Melatonin-treated Mice Research Articles

Melatonin Ameliorates Sevoflurane Anesthesia-Induced Deficits in Learning and Memory of Aged Mice Through Nrf2 Signaling Related Ferroptosis.

Our research aimed at investigating the protective effects in aged mice exposed to sevoflurane anesthesia. To assess learning and memory abilities and exploratory behavior, the novel object recognition (NOR) test, Morris water maze (MWM) test, and open field test were employed. Commercial kits were used to measure levels of malondialdehyde, nicotinamide adenine dinucleotide phosphate oxidase activity, superoxide dismutase activity, catalase activity, and iron. The messenger RNA and protein levels of ferritin heavy chain 1, nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1, and glutathione peroxidase 4 in the hippocampus were detected. Treatment with melatonin significantly ameliorated the decrease in exploration time of novel objects and the discrimination index induced by sevoflurane anesthesia. Melatonin also reduced escape latencies and increased the time spent in the target quadrant in the MWM test. In the open field test, melatonin-treated mice exhibited greater exploratory activity, including longer distances traveled and a higher number of rearing events. Further, melatonin treatment markedly decreased the levels of oxidative stress markers and iron in the hippocampus of aged mice exposed to sevoflurane anesthesia. However, the beneficial effects of melatonin were significantly attenuated following treatment with the Nrf2 inhibitor ML385. Our results suggest that melatonin could alleviate learning and memory impairment induced by sevoflurane anesthesia in aged mice through its antioxidant properties, partially through the Nrf2 pathway.

Melatonin inhibits the formation of intraplaque neovessels in ApoE−/− mice via PPARγ- RhoA-ROCK pathway

BackgroundAccording to former research, the atherosclerotic plaque is thought to be aggravated by intraplaque neovessels (IPN) and intraplaque hemorrhage (IPH). Intriguingly, a lower incidence of IPH was found in plaque treated with melatonin. In this study, we attempted to investigate the impact and underlying mechanism regarding the influences of melatonin upon IPN. MethodsA mouse model was established by subjecting the high fat diet (HFD)-fed ApoE−/− mice to tandem stenosis (TS) surgery with melatonin and GW9662, a PPARγ antagonist, being given by gavage. In vitro experiment was conducted with HUVECs exposing to according treatments of VEGF, melatonin, GW9662, or Y27632. ResultsPlaque and IPN were attenuated by treatment with melatonin, which was then reversed by blocking PPARγ. Western blotting results showed that melatonin increased PPARγ and decreased RhoA/ROCK signaling in carotid artery. Elevated RhoA/ROCK signaling was observed in melatonin-treated mice when PPARγ was blocked. In accordance with it, experiments using protein and mRNA from HUVECs revealed that melatonin inhibited the RhoA/ROCK signaling by enhancing PPARγ. According to in vitro study, melatonin was able to inhibit cell migration and angiogenesis, which was aborted by GW9662. Blockage of ROCK using Y27632 was able to cease the effect of GW9662 and restored the suppression on cell migration and angiogenesis by melatonin. ConclusionsOur study demonstrates that melatonin is able to curb development of plaque and IPN formation by inhibiting the migration of endothelial cells via PPARγ- RhoA-ROCK pathway. That provides a therapeutic potential for both melatonin and PPARγ agonist targeting IPN, IPH, and atherosclerotic plaque.

Anti-PANoptosis is involved in neuroprotective effects of melatonin in acute ocular hypertension model.

Acute ocular hypertension (AOH) is the most important characteristic of acute glaucoma, which can lead to retinal ganglion cell (RGC) death and permanent vision loss. So far, approved effective therapy is still lacking in acute glaucoma. PANoptosis (pyroptosis, apoptosis, and necroptosis), which consists of three key modes of programmed cell death-apoptosis, necroptosis, and pyroptosis-may contribute to AOH-induced RGC death. Previous studies have demonstrated that melatonin(N-acetyl-5-methoxytryptamine) exerts a neuroprotective effect in many retinal degenerative diseases. However, whether melatonin is anti-PANoptotic and neuroprotective in the progression of acute glaucoma remains unclear. Thus, this study aimed to explore the role of melatonin in AOH retinasand its underlying mechanisms. The results showed that melatonin treatment attenuated the loss of ganglion cell complex thickness, retinal nerve fiber layer thickness, and RGC after AOH injury, and improved the amplitudes of a-wave, b-wave, and oscillatory potentials in the electroretinogram. Additionally, the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells was decreased, and the upregulation of cleaved caspase-8, cleaved caspase-3, Bax, and Bad and downregulation of Bcl-2 and p-Bad were inhibited after melatonin administration. Meanwhile, both the expression and activation of MLKL, RIP1, and RIP3, along with the number of PI-positive cells, were reduced in melatonin-treated mice, and p-RIP3 was in both RGC and microglia/macrophage after AOH injury. Furthermore, melatonin reduced the expression of NLRP3, ASC, cleaved caspase-1, gasdermin D (GSDMD), and cleaved GSDMD, and decreased the number of Iba1/interleukin-1β-positive cells. In conclusion, melatonin ameliorated retinal structure, prevented retinal dysfunction after AOH, and exerted a neuroprotective effect via inhibition of PANoptosis in AOH retinas.

