatonin inhibited IAV infection, improved lung function and lung damages of AECOPD via suppressing IL-1β/STAT1-driven macrophage M1 polarization and apoptosis in a MTs-dependent manner. Melatonin may be considered as a potential therapeutic agent for influenza virus infection-induced AECOPD. Graphical Abstract Schematic mechanisms underlying the regulatory effects of melatonin on macrophage polarization and apoptosis in IAV infection plus cigarette stimulation-induced AECOPD model.Keywords:
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Major biochemical activities of the pineal gland include melatonin biosynthesis and 12-lipoxygenation. In this paper, we provide evidence in vivo that melatonin regulates 12-lipoxygenation via 12-lipoxygenase (LOX) expression. The relationship between these two biochemical activities was established by monitoring levels of endogenous melatonin and a 12-LOX metabolite, 12-hydroxyeicosatetraenoic acid (12-HETE), in the rat pineal gland both during the light-dark cycle and after isoproterenol injection using GC/MS with negative ion chemical ionization. As pineal melatonin production reflected a typical diurnal variation, 12-HETE levels showed an off-phase diurnal pattern in relation to melatonin levels. Intravenous administration of isoproterenol, which has been shown to elevate melatonin production, decreased the 12-HETE level significantly. The reduction of 12-HETE levels during the dark phase and after isoproterenol injection was accompanied by decreases in 12-LOX mRNA and protein levels. Direct administration of melatonin to rats by intravenous injection decreased pineal 12-LOX protein levels significantly, indicating that melatonin plays a role in down-regulating 12-LOX expression. When pineal glands were incubated with exogenous melatonin in culture, time-dependent reduction of 12-LOX protein levels was observed. The melatonin-induced reduction in 12-LOX protein was abolished in the presence of the melatonin receptor antagonist luzindole, establishing further the role of melatonin in this process. Incubation of pineal homogenates with exogenous melatonin partially inhibited 12-LOX activity. Taken together, an inverse relationship exists in the endogenous production of 12-HETE, 12-LOX mRNA and protein with respect to melatonin production in the rat pineal gland. Melatonin decreased both 12-LOX mRNA and protein levels in addition to 12-LOX enzyme activity, indicating that melatonin is an endogenous modulator of pineal 12-lipoxygenation.
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Melatonin, the chief secretory product of the pineal gland, has been shown to protect the heart against ischemia-reperfusion injury. This was attributed to its free radical scavenging and broad-spectrum antioxidant properties. The possibility that melatonin may act via its receptor and intracellular signaling, has not yet been addressed in this regard. In all previous studies, only the acute effects of melatonin on the heart, were evaluated. The aims of the present study were to: (i) compare the acute and long-term effects of melatonin on infarct size and functional recovery of the ischemic heart, and (ii) evaluate the role of the melatonin receptor in cardioprotection. For evaluation of the short-term effects of melatonin on contractile recovery and infarct size, the isolated perfused working rat heart was subjected to 20 min global ischemia or 35 min regional ischemia respectively, and melatonin (25-50 microm) administered either before and during reperfusion, or before ischemia or during reperfusion after ischemia. The melatonin receptor was manipulated using luzindole and N-acetyltryptamine. The long-term effects of melatonin were evaluated 24 hr after melatonin administration (2.5 or 5.0 mg/kg, i.p.) or after oral administration for 7 days (20 or 40 microg/mL). Infarct size and mechanical recovery during reperfusion of the working heart were used as endpoints. Melatonin (50 microm), when administered either before and during reperfusion after ischemia or during reperfusion only, significantly improved cardiac output and work performance and reduced infarct size compared with untreated controls. Luzindole (5 microm), a melatonin receptor antagonist, abolished these cardioprotective effects. Long-term administration of melatonin (i.p. or orally for 7 days) caused a significant reduction in infarct size of hearts subjected to 35 min regional ischemia. The cardioprotection persisted for 2-4 days after discontinuation of treatment. In summary, the results obtained suggest that melatonin induces short- as well as long-term protection and that the melatonin receptor is also involved in its cardioprotective actions.
