Melatonin,an endocrine hormone synthesized by the pineal gland,plays an important role in the reproduction.The growth and development of follicles is the basis of female mammalian fertility.Follicles have a high concentration of melatonin.Melatonin receptors exist on ovarian granulosa cells,follicle cells,and oocytes.It regulates the growth and development of these cells and the maturation and atresia of follicles,affecting female fertility.This paper reviews the protective effects and regulatory mechanisms of melatonin on the development of ovarian follicles,granulosa cells,and oocytes and makes an outlook on the therapeutic potential of melatonin for ovarian injury,underpinning the clinical application of melatonin in the future.
Animals ; Female ; Melatonin/pharmacology* ; Ovarian Follicle ; Oocytes ; Granulosa Cells/physiology* ; Mammals

OBJECTIVE: To investigate the effects of melatonin (MT) on bone mass and serum inflammatory factors in rats received ovariectomy (OVX) and to investigate the effects of MT on the levels of inflammatory factors in culture medium and osteogenic ability of bone marrow mesenchymal stem cells (BMSCs) stimulated by lipopolysaccharide. METHODS: Fifteen 12-week-old Sprague Dawley (SD) rats were randomly divided into 3 groups. The rats in Sham group only received bilateral lateral abdominal incision and suture, the rats in OVX group received bilateral OVX, and the rats in OVX+MT group received 100 mg/(kg·d) MT oral intervention after bilateral OVX. After 8 weeks, the levels of serum inflammatory factors [interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α)] were detected using ELISA assay. Besides, the distal femurs were detected by Micro-CT to observe changes in bone mass and microstructure, and quantitatively measured bone volume fraction, trabecular thickness, and trabecular number. The BMSCs were extracted from the femurs of three 3-week-old SD rats using whole bone marrow culture method and passaged. The 3rd-5th passage BMSCs were cultured with different concentrations of MT (0, 1, 10, 100, 1 000 µmol/L), and the cell viability was then detected using cell counting kit 8 (CCK-8) to select the optimal concentration of MT for subsequent experiments. Cells were devided into osteogenic induction group (group A) and osteogenic induction+1/5/10 μg/mL lipopolysaccharide group (group B-D). The levels of inflammatory factors (IL-1β, IL-6 and TNF-α) in cell culture medium were detected using ELISA assay after corresponding intervention. According to the results of CCK-8 method and ELISA detection, the cells were intervened with the most significant concentration of lipopolysaccharide for stimulating inflammation and the optimal concentration of MT with osteogenic induction, defining as group E, and the cell culture medium was collected to detect the levels of inflammatory factors by ELISA assay. After that, alkaline phosphatase (ALP) staining and alizarin red staining were performed respectively in groups A, D, and E, and the expression levels of osteogenic related genes [collagen type Ⅰ alpha 1 chain (Col1a1) and RUNX family transcription factor 2 (Runx2)] were also detected by real time fluorescence quantitative PCR (RT-qPCR). RESULTS: ELISA and Micro-CT assays showed that compared with Sham group, the bone mass of the rats in the OVX group significantly decreased, and the expression levels of serum inflammatory factors (IL-1β, IL-6, and TNF-α) in OVX group significantly increased (P<0.05). Significantly, the above indicators in OVX+MT group were all improved (P<0.05). Rat BMSCs were successfully extracted, and CCK-8 assay showed that 100 µmol/L was the maximum concentration of MT that did not cause a decrease in cell viability, and it was used in subsequent experiments. ELISA assays showed that compared with group A, the expression levels of inflammatory factors (IL-1β, IL-6, and TNF-α) in the cell culture medium of groups B-D were significantly increased after lipopolysaccharide stimulation (P<0.05), and in a concentration-dependent manner. Moreover, the expression levels of inflammatory factors in group D were significantly higher than those in groups B and C (P<0.05). After MT intervention, the expression levels of inflammatory factors in group E were significantly lower than those in group D (P<0.05). ALP staining, alizarin red staining, and RT-qPCR assays showed that compared with group A, the percentage of positive area of ALP and alizarin red and the relative mRNA expressions of Col1a1 and Runx2 in group D significantly decreased, while the above indicators in group E significantly improved after MT intervention (P<0.05). CONCLUSION MT may affect the bone mass of postmenopausal osteoporosis by reducing inflammation in rats; MT can reduce the inflammation of BMSCs stimulated by lipopolysaccharide and weaken its inhibition of osteogenic differentiation of BMSCs.
Female ; Rats ; Animals ; Tumor Necrosis Factor-alpha ; Osteogenesis ; Rats, Sprague-Dawley ; Core Binding Factor Alpha 1 Subunit ; Melatonin/pharmacology* ; Interleukin-6/genetics* ; Lipopolysaccharides/pharmacology* ; Coloring Agents ; Inflammation