Melatonin Attenuates Vascular Smooth Muscle Contraction Through the γ-Secretase/Notch Intracellular Domain/Myocardin Pathway.

Inositol 1, 4, 5-trisphosphate (IP3) signaling-mediated calcium release drives the contraction of vascular smooth muscles and hence regulates blood vessel volume and blood pressure. Melatonin supplementation has been suggested to be beneficial for hypertension. To determine whether the blood pressure-lowering effect of melatonin was accounted for by IP3 signaling, we evaluated the vasoconstriction response and IP3 signaling in isolated mouse thoracic aortic rings during melatonin incubation. C57BL/6 mice were given intraperitoneal injections daily with melatonin, and the systolic blood pressure and contractility of aortic rings from melatonin-treated mice were decreased, and the contraction suppression effect of melatonin was attributed to the impaired expression of contractile proteins in vascular smooth muscle cells rather than IP3 signaling. Our results further showed that melatonin increased the expression of γ-secretase, which could cleave and release the notch intracellular domain, and the notch intracellular domain prevented the transcription of contractile genes by interfering with the interaction between serum response factor and myocardin, the master regulator of contractile protein. In this article, we report a novel mechanism by which melatonin regulates smooth muscle contractility that does not depend on IP3 signaling.

Melatonin exerts anti-angiogenic and anti-inflammatory effects in alkali-burned corneas.

BackgroundCorneal neovascularization (CNV) caused by alkali burn injury is tightly associated with an inflammatory reaction and can lead to vision loss. Melatonin is involved in anti-inflammation and anti-angiogenesis, but its role in CNV has not yet been investigated.MethodsWe induced CNV using sodium hydroxide (NaOH) and compared the reactions of vehicle control and melatonin-treated male C57BL/6 mice at 7 and 14 days following the corneal burn. The infiltration of inflammatory cells and the expression of proangiogenic factors, chemokines, and inflammation-related molecules were quantified via immunohistochemical (IHC) analysis and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), respectively. Murine peritoneal macrophages were used in vitro to further verify the effect of melatonin in inflammatory CNV.ResultsCompared with the vehicle control mice, the melatonin-treated mice showed significant inhibition of angiogenesis and reduction of corneal epithelial defects in alkali-burned corneas. Concomitantly, the infiltration of inflammatory cells and F4/80+ cells were dramatically reduced after melatonin treatment. The messenger RNA (mRNA) expression of proangiogenic factors [vascular endothelial growth factors (VEGF) and matrix metalloproteinase-9 (MMP-9)], monocyte chemotactic protein-1 (MCP-1), nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), and pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) and IL-6] were down-regulated in the melatonin-treated mice. Moreover, melatonin inhibited the expression of these factors in murine peritoneal macrophages.ConclusionsMelatonin inhibits the neovascular and inflammatory responses in corneal alkali burn injury, suggesting that it may be a potential therapy for CNV.

Melatonin Mitigates Oxazolone-Induced Colitis in Microbiota-Dependent Manner.