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Abstract Background Cardiac ischaemia/reperfusion (I/R) injury has been an economic and health burden worldwide. Previous studies have reported the beneficial effects of melatonin when given prior to cardiac ischaemia in animals with cardiac I/R injury. However, the effects of melatonin on the hearts when it is given after ischaemia or at the onset of reperfusion, which is more relevant to the clinical setting, is not known. Moreover, the mechanisms responsible for the potential benefits of melatonin and the roles of melatonin receptors on the heart during cardiac I/R injury have not been fully investigated. Purpose We tested the hypothesis that in rats with cardiac I/R injury, melatonin exerts cardioprotective effects even when it is given after ischaemia via an activation of both melatonin receptors 1 (MT1) and 2 (MT2), leading to decreased mitochondrial dysfunction, mitochondrial dynamics imbalance, excessive mitophagy, cardiomyocyte death and finally resulting in decreased infarct size and improved left ventricular (LV) function. Methods Male Wistar rats were subjected to cardiac I/R (30 min of LAD ligation and 120 min of reperfusion). These rats were divided into 4 interventions (n=12/group) including vehicle, pretreatment with melatonin, melatonin treatment during ischaemia, or at the onset of reperfusion. Melatonin was given to the rats at the dose of 10 mg/kg via intravenous injection. In addition, either a non-specific melatonin receptor blocker (Luzindole) or specific MT2 blocker (4-PPDOT) at 1 mg/kg was given intravenously to 2 additional sets of rats (n=12/set) prior to melatonin and cardiac I/R induction. At the end of cardiac I/R, infarct size, LV function, and molecular mechanisms were determined. Furthermore, in vitro experiment was conducted in MT1 or MT2 silenced H9C2 cell with hypoxia/reoxygenation (H/R) to investigate the mechanism underlying cardioprotective effects of melatonin during cardiac I/R. Results Rats in all melatonin-treated groups had similarly reduced cardiac I/R injury as indicated by reduced infarct size (Fig. 1A), arrhythmia score. Melatonin-treated rats also had decreased mitochondrial ROS production, mitochondrial depolarization and swelling, decreased p-Drp1/Drp1 ratio (Fig. 1B) and increased Mfn1, Mfn2, and OPA1, and decreased apoptosis, leading to increased %LVEF. Luzindole and 4-PPDOT abolished these protective effects of melatonin (Fig. 1A). In in vitro study, melatonin increased %cell viability (Fig. 1C), reduced mitochondrial dynamics imbalance and cardiomyocyte apoptosis in H9C2 cells with H/R. However, these beneficial effects of melatonin were abrogated only in MT2 silenced H9C2 cell with H/R. Conclusion Melatonin exerted both preventive and treatment effects in reducing cardiac I/R injury. Its cardioprotective effects were dependent upon the activation of MT2 receptor. Figure 1 Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): National Science and Technology Development Agency of Thailand
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This review summarizes the involvement of centrally and peripherally applied melatonin, a major hormone of pineal gland, in the mechanism of gastric mucosal integrity, gastroprotection and ulcer healing. Melatonin was originally shown to attenuate gastric mucosal lesions but the controversy exists in the literature as to whether melatonin derived from the pineal gland, considered as the major source of this indole or rather that locally generated from L-tryptophan within gastric mucosa, plays predominant role in the mechanism of gastrointestinal integrity. Both, intragastric (i.g.) and intracerebroventricular (i.c.v.) administration of melatonin and its precursor, L-tryptophan to rats without or with removed pineal gland by pinealectomy attenuates in the dose-dependent manner the formation of on gastric lesions induced by topical irritants and water immersion restraint stress (WRS). Melatonin accelerated the gastric ulcer healing and this was accompanied by the rise in gastric blood flow (GBF), the plasma melatonin and gastrin levels, the mucosal generation of PGE(2) and luminal NO content. Pinealectomy, which suppresses the plasma melatonin levels, markedly aggravated the gastric lesions induced by WRS. Concurrent supplementation of pinealectomized animals with melatonin or L-tryptophan, the melatonin precursor, attenuated the lesions induced by WRS. Treatment with luzindole, an antagonist of Mel(2) receptors, or with L-NNA, the NO-synthase inhibitor, significantly attenuated melatonin- and L-tryptophan-induced protection and the acceleration of ulcer healing and the accompanying increase in the GBF and luminal content of NO. We conclude that 1) exogenous melatonin and that released from the L-tryptophan attenuate lesions induced by topical irritant such as ethanol and WRS via interaction with MT(2) receptors and due to an enhancement of gastric microcirculation, probably mediated by NO and PG derived from cNOS, iNOS and COX-2 overexpression and activity, and 2) the pineal gland plays an important role in the limitation of WRS-induced gastric lesions and acceleration of ulcer healing via releasing melatonin predominately at night time, that exerts gastroprotective and ulcer healing actions.