The aim of this study was to investigate the role of selenoprotein M (SelM) in endoplasmic reticulum stress and apoptosis in nickel-exposed mouse hearts and to explore the detoxifying effects of melatonin. At 21 d after intraperitoneal injection of nickel chloride (NiCl₂) and/or melatonin into male wild-type (WT) and SelM knockout (KO) C57BL/6J mice, NiCl₂ was found to induce changes in the microstructure and ultrastructure of the hearts of both WT and SelM KO mice, which were caused by oxidative stress, endoplasmic reticulum stress, and apoptosis, as evidenced by decreases in malondialdehyde (MDA) content and total antioxidant capacity (T-AOC) activity. Changes in the messenger RNA (mRNA) and protein expression of genes related to endoplasmic reticulum stress (activating transcription factor 4 (ATF4), inositol-requiring protein 1 (IRE1), c-Jun N-terminal kinase (JNK), and C/EBP homologous protein (CHOP)) and apoptosis (B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, Caspase-9, and Caspase-12) were also observed. Notably, the observed damage was worse in SelM KO mice. Furthermore, melatonin alleviated the heart injury caused by NiCl₂ in WT mice but could not exert a good protective effect in the heart of SelM KO mice. Overall, the findings suggested that the antioxidant capacity of SelM, as well as its modulation of endoplasmic reticulum stress and apoptosis, plays important roles in nickel-induced heart injury.
Animals ; Male ; Mice ; Antioxidants/pharmacology* ; Apoptosis ; Endoplasmic Reticulum Stress ; Melatonin/pharmacology* ; Mice, Inbred C57BL ; Nickel/adverse effects* ; Selenoproteins/genetics* ; Heart/drug effects*

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*

OBJECTIVE: To investigate the effect of melatonin (MLT) on the proliferation and apoptosis of human multiple myeloma cell line RPMI 8226 and its possible mechanism. METHODS: RPMI 8226 cells were cultured in vitro, and different concentrations of MLT were treated on RPMI 8226 cells. The effects of MLT on RPMI 8226 cell proliferation were detected by CCK-8 assay and methylcellulose cloning assay, and the effects of MLT on cell apoptosis were detected by AnnexinV-FITC /PI, flow cytometry. Western blot was used to determine the expression of apoptosis and endoplasmic reticulum stress-related proteins in each group, and CCK-8 assay was used to determine the effect of MLT combined with bortezemib on the viability of RPMI 8226 cells. RESULTS: MLT inhibited the proliferation of RPMI 8226 cells in a dose- and time-dependent manner (r=-0.9777，r=-0.9951). With the increase of MLT concentration, the number of clones decreased, the apoptosis of RPMI 8226 cells increased (P<0.05), the expression of anti-apoptotic protein XIAP decreased, the expression of apoptotic proteins Bax and Caspase3 increased, and the expression of endoplasmic reticulum stress-related proteins increased. Compared with the control group, the survival of RPMI 8226 cells in the MLT and BTZ combined group significantly decreased (P<0.01). CONCLUSION MLT can inhibit the proliferation of RPMI 8226 cells, promote the apoptosis of RPMI 8226 cells, and enhance the anti-tumor effect of BTZ on RPMI 8226 cells. The mechanism may be related to endoplasmic reticulum stress.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Endoplasmic Reticulum Stress ; Humans ; Melatonin/pharmacology* ; Multiple Myeloma/pathology* ; Sincalide/pharmacology*