Levels of type 2 cytokines are elevated in the blood and intestinal tissues of ulcerative colitis (UC) patients in the active phase; this phenomenon indicates the participation of type 2 immune response in UC progression. The beneficial effects of melatonin in dextran sodium sulfate (DSS) and 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis models have been illustrated, but its role in the oxazolone (Oxa)-induced colitis model (driven by type 2 immune response) remains relatively unknown. We investigated the relationship between melatonin concentration and the severity of UC, revealing a significantly negative correlation. Subsequently, we investigated the effects of melatonin in Oxa-induced colitis mice and the potential underlying mechanisms. Administration of melatonin significantly counteracted body weight loss, colon shortening, and neutrophil infiltration in Oxa-induced colitis mice. Melatonin treatment mitigated Oxa-induced colitis by suppressing type 2 immune response. In addition, melatonin attenuated intestinal permeability by enhancing the expression of ZO-1 and occludin in colitis mice. Interestingly, the protective effect of melatonin was abolished when the mice were co-housed, indicating that the regulation of gut microbiota by melatonin was critical in alleviating Oxa-induced colitis. Subsequently, 16S rRNA sequencing was performed to explore the microbiota composition. Decreased richness and diversity of intestinal microbiota at the operational taxonomic unit (OTU) level resulted from melatonin treatment. Melatonin also elevated the abundance of Bifidobacterium, a well-known probiotic, and reduced proportions of several harmful bacterial genera, such as Desulfovibrio, Peptococcaceae, and Lachnospiraceae. Fecal microbiota transplantation (FMT) was used to explore the role of microbiota in the function of melatonin in Oxa-induced colitis. Microbiota transplantation from melatonin-treated mice alleviated Oxa-induced colitis, suggesting that the microbiome participates in the relief of Oxa-induced colitis by melatonin. Our findings demonstrate that melatonin ameliorates Oxa-induced colitis in a microbiota-dependent manner, suggesting the therapeutic potential of melatonin in treating type 2 immunity-associated UC.

Oral administration of melatonin increases plasma calcium and magnesium and improves bone metabolism in aged male mice

We previously reported that the oral administration of melatonin from 4 to 20 months to male mice improved femoral bone strength and bone density during the aging. Additionally, melatonin receptor, MT2, was immunologically detected in both osteoblasts and osteoclasts of the mouse femoral bone. Thus, melatonin can act on both osteoblasts and osteoclasts to maintain bone strength during the aging process. Here, we analyzed plasma calcium (Ca2+), magnesium (Mg2+), and inorganic phosphorus ([PO4]3-) in 20-month-old male mice with or without administration melatonin (15-20 mg/kg/day) in drinking water. We found that plasma Ca2+ and Mg2+ levels in melatonin-treated mice increased significantly as compared with control mice. In [PO4]3-, melatonin administration tended to increase its plasma level, but did not reach statistical significance. The potential association between these divalent ions and metabolism markers of femoral bone was also examined. In the femoral diaphysis, the plasma Ca2+ and Mg2+ concentrations were positively correlated with periosteal and endosteal circumference which were significantly associated with the Strength Strain Index. Therefore, melatonin treatment enlarged femoral diaphysis and enhanced bone strength by increasing mineral depositions. In addition, the plasma melatonin levels were significantly positive correlation with total bone density and critical thickness in the femoral diaphysis. Since we had not observed the primary trabecular bone and osteoclasts in 20-month-old mice previously, it is suggested that plasma Ca2+ and Mg2+ are not elevated due to bone resorption. The increased plasma Ca2+ and Mg2+ by melatonin may originate from the intestinal absorption of these ions since melatonin binds to the vitamin D3 receptor, its activation is known to promote the intestinal absorption of Ca2+.

Quantitative proteomic analysis of the effects of melatonin treatment for mice suffered from small intestinal damage induced by γ-ray radiation

Purpose Intestinal damage induced by radiation exposure is a major clinic concern of radiotherapy for patients with abdominal or pelvic tumor. Melatonin (N-acetyl-5-methoxytryptamine) is likely be an ideal radioprotector to protect individuals from radiation exposure. The study aimed to define the role of melatonin in small intestinal damage caused by abdominal irradiation (ABI). Materials and methods 30-day survival rate and pathological histology of the intestines from melatonin-treated mice after 13 Gy ABI exposure was first detected. Next, quantitative proteomics analysis of the small intestines tissue was examined and GO term and KEGG pathways analysis were performed. Results Melatonin treatment before ABI exposure significantly increased 30-day survival rate to 83% and ameliorated damage to the intestinal epithelial cells. Melatonin significantly altered the proteins profile of the small intestines following irradiation. For the irradiated mice treated with melatonin in comparison with the irradiated mice, the enriched GO terms were mainly involved in defense response to other organism (BP, GO: 0098542), response to other organism (BP, GO: 0051707), anion transmembrane transporter activity (MF, GO: 0008509), and secondary active transmembrane transporter activity (MF, GO: 0015291). In the process of antioxidant activity (MF, GO: 0016209), melatonin treatment prior to radiation exhibited high protein levels of Sod3 and Gpx3. The markedly KEGG pathways for melatonin treatment prior to radiation mainly included protein digestion and absorption (ko 04974) and mineral absorption (ko 04978). p53 signaling pathway and DNA repair pathways were enriched in melatonin treated mice. The amount of radiation-induced DNA damage and the cell apoptosis of the small intestines was decreased in the melatonin-treated mice. Conclusions Melatonin may protect small intestines from radiation damage through increasing DNA repair and decreasing cell apoptosis of the small intestines. Our data provided perspective for the study of melatonin in mitigating ABI-caused intestinal damage.