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Abstract: Melatonin deficiency has been postulated as an etiologic factors in adolescent idiopathic scoliosis (AIS). In previous studies, melatonin was shown to regulate skeletal growth and bone formation in both humans and rats. Although it remains controversial whether there are differences in serum melatonin level between AIS and control subjects, melatonin signaling pathway dysfunction in osteoblasts has been reported in patients with AIS. Recently, our group found that melatonin receptor 1B (MT2) gene polymorphism was associated with the occurrence of AIS. Hence, the present study investigated the effect of melatonin on AIS osteoblasts. In vitro assays were performed with osteoblasts isolated from 17 severe AIS girls and nine control subjects. The osteoblasts were exposed to different concentrations of melatonin for 3 days. The effects of melatonin on cell proliferation (as evidenced by MTT assay) and differentiation (demonstrated by alkaline phosphatase activity) were determined. In the control group, melatonin significantly stimulated osteoblasts to proliferate and differentiate. However, in the AIS group, the stimulatory effects of melatonin were not discernible. Importantly, this finding demonstrated that there is a significant difference between AIS and control osteoblasts in functional response toward melatonin. Melatonin-stimulated proliferation of control osteoblasts was inhibited by the MT2 antagonist, 4-phenyl-2-propionamidotetraline, as well as by luzindole, a nonselective melatonin receptor antagonist, suggesting that MT2 is associated with the proliferative action of melatonin. The lack of response in AIS osteoblasts might be because of dysfunction of the melatonin signaling pathway, which may contribute to the low bone mineral density and abnormal skeletal growth observed in patients with AIS.
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Objective: Agomelatine is a synthetic analog of melatonin with selective agonist action on melatonin MT1 and MT2 receptors and antagonistic effect on serotonin 5HT2c receptors. Similar to melatonin, it also has antinociceptive properties. There are only a few studies comparing the antinociceptive effects of agomelatine to melatonin. Methods: Male Wistar albino rats were grouped into seven treatment groups and a control group (8 animals each). Treatment groups received intraperitoneal injections of agomelatine 25 mg/kg, 35 mg/kg and 50 mg/kg; agomelatine 35 mg/kg + luzindole 10 mg/kg; melatonin 60 mg/kg and 90 mg/kg and melatonin 60 mg/kg + sertraline 10 mg/kg. Animals were placed on a hot plate and time to pain avoidance behavior was measured. Results: Agomelatine 25 mg/kg, 35 mg/kg and 50 mg/kg were significantly superior to placebo (all p < 0. 0001); agomelatine 35 mg/kg and 50 mg/kg were significantly superior to melatonin 60 mg/kg (p < 0.05 and p < 0.01, respectively) and melatonin 90 mg/kg (p < 0.001 and p < 0.001, respectively) in prolonging reaction times. Agomelatine 50 mg/kg was superior to melatonin 60 mg/kg + sertraline 10 mg/kg (p < 0,05); on the contrary; agomelatine 25 mg/kg was significantly inferior to this combination in prolonging reaction times (p = 0,041). Melatonin 90 mg/kg but not 60 mg/kg was significantly superior to placebo in prolonging reaction times (p < 0.0001). Both agomelatine 50 mg/kg and 35 mg/kg were superior to agomelatine 25 mg/kg (p < 0.0001 and p = 0.002, respectively), but the difference between agomelatine 50 mg/kg and 35 mg/kg was not significant. Conclusions: We demonstrated that agomelatine is superior to melatonin on pain behavior in a dose -dependent manner using an experimental pain model. The a ddition of a serotonergic agent increased the antinociceptive effect of melatonin in regard to agomelatine, but high doses of agomelatine were still more effective, suggesting a dose -dependent effect of agomelatine on 5-HT2c receptors.
