OBJECTIVE: To investigate the intervention of melatonin (MT) in the expression of circadian genes in patients with pulmonary fibrosis and to analyze the mechanism by which it alleviates the progression of pulmonary fibrosis. METHODS: By utilizing the Gene Expression Omnibus (GEO) database, we identified differentially expressed circadian genes between patients with pulmonary fibrosis and controls. We analyzed the correlation between circadian genes and pulmonary function as well as genes related to pulmonary fibrosis. A bleomycin-induced mouse model of pulmonary fibrosis (BLM group) was constructed to observe the expression differences of PER2 and CRY2 by sequencing and immunohistochemical staining in the BLM group and after MT intervention (BLM+MT group). Hematoxylin and eosin (HE) staining and Masson staining were used to observe the effects of MT on fibrosis. We used Western blot to detect the expression of P-smad2/3 in lung epithelial cells induced by transforming growth factor β (TGF-β). Reverse transcription quantitative real-time PCR technology was employed to investigate the rhythmic expression changes of circadian genes in the control group, TGF-β group, and TGF-β+MT group. Finally, luzindole, a MT receptor antagonist, was used to intervene in TGF-β+MT group, and Western blot was used to explore the receptor dependence of MT in alleviating TGF-β-induced epithelial-mesenchymal transition. RESULTS: (1) Analysis of the GEO dataset (GSE) revealed a negative correlation between circadian genes PER2 and CRY2 and the expression of TGF-β, and a positive correlation with pulmonary function indicators in patients. (2) Transcriptome sequencing analysis of lung tissue in BLM group found that the expression of PER2 and CRY2 was significantly reduced compared with the normal group. Histopathological staining results showed that the lung tissue structure of the normal group was intact and clear, with thin alveolar septa; in the BLM group, there was a large increase in collagen fibers and disordered alveolar structure; compared with the BLM group, the BLM+MT group had reduced collagen fiber proliferation and inflammatory cell infiltration; the expression of PER2 and CRY2 in the BLM group was lower than in the normal group, and the expression in the BLM+MT group was increased compared with the BLM group. (3) In vitro lung epithelial cell experiments with TGF-β intervention showed that compared with the control group, the expression of P-smad2/3 increased in the TGF-β group, and MT intervention inhibited the inducing effect of TGF-β on P-smad2/3, while intervention with the MT receptor antagonist reversed this phenomenon. The results indicated that MT could inhibit the activation of the TGF-β pathway, and this process was dependent on MT receptors. (4) The 48-hour rhythm experiment in lung epithelial cells showed that the mRNA rhythm of PER2 and CRY2 in the TGF-β+MT group was close to 24 hours and showed a trend towards restoring the rhythm of the control group, while the addition of the MT receptor blocker tended to make the rhythm duration and amplitude of both groups approach that of the TGF-β group. CONCLUSION MT, by binding to its receptors, can restore the periodic expression of the circadian genes PER2 and CRY2, thereby inhibiting the activation of the TGF-β classical pathway and suppressing the pathological process of epithelial-mesenchymal transition in pulmonary fibrosis. This finding provides new molecular targets and potential therapeutic strategies for the treatment of pulmonary fibrosis.
Melatonin/pharmacology* ; Animals ; Mice ; Pulmonary Fibrosis/chemically induced* ; Bleomycin ; Humans ; Transforming Growth Factor beta/metabolism* ; Period Circadian Proteins/metabolism* ; Smad3 Protein/genetics* ; Disease Models, Animal ; Lung/pathology* ; Cryptochromes/metabolism* ; Smad2 Protein/genetics* ; Epithelial Cells/metabolism* ; Mice, Inbred C57BL

Sleep-wake disorder is one of the most common nonmotor symptoms of Parkinson's disease (PD). Melatonin has the potential to improve sleep-wake disorder, but its mechanism of action is still unclear. Our data showed that melatonin only improved the motor and sleep-wake behavior of a zebrafish PD model when melatonin receptor 1 was present. Thus, we explored the underlying mechanisms by applying a rotenone model. After the PD zebrafish model was induced by 10 nmol/L rotenone, the motor and sleep-wake behavior were assessed. In situ hybridization and real-time quantitative PCR were used to detect the expression of melatonin receptors and lipid-metabolism-related genes. In the PD model, we found abnormal lipid metabolism, which was reversed by melatonin. This may be one of the main pathways for improving PD sleep-wake disorder.
Animals ; Zebrafish ; Melatonin/pharmacology* ; Lipid Metabolism/drug effects* ; Disease Models, Animal ; Rotenone/pharmacology* ; Sleep Wake Disorders/metabolism* ; Parkinson Disease/metabolism* ; Motor Activity/drug effects* ; Sleep/drug effects*