Melatonin inhibits growth of B16 melanoma in C57BL/6 mice

Melatonin (N-acetyl-5-methoxytryptamine) has oncostatic properties in a wide variety of tumors. In melanoma, melatonin displayed growth suppressive effects in cultured cell lines and tumors. Thus far, however, there is no evidence of orally administrated melatonin reducing melanoma tumor growth. Therefore, the current study investigated the preventive effect of melatonin on C57BL/6 mice injected with B16-F10 murine metastatic melanoma cells. The animals were divided into two groups; control (vehicle) and melatonin pre-treated with oral melatonin in the drinking water (10 mg/kg/day) for 15 days. Grossly, the control animals had a significant exponential increase in tumor size until day 33, and all control animals were dead by day 38; conversely, melatonin pre-treated mice demonstrated delayed tumour appearance as well as decreased tumour volume and increased survival rates. PCNA immunostaining corroborated these data and demonstrated a significant reduction in the number of proliferating cells in the melatonin-treated mice (P < 0.005). Interestingly, histopathological analysis revealed the presence of undifferentiated and pleomorphic cells associated with higher mitotic rate in the control group, while epithelioid-shaped cells, sometimes containing melanin were clearly identified in melatonin-treated animals. Mitochondrial parameters measurement showed greater PTP opening and increased mitochondrial nitrite level associated in melatonin-pretreated animals. Finally, the decreased P-ERK1,2 cytoplasmic expression in melatonin mice compared with the controls supports the conclusion that the MAPK signalling pathway is repressed by melatonin in B16-F10 melanoma. Collectively, these results suggest for the first time that orally-administered melatonin reduces malignant melanoma progression in vivo and increases the percent of survival by lowering tumor cells proliferation due to mitochondrial dependent cytotoxicity and decreased P-ERK1,2 expression. This study demonstrates the chemopreventive potential of melatonin against malignant melanoma in C57BL/6 mice.

Melatonin protects against apoptosis of megakaryocytic cells via its receptors and the AKT/mitochondrial/caspase pathway.

Clinical studies have shown that melatonin lowers the frequency of thrombocytopenia in patients with cancer undergoing radiotherapy or chemotherapy. Here, we investigated the mechanisms by which melatonin promotes platelet formation and survival. Our results show that melatonin exerted protective effects on serum-free induced apoptosis of CHRF megakaryocytes (MKs). Melatonin promoted the formation of MK colony forming units (CFUs) in a dose-dependent manner. Using doxorubicin-treated CHRF cells, we found that melatonin rescued G2/M cell cycle arrest and cell apoptosis induced by doxorubicin. The expression of p-AKT was increased by melatonin treatment, an effect that was abolished by melatonin receptor blocker. In addition, we demonstrated that melatonin enhanced the recovery of platelets in an irradiated mouse model. Megakaryopoiesis was largely preserved in melatonin-treated mice. We obtained the same results in vivo from bone marrow histology and CFU-MK formation assays. Melatonin may exert these protective effects by directly stimulating megakaryopoiesis and inhibiting megakaryocyte apoptosis through activation of its receptors and AKT signaling.

Protective action of melatonin on diabetic rat testis at cellular, hormonal and immunohistochemical levels

The aim of the present study was to analyze the effects of melatonin treatment on diabetic rat testes. Fifty albino rats were divided into the following groups: CG: control group; GD: placebo-induced and placebo-treated mice; GDI: insulin-induced and post-confirmation diabetes-induced rats; GDM: diabetes-induced and melatonin-treated post-confirmation mice and GDMS: diabetes-induced and melatonin-treated mice simultaneously. Melatonin was administered at a dose of 10 mg/kg in drinking water every day for 20 days at night. Diabetes was induced by streptozotocin (60 mg/kg) and confirmed after the fifth day of induction. Insulin was administered at 5 IU (international units)/day at different times of the day for 20 days. The testes were submitted to histopathological, morphometric, immunohistochemical and oxidative stress analysis. Melatonin moderately decreased glycemic levels, protected weight loss and morphometric changes in the testicles, increased antioxidant enzyme levels and stabilized plasma testosterone and androgen receptor levels and decreased inflammatory markers in the testicles. Showing its potential to mitigate diabetes effects.