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Major biochemical activities of the pineal gland include melatonin biosynthesis and 12-lipoxygenation. In this paper, we provide evidence in vivo that melatonin regulates 12-lipoxygenation via 12-lipoxygenase (LOX) expression. The relationship between these two biochemical activities was established by monitoring levels of endogenous melatonin and a 12-LOX metabolite, 12-hydroxyeicosatetraenoic acid (12-HETE), in the rat pineal gland both during the light-dark cycle and after isoproterenol injection using GC/MS with negative ion chemical ionization. As pineal melatonin production reflected a typical diurnal variation, 12-HETE levels showed an off-phase diurnal pattern in relation to melatonin levels. Intravenous administration of isoproterenol, which has been shown to elevate melatonin production, decreased the 12-HETE level significantly. The reduction of 12-HETE levels during the dark phase and after isoproterenol injection was accompanied by decreases in 12-LOX mRNA and protein levels. Direct administration of melatonin to rats by intravenous injection decreased pineal 12-LOX protein levels significantly, indicating that melatonin plays a role in down-regulating 12-LOX expression. When pineal glands were incubated with exogenous melatonin in culture, time-dependent reduction of 12-LOX protein levels was observed. The melatonin-induced reduction in 12-LOX protein was abolished in the presence of the melatonin receptor antagonist luzindole, establishing further the role of melatonin in this process. Incubation of pineal homogenates with exogenous melatonin partially inhibited 12-LOX activity. Taken together, an inverse relationship exists in the endogenous production of 12-HETE, 12-LOX mRNA and protein with respect to melatonin production in the rat pineal gland. Melatonin decreased both 12-LOX mRNA and protein levels in addition to 12-LOX enzyme activity, indicating that melatonin is an endogenous modulator of pineal 12-lipoxygenation.
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Abstract Aim: To determine the receptor subtypes of melatonin in estrogen receptor‐positive endometrial cancer cell line, Ishikawa, and the influence of melatonin on chemosensitivity. Methods: To confirm the subtype of melatonin on Ishikawa cells, cells were treated with melatonin alone and with antagonists against melatonin receptor luzindole and 4‐phenyl‐2‐propionamidotetralin (4‐P‐PDOT). Expression of MT1/MT2 mRNA was analyzed by reverse transcriptase‐polymerase chain reaction (RT‐PCR). Immunocytochemistry of MT1/MT2 was also performed. The effect of melatonin against expression of MT1, MT2, and ERα‐receptors mRNA was compared with RT‐PCR. To determine whether melatonin enhances the effect of anticancer agents, chemosensitivity test was performed with or without melatonin. Results: Our study revealed that Ishikawa cells express MT1 by both RT‐PCR and immunocytochemistry. In contrast, expression of MT2 mRNA was not found. Furthermore, ERα mRNA expression was attenuated at melatonin level of 1 × 10 −9 M. Chemosensitivity test revealed that melatonin enhanced anti‐tumor effects of paclitaxel among anticancer drugs tested. Conclusion: Based on the above results, MT1 receptor, but not MT2, is expressed in Ishikawa cells. It was also revealed that the cytostatic effect of melatonin is partly an action mediated by MT1 receptor, and attenuation of ERα expression was predicted as the mechanism of action. Clinical application of melatonin to biochemotherapy might be also expected.
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