Melatonin receptor 1B (MT2, encoded by the MTNR1B gene), a high-affinity receptor for melatonin, is associated with glucose homeostasis including glucose uptake and transport. The rs10830963 variant in the MTNR1B gene is linked to glucose metabolism disorders including gestational diabetes mellitus (GDM); however, the relationship between MT2-mediated melatonin signaling and a high birth weight of GDM infants from maternal glucose abnormality remains poorly understood. This article aims to investigate the relationship between rs10830963 variants and GDM development, as well as the effects of MT2 receptor on glucose uptake and transport in trophoblasts. TaqMan-MGB (minor groove binder) probe quantitative real-time polymerase chain reaction (qPCR) assays were used for rs10930963 genotyping. MT2 expression in the placenta of GDM and normal pregnant women was detected by immunofluorescence, western blot, and qPCR. The relationship between MT2 and glucose transporters (GLUTs) or peroxisome proliferator-activated receptor γ (PPARγ) was established by western blot, and glucose consumption of trophoblasts was measured by a glucose assay kit. The results showed that the genotype and allele frequencies of rs10830963 were significantly different between GDM and normal pregnant women (P<0.05). The fasting, 1-h and 2-h plasma glucose levels of G-allele carriers were significantly higher than those of C-allele carriers (P<0.05). Besides, the protein and messenger RNA (mRNA) expression of MT2 in the placenta of GDM was significantly higher than that of normal pregnant women (P<0.05). Melatonin could stimulate glucose uptake and GLUT4 and PPARγ protein expression in trophoblasts, which could be attenuated by MT2 receptor knockdown. In conclusion, the rs10830963 variant was associated with an increased risk of GDM. The MT2 receptor is essential for melatonin to raise glucose uptake and transport, which may be mediated by PPARγ.
Female ; Humans ; Pregnancy ; Blood Glucose/metabolism* ; Diabetes, Gestational/metabolism* ; Glucose/metabolism* ; Melatonin/metabolism* ; Polymorphism, Genetic ; PPAR gamma ; Receptor, Melatonin, MT2/genetics*

OBJECTIVE: To investigate the effects of melatonin on the growth and metastasis of MDA-MB-231 breast cancer cells and explore the mechanism. METHODS: MDA-MB-231 cells were treated with 1, 3 or 5 mmol/L melatonin, and the changes in cell proliferation were examined using CCK-8 assay. Colony-forming assay and wound healing assay were used to assess the effects of melatonin treatmnent on colony-forming ability and migration of the cells. Flow cytometry and immunofluoresnce assay were employed to examine apoptosis and positive staining for autophagy-related proteins in the cells treated with 3 mmol/L melatonin. The effects of melatonin treatment alone or in combination with 3-methyladenine (3-MA) on the expressions of the proteins associated with autophagy (LC3, P62 and Beclin1), apoptosis (Bcl2 and Bax) and epithelial-mesenchymal transition (E-cadherin and Snail) were examined with Western blotting. RESULTS: Melatonin treatment significantly inhibited the proliferation of breast cancer cells in a concentration- and time-dependent manner (P < 0.05), suppressed colony-forming ability and migration (P < 0.01), and promoted apoptosis of the cells (P < 0.01). Melatonin treatment alone significantly increased the expressions of Bax (P < 0.05), E-cadherin, LC3-II/LC3-I, and Beclin1 and lowered the expressions of Bcl2 (P < 0.05), Snail, P62 (P < 0.05), and Bcl2/Bax ratio (P < 0.01) in the cells, and caused enhanced positive staining of Beclin1 protein and attenuated staining of P62 protein. Compared with melatonin treatment alone, melatonin treatment combined with 3-MA significantly decreased the expressions of Beclin1 (P < 0.001), LC3-II/LC3-I (P < 0.05), Bax (P < 0.01), and E-cadherin (P < 0.001) and increased the expressions of Bcl2 (P < 0.05), Snail, and Bcl2/Bax ratio (P < 0.01). CONCLUSION Melatonin can induce autophagy of MDA-MB-231 breast cancer cells to inhibit cell proliferation and metastasis and promote cell apoptosis, and suppressing autophagy can weaken the inhibitory effect of melatonin on the growth and metastasis of breast cancer cells.
Autophagy ; Autophagy-Related Proteins/metabolism* ; Breast Neoplasms ; Cell Line, Tumor ; Female ; Humans ; Melatonin/pharmacology*