Melatonin alleviates cardiac fibrosis via inhibiting lncRNA MALAT1/miR-141-mediated NLRP3 inflammasome and TGF-β1/Smads signaling in diabetic cardiomyopathy.

Melatonin is a hormone produced by the pineal gland, and it has extensive beneficial effects on various tissue and organs; however, whether melatonin has any effect on cardiac fibrosis in the pathogenesis of diabetic cardiomyopathy (DCM) is still unknown. Herein, we found that melatonin administration significantly ameliorated cardiac dysfunction and reduced collagen production by inhibiting TGF-β1/Smads signaling and NLRP3 inflammasome activation, as manifested by downregulating the expression of TGF-β1, p-Smad2, p-Smad3, NLRP3, ASC, cleaved caspase-1, mature IL-1β, and IL-18 in the heart of melatonin-treated mice with diabetes mellitus (DM). Similar beneficial effects of melatonin were consistently observed in high glucose (HG)-treated cardiac fibroblasts (CFs). Moreover, we also found that lncRNA MALAT1 (lncR-MALAT1) was increased along with concomitant decrease in microRNA-141 (miR-141) in DM mice and HG-treated CFs. Furthermore, we established NLRP3 and TGF-β1 as target genes of miR-141 and lncR-MALAT1 as an endogenous sponge or ceRNA to limit the functional availability of miR-141. Finally, we observed that knockdown of miR-141 abrogated anti-fibrosis action of melatonin in HG-treated CFs. Our findings indicate that melatonin produces an antifibrotic effect via inhibiting lncR-MALAT1/miR-141-mediated NLRP3 inflammasome activation and TGF-β1/Smads signaling, and it might be considered a potential agent for the treatment of DCM.

Melatonin inhibits inflammasome-associated activation of endothelium and macrophages attenuating pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a pathophysiological syndrome associated with pulmonary/systemic inflammation. Melatonin relieves PAH, but the molecular mode of action remains unclear. Here, we investigated the role of melatonin in normalizing vascular homeostasis. Light-time mean serum melatonin concentration was lower in patients with PAH than in normal controls [11.06 ± 3.44 (7.13-15.6) vs. 14.55 ± 1.28 (8.0-19.4) pg/mL], which was negatively correlated with increased serum levels of interleukin-1β (IL-1β) in patients with PAH. We showed that inflammasomes were activated in the PAH mice model and that melatonin attenuated IL-1β secretion. On one hand, melatonin reduced the number of macrophages in lung by inhibiting the endothelial chemokines and adhesion factors. Moreover, use of Il1r-/- mice, Caspase1/11-/- mice, and melatonin-treated mice revealed that melatonin reduced hypoxia-induced vascular endothelial leakage in the lung. On the other hand, we verified that melatonin reduced the formation of inflammasome multiprotein complexes by modulating calcium ions in macrophages using a live cell station, and melatonin decreased inositol triphosphate and increased cAMP. Furthermore, knockdown of melatonin membrane receptors blocked melatonin function, and a melatonin membrane receptors agonist inactivated inflammasomes in macrophages. Melatonin attenuated inflammasome-associated vascular disorders by directly improving endothelial leakage and decreasing the formation of inflammasome multiprotein complexes in macrophages. Taken together, our data provide a theoretical basis for applying melatonin clinically, and inflammasomes may be a possible target of PAH treatment.

Melatonin promotes osteoblastic differentiation and regulates PDGF/AKT signaling pathway

Melatonin has been reported to participate in bone metabolism in recent studies. However, the underlying mechanism in melatonin-mediated osteoblastic differentiation remains largely unknown. The aim of this study is to investigate the role of melatonin in osteoblastic differentiation. In the present study, additional melatonin significantly promoted osteoblastic differentiation of MC3T3-E1 cells as evidenced by increased messenger RNA (mRNA) levels of osteogenic markers, alkaline phosphatase (ALP), collagen type I α1 chain, osteocalcin, and runt-related transcription factor 2 (Runx2). It was noteworthy that the expression level of platelet-derived growth factor subunit B (PDGFB) and content of its homodimer PDGF-BB were remarkably increased after melatonin administration. Moreover, the mRNA levels of phosphorylated PDGFRβ (PDGF receptor β) and Akt, a serine/threonine-specific protein kinase, were significantly upregulated in melatonin-treated MC3T3-E1 cells determined by a real-time polymerase chain reaction. Besides, by performing alizarin red staining, osteoblastic differentiation of MC3T3-E1 cells was conspicuously promoted by melatonin, which could be partially attenuated by crenolanib, a PDGFR inhibitor. Similarly, results from immunofluorescence and western blot assay showed that melatonin-induced upregulation of Runx2 and phosphorylated Akt was suppressed by crenolanib. Akt inhibition by MK-2206 also suppressed osteoblastic differentiation. Furthermore, by in vivo assay, additional melatonin promoted osteoblastic differentiation in mice with femoral fracture, and obvious callus formation was observed in melatonin-treated mice 5 weeks after fracture. Melatonin supplement also inhibited osteoclastic differentiation in mice. All statistical analysis was performed using GraphPad Prism and a P < 0.05 was deemed to be significant. To summarize, we demonstrate that melatonin promotes osteoblastic differentiation in MC3T3-E1 cells and enhances fracture healing in mouse femoral fracture model and regulates PDGF/AKT signaling pathway.

Melatonin regulates the activities of ovary and delays the fertility decline in female animals via MT1/AMPK pathway.

Female fertility irreversibly declines with aging, and this is primarily associated with the decreased quality and quantity of oocytes. To evaluate whether a long-term of melatonin treatment would improve the fertility of aged mice, different concentrations of melatonin (10-3 , 10-5 , 10-7 mol/L) were supplemented into drinking water. Melatonin treatments improved the litter sizes of mice at the age of 24weeks. Mice treated with 10-5 mol/L melatonin had the largest litter size among other concentrations. At this optimal concentration, melatonin not only significantly increased the total number of oocytes but also their quality, having more oocytes with normal morphology that could generate more blastocyst after in vitro fertilization in melatonin (10-5 mol/L)-treated group than that in the controls. When these blastocysts were transferred to recipients, the litter size was also significantly larger in melatonin treated mice than that in controls. The increases in TAOC and SOD level and decreases in MDA were detected in ovaries and uterus from melatonin-treated mice compared to the controls. Melatonin reduced ROS level and maintained mitochondrial membrane potential in the oocytes cultured in vitro. Mechanistically studies revealed that the beneficial effects of melatonin on oocytes were mediated by MT1 receptor and AMPK pathway. Thereafter, MT1 knocking out (MT1-KO) were generated and shown significantly reduced number of oocytes and litter size. The expression of SIRT1, C-myc, and CHOP were downregulated in the ovary of MT1-KO mice, but SIRT1 and p-NF-kB protein level were elevated in response to disturbed redox balance. The results have convincingly proven that melatonin administration delays ovary aging and improves fertility in mice via MT1/AMPK pathway.

Melatonin reprogramming of gut microbiota improves lipid dysmetabolism in high-fat diet-fed mice.

Melatonin has been shown to improve lipid metabolism and gut microbiota communities in animals and humans; however, it remains to know whether melatonin prevents obesity through gut microbiota. Here, we found that high-fat diet promoted the lipid accumulation and intestinal microbiota dysbiosis in mice, while oral melatonin supplementation alleviated the lipid accumulation and reversed gut microbiota dysbiosis, including the diversity of intestinal microbiota, relative abundances of Bacteroides and Alistipes, and functional profiling of microbial communities, such as energy metabolism, lipid metabolism, and carbohydrate metabolism. Interestingly, melatonin failed to alleviate the high-fat-induced lipid accumulation in antibiotic-treated mice; however, microbiota transplantation from melatonin-treated mice alleviated high-fat diet-induced lipid metabolic disorders. Notably, short-chain fatty acids were decreased in high-fat diet-fed mice, while melatonin treatment improved the production of acetic acid. Correlation analysis found a marked correlation between production of acetic acid and relative abundances of Bacteroides and Alistipes. Importantly, sodium acetate treatment also alleviated high-fat diet-induced lipid metabolic disorders. Taken together, our results suggest that melatonin improves lipid metabolism in high-fat diet-fed mice, and the potential mechanisms may be associated with reprogramming gut microbiota, especially, Bacteroides and Alistipes-mediated acetic acid production. Future studies are needed for patients with metabolic syndrome to fully understand melatonin's effects on body weight and lipid profiles and the potential mechanism of gut microbiota.

Melatonin modulates dysregulated circadian clocks in mice with diethylnitrosamine-induced hepatocellular carcinoma.