Depression is a debilitating psychiatric disorder with a huge socioeconomic burden, and its treatment relies on antidepressants including selective serotonin reuptake inhibitors (SSRIs). Recently, the melatonergic system that is closely associated with the serotonergic system has been implicated in the pathophysiology and treatment of depression. However, it remains unknown whether combined treatment with SSRI and melatonin has synergistic antidepressant effects. In this study, we applied a sub-chronic restraint stress paradigm, and evaluated the potential antidepressant effects of combined fluoxetine and melatonin in adult male mice. Sub-chronic restraint stress (6 h/day for 10 days) induced depression-like behavior as shown by deteriorated fur state, increased latency to groom in the splash test, and increased immobility time in the forced-swim test. Repeated administration of either fluoxetine or melatonin at 10 mg/kg during stress exposure failed to prevent depression-like phenotypes. However, combined treatment with fluoxetine and melatonin at the selected dose attenuated stress-induced behavioral abnormalities. Moreover, we found that the antidepressant effects of combined treatment were associated with the normalization of brain-derived neurotrophic factor (BDNF)-tropomyosin receptor kinase B (TrkB) signaling in the hippocampus, but not in the prefrontal cortex. Our findings suggest that combined fluoxetine and melatonin treatment exerts synergistic antidepressant effects possibly by restoring hippocampal BDNF-TrkB signaling.
Animals ; Antidepressive Agents ; pharmacology ; Behavior, Animal ; drug effects ; Brain-Derived Neurotrophic Factor ; drug effects ; metabolism ; Depression ; Drug Synergism ; Drug Therapy, Combination ; Fluoxetine ; pharmacology ; Hippocampus ; drug effects ; metabolism ; Male ; Melatonin ; pharmacology ; Membrane Glycoproteins ; drug effects ; metabolism ; Mice, Inbred C57BL ; Protein-Tyrosine Kinases ; drug effects ; metabolism ; Restraint, Physical ; Signal Transduction ; drug effects

OBJECTIVETo investigate the effects of Liuwei Dihuang (LWDH) decoction on serotonine (5-HTs), melatonin and the activity of the rate-limiting enzymes ANNAT and HIOMT in cultured human melanocytes and in melanocytes co-cultured with keratinocytes.

METHODSCCK-8 assay was used to assess the proliferation of melanocytes and melanocytes co-cultured with keratinocytes after treatment with the serum from rabbits fed with LWDH decoction. High-performance liquid chromatography was used to determine 5-HT and melatonin contents, and real-time fluorescent PCR was employed to evaluate the ANNAT and HIOMT activities in the cell cultures.

RESULTSThe serum from rabbits fed with LWDH Decoction at low doses did not affect the proliferation of melanocytes co-cultured with keratinocytes, but at the concentrations of 20%-40%, the serum significantly inhibited the proliferation of melanocytes, and the effect was optimal with a concentration of 40% (P<0.05). 5-HT and melatonin contents in the cell culture decreased as the serum concentration increased (P<0.05), which was the most obvious with a serum concentration of 40% (P<0.01). Exposure of the cells to low and moderate doses of the serum caused a dose-dependent decrease in AANAT activity (P<0.05), but the serum produced no significant changes in the level of HIOMT mRNA expression in the cells.

COUCLUSIONSThe serotoninergic/melatoninergic system mediate the regulation of melanin metabolism by LWDH Decoction, the mechanism of which may involve 5-HTs, melatonin and ANNAT.

Animals ; Cells, Cultured ; Coculture Techniques ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Keratinocytes ; Melanins ; metabolism ; Melanocytes ; drug effects ; metabolism ; Melatonin ; metabolism ; Rabbits ; Serotonin ; metabolism ; Serum ; chemistry

OBJECTIVETo investigate the expressions of P38 mitogen-activated protein kinases (P38 MAPK), JNK mitogen-activated protein kinases (JNK MAPK) and the therapeutical effects of melatonin in the renal tissue of acute acuteparaquat-induced rats.

METHODSSeventy-eight healthy adult Sprague-Dawley (SD) rats (39 male, 39 female) were randomly divided into three groups: (1) Control group (group A): 6 rats, (2) Poisoned group (group B): 36 rats, (3) Therapeutical group (group C): 36 rats. At 1, 3, 5, 7, 10 and 14 days after poisoning, six rats in Group B and group C were used to assess renal pathological changes and the expression of P38 MAPK, JNK MAPK in kidney were evaluated by immunohistochemistry.