Disruption of circadian rhythms, which are regulated by the circadian clock machinery, plays an important role in different long-term diseases including hepatocellular carcinoma (HCC). Melatonin has been reported to alleviate promotion and progression of HCC, but the potential contribution of circadian clock modulation is unknown. We investigated the effects of melatonin in mice which received diethylnitrosamine (DEN) (35mg/kg body weight ip) once a week for 8weeks. Melatonin was given at 5 or 10mgkg-1 d-1 ip beginning 4weeks after the onset of DEN administration and ending at the sacrifice time (10, 20, 30, or 40weeks). Liver expression of Bmal1, Clock, Npas2, Rorα, and Sirt1 increased, whereas Cry1, Per1, Per2, Per3, CK1ε, Rev-erbα, and Rev-erbβ decreased following DEN administration. Melatonin treatment prevented changes in the expression of clock genes, and this effect was accompanied by an upregulation of the MT1 receptor and reduced levels of the hypoxia-inducible factors Hif-1α and Hif-2α. An increased expression of p21, p53, and PARP1/2, a higher Bax/Bcl-2 ratio, and a lower expression of Cyclin D1, CDK6, HSP70, HSP90, and GRP78 proteins were also observed in melatonin-treated mice. Melatonin significantly potentiated the suppression of proliferation and cell cycle arrest induced by the synthetic REV-ERB agonist SR9009 in human Hep3B cells, and BMAL1 knocking down attenuated the pro-apoptotic and antiproliferative effect of melatonin. Results support a contribution of changes in the circadian clock components to the beneficial effects of melatonin in HCC and highlight the usefulness of strategies modulating the circadian machinery in hepatocarcinogenesis.

Melatonin Ameliorates the Progression of Atherosclerosis via Mitophagy Activation and NLRP3 Inflammasome Inhibition.

The NLRP3 (nucleotide-binding domain and leucine-rich repeat pyrin domain containing 3) inflammasome-mediated inflammatory responses are critically involved in the progression of atherosclerosis (AS), which is the essential cause for cardiovascular diseases. Melatonin has anti-inflammatory properties. However, little is known about the potential effects of melatonin in the pathological process of AS. Herein, we demonstrate that melatonin suppressed prolonged NLRP3 inflammasome activation in atherosclerotic lesions by reactive oxygen species (ROS) scavenging via mitophagy in macrophages. The atherosclerotic mouse model was induced with a high-fat diet using ApoE−/− mice. Melatonin treatment markedly attenuated AS plaque size and vulnerability. Furthermore, melatonin decreased NLRP3 inflammasome activation and the consequent IL-1β secretion within atherosclerotic lesions. Despite the unchanged protein expression, the silent information regulator 3 (Sirt3) activity was elevated in the atherosclerotic lesions in melatonin-treated mice. In ox-LDL-treated macrophages, melatonin attenuated the NLRP3 inflammasome activation and the inflammatory factors secretion, while this protective effect was abolished by either Sirt3 silence or autophagy inhibitor 3-MA. Mitochondrial ROS (mitoROS), which was a recognized inducer for NLRP3 inflammasome, was attenuated by melatonin through the induction of mitophagy. Both Sirt3-siRNA and autophagy inhibitor 3-MA partially abolished the beneficial effects of melatonin on mitoROS clearance and NLRP3 inflammasome activation, indicating the crucial role of Sirt3-mediated mitophagy. Furthermore, we demonstrated that melatonin protected against AS via the Sirt3/FOXO3a/Parkin signaling pathway. In conclusion, the current study demonstrated that melatonin prevented atherosclerotic progression, at least in part, via inducing mitophagy and attenuating NLRP3 inflammasome activation, which was mediated by the Sirt3/FOXO3a/Parkin signaling pathway. Collectively, our study provides insight into melatonin as a new target for therapeutic intervention for AS.