RESULTSCompared with control group, the expression of P38MAPK in renal tissue of poisoned group significantly rose at the first day, reached the peak at the 10th day and afterwards decreased slowly. Expression of JNK MAPK reached the peak at the first day, and kept at relatively high levels up to the 14th day. Melatonin weakened markedly the expressions of P38 MAPK and JNK MAPK in renal tissue of acute acuteparaquat-induced rats.

CONCLUSIONP38 MAPK and JNK MAPK play an important role in renal injury of acute paraquat -poisoning rats. Melatonin takes a significant effect on the activation of them.

Animals ; Female ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Kidney ; drug effects ; metabolism ; physiopathology ; Male ; Melatonin ; therapeutic use ; Paraquat ; poisoning ; Rats ; Rats, Sprague-Dawley ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo explore the effects of Kaixin Powder (, KXP) on melatonin receptor (MR) expression and (125)I-Mel binding affinity in a depression rat model.

METHODSSeventy-two male Wistar rats were divided into six groups: a blank control group, model group, ramelteon group, KXP high-dosage group (HKXP), medium-dosage group (MKXP) and low-dosage group (LKXP). To establish the depression model, all groups except the blank control group were singly housed and exposed to chronic unpredictable mild stress. Weight gain, sucrose consumption and the open-field test were used to evaluate induction of depression. KXP at 260, 130 and 65 mg/(kg•d) was also respectively administered to the rats in the HKXP, MKXP and LKXP groups for 21 days. Ramelteon [0.83 mg/(kg•d)] was given to the positive drug control group. An equivalent volume of physiological saline was given to the blank and model groups. The liquid chip method was used to measure the concentration of plasma melatonin (MT). Mel1a (MT1) and Mel1b (MT2) expression levels were determined by Western blotting. In addition, a radioactive ligand-binding assay was used to analyze the specific binding properties and dynamic characteristics between MR and (125)I-Mel.

RESULTSThe results of weight gain, sucrose consumption and the open-field test showed that our model successfully produced depressive symptoms and depressive-like behavior. The concentration of plasma MT in the model group decreased significantly at night but increased in the MKXP group (P<0.05). The HKXP group showed significantly increased expression of MT1 (P<0.05); however, the expression of MT2 in all groups exhibited no significant differences (P>0.05). The maximum binding capacity (B(max)) for specific binding between MR and 125I-Mel in the MKXP group was significantly higher than that in the model group (P<0.05), but no significant differences were found in the equilibrium dissociation constant (K(d)) of each group (P>0.05).

CONCLUSIONSKXP may have a similar effect as ramelteon. KXP improved depressive-like behavior by increasing the concentration of plasma MT and MT1 expression, thereby increasing three B(max) of MR to achieve the desired antidepressant effect.

Animals ; Brain ; drug effects ; metabolism ; pathology ; Depression ; blood ; drug therapy ; metabolism ; Disease Models, Animal ; Drinking Behavior ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Gene Expression Regulation ; drug effects ; Indenes ; Iodine Radioisotopes ; Male ; Melatonin ; blood ; metabolism ; Rats, Wistar ; Receptors, Melatonin ; genetics ; metabolism ; Weight Gain ; drug effects

In order to investigate the role of melatonin in inhibiting the proliferation of murine gastric cancer and the underlying molecular mechanism, we performed an in vivo study by inoculating murine foregastric carcinoma (MFC) cells in mice, and then tumor-bearing mice were treated with different concentrations of melatonin (i.p.). The changes of Bcl-2, Bax, p21 and p53 expressions in tumor tissue were detected by using real-time fluorescence quantitative RT-PCR and Western blot. We found that: (1) melatonin resulted in reductions of tumor's volume and weight in the gastric cancer-bearing mice and thus showed anti-cancer effect; (2) melatonin reduced Bcl-2 expression, but increased the expression of Bax, p53 and p21 in tumor tissue. Our results suggest that melatonin could inhibit the growth of tumors in gastric cancer-bearing mice through accelerating the apoptosis of tumor cells.
Animals ; Apoptosis ; Melatonin ; pharmacology ; Mice ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Stomach Neoplasms ; drug therapy ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; bcl-2-Associated X Protein ; metabolism

Animals ; Antioxidants ; metabolism ; Diabetes Mellitus, Experimental ; metabolism ; Melatonin ; metabolism ; Physical Conditioning, Animal ; Rats