Effects of melatonin administration on embryo implantation and offspring growth in mice under different schedules of photoperiodic exposure

BackgroundEmbryo implantation is crucial for animal reproduction. Unsuccessful embryo implantation leads to pregnancy failure, especially in human-assisted conception. Environmental factors have a profound impact on embryo implantation. Because people are being exposed to more light at night, the influence of long-term light exposure on embryo implantation should be explored.MethodsThe effects of long photoperiodic exposure and melatonin on embryo implantation and offspring growth were examined. Long photoperiodic exposure (18:6 h light:dark) was selected to resemble light pollution. Melatonin (10−2, 10−3, 10−4, 10−5 M) was added to the drinking water of mice starting at Day 1 (vaginal plugs) until delivery.ResultsMelatonin treatment (10−4,10−5 M) significantly increased litter sizes compared to untreated controls (12.9 ± 0.40 and 12.2 ± 1.01 vs. 11.5 ± 0.43; P < 0.05). The most effective concentration of melatonin (10−4 M) was selected for further investigation. No remarkable differences were found between melatonin-treated mice and controls in terms of the pups’ birth weights, weaning survival rates, and weaning weights. Long photoperiodic exposure significantly reduced the number of implantation sites in treated mice compared to controls (light/dark, 12/12 h), and melatonin rescued this negative effect. Mechanistic studies revealed that melatonin enhanced the serum 17β-estradiol (E2) levels in the pregnant mice and upregulated the expression of the receptors MT1 and MT2 and p53 in uterine tissue. All of these factors may contribute to the beneficial effects of melatonin on embryo implantation in mice.ConclusionMelatonin treatment was associated with beneficial effects in pregnant mice, especially those subjected to long photoperiodic exposure. This was achieved by enhanced embryo implantation. At the molecular level, melatonin administration probably increases the E2 level during pregnancy and upregulates p53 expression by activating MT1/2 in the uterus. All of the changes may improve the microenvironment of the uterus and, thus, the outcomes of pregnancy.

Abstract 884: Detection of intratumoral heterogeneity using MR-defined tumor habitats in breast cancer model under melatonin treatment

Abstract Breast cancer shows high intratumoral heterogeneity, which can be visualized as localized sub-populations of tumor cells with different malignancy potential and treatment responsiveness. These regions are like “habitats” that contain different combinations of environmental selection forces, such as hypoxia, acidosis, or nutrient availability. Thus, agents that can perturb these forces may be useful in limiting cancer progression. Studies have shown that melatonin can affect tumor metabolism and we hypothesize that this may be a mechanism by which the disruption of circadian melatonin production by light exposure at night (LAN) promotes breast cancer initiation and progression. The aim of this study was to evaluate the effect of melatonin on intratumoral heterogeneity measured by non-invasive MR imaging in a syngeneic mouse model of breast cancer (4T1). To visualize habitats, clusters were generated from combined MR images generated by different pulse sequences. These were co-registered with histology using tumor specific 3-D printed cradles to classify these tumor habitats. Methods: Mice were exposed to LAN (Control) or LAN plus melatonin. The melatonin metabolite 6-sulfatoxymelatonin (6-SMT) was assessed in urine as a measure of melatonin production. Mice were imaged in a 7T MRI system with: 1) anatomical T2 images, 2) T2*, 3) diffusion-weighted MRI (DWI); and 4) T1 weighted dynamic contrast enhanced (DCE). DCE maps were obtained through semi-quantitative analysis of the pre- and post-contrast agent bolus time-series on a pixel-by-pixel basis. These were combined to the corresponding T2, T2* and Apparent Diffusion Coefficient (ADC) values to generate parameter maps. Parameter maps were used to classify multiple clusters, based on a Gaussian mixture model. Following euthanasia, a 3D printed mold based on the T2 tumor isosurface was created in order to co-register the MRI and histology. Tumors were stained with H&amp;E and for pimonidazole (hypoxia) by immunohistochemistry. Results: Melatonin treatment increased the 6-SMT in urine of mice exposed to LAN and statistically reduced tumor growth by day 15 (p&amp;lt;0.05). Distinct hypoxic habitats have been observed by MRI. Habitat clusters with low values in ADC (high cellularity) and low contrast enhancement were associated with positive staining of pimonidazole. Initial results suggest that the melatonin-treated mice had more homogeneous tumors, although additional numbers will be needed to reach statistical significance. Conclusion: These data indicate that the spatial heterogeneity of breast tumors, corroborated by histological features, may be used as a non-invasive imaging method for monitoring the changes in intratumoral heterogeneity following cancer therapy. Citation Format: Bruna V. Jardim-Perassi, William Dominguez-Viqueira, Asmaa El-Kenawi, Dominique F. Abrahams, Mikalai Budzevich, Epifanio Ruiz, Suning Huang, Pedro M. Enriquez-Navas, Gary Martinez, Debora Aparecida Pires de Campos Zuccari, Robert J. Gillies. Detection of intratumoral heterogeneity using MR-defined tumor habitats in breast cancer model under melatonin treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 884. doi:10.1158/1538-7445.AM2017-884