OBJECTIVE: To study the effect of different melatonin treatment regimens on long-term behavior and white matter damage in neonatal rats with hypoxic-ischemic brain damage (HIBD), and to seek an optimal melatonin treatment regimen. METHODS: Healthy Sprague-Dawley rats, aged 7 days, were randomly divided into four groups: sham-operation, HIBD, single-dose immediate treatment (SDIT), and 7-day continuous treatment (7DCT), with 8 rats in each group. A neonatal rat model of HIBD was prepared according to the classical Rice-Vannucci method. On day 21 after HIBD, the Morris water maze test was used to evaluate spatial learning and memory abilities. On day 70 after HIBD, immunofluorescence assay was used to measure the expression of neuronal nuclear antigen (NeuN) in the cerebral cortex and the hippocampal CA1 region of neonatal rats, and double-label immunofluorescence was used to measure the expression of myelin basic protein (MBP) and neurofilament 200 (NF200) in the corpus striatum and the corpus callosum. RESULTS: The results of the Morris water maze test showed that the SDIT and 7DCT groups had a significantly shorter mean escape latency than the HIBD group, and the 7DCT group had a significantly shorter mean escape latency than the SDIT group ( CONCLUSIONS Both SDIT and 7DCT can improve long-term behavior and reduce white matter damage in neonatal rats with HIBD, and 7DCT is more effective than SDIT.
Animals ; Animals, Newborn ; Hypoxia-Ischemia, Brain/drug therapy* ; Melatonin/pharmacology* ; Rats ; Rats, Sprague-Dawley ; White Matter

OBJECTIVE: To investigate the protective effect of melatonin against myocardial ischemia reperfusion (IR) injury in isolated rat hearts and explore the underlying mechanisms. METHODS: The isolated hearts from 40 male SD rats were randomly divided into 4 groups (=10): the control group, where the hearts were perfused with KH solution for 175 min; IR group, where the hearts were subjected to global ischemia for 45 min followed by reperfusion for 120 min; IR+melatonin (Mel+IR) group, where melatonin (5 μmol/L) was administered to the hearts 1 min before ischemia and during the first 5 min of reperfusion, followed by 115 min of reperfusion; and IR+2, 3-butanedione monoxime (IR+BDM) group, where the hearts were treated with BDM (20 mmol/L) in the same manner as melatonin treatment. Myocardial injury in the isolated hearts was assessed based on myocardial injury area, caspase-3 activity, and expressions of cytochrome C and cleaved caspase-3 proteins. Cardiac contracture was assessed using HE staining and by detecting lactate dehydrogenase (LDH) activity and the content of cardiac troponin I (cTnI) in the coronary outflow, measurement of left ventricular end-diastolic pressure (LVEDP) and electron microscopy. The content of ATP in the cardiac tissue was also determined. RESULTS: Compared with those in the control group, the isolated hearts in IR group showed significantly larger myocardial injury area and higher caspase-3 activity and the protein expressions of cytochrome C and cleaved caspase-3 with significantly increased LDH activity and cTnI content in the coronary outflow and elevated LVEDP at the end of reperfusion; HE staining showed obvious fractures of the myocardial fibers and the content of ATP was significantly decreased in the cardiac tissue; electron microscopy revealed the development of contraction bands. In the isolated hearts with IR, treatment with Mel or BDM significantly reduced the myocardial injury area, caspase-3 activity, and protein expressions of cytochrome C and cleaved caspase-3, obviously inhibited LDH activity, lowered the content of cTnI and LVEDP, reduced myocardial fiber fracture, and increased ATP content in the cardiac tissue. Both Mel and BDM inhibited the formation of contraction bands in the isolated hearts with IR injury. CONCLUSIONS Mel can alleviate myocardial IR injury in isolated rat hearts by inhibiting cardiac contracture, the mechanism of which may involve the upregulation of ATP in the cardiac myocytes to lessen the tear of membrane and reduce cell content leakage.
Animals ; Heart ; drug effects ; Male ; Melatonin ; pharmacology ; therapeutic use ; Muscle Contraction ; drug effects ; Myocardial Reperfusion Injury ; drug therapy ; Myocytes, Cardiac ; drug effects ; Rats ; Rats, Sprague-Dawley

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 study the effect of melatonin on the gastrointestinal motility and plasma levels of the stress hormone in overtraining rats.

METHODThirty adult SD rats were randomly divided into three groups (n = 10): control group, over-training group, melatonin intervention group. 30 min before each training, rats in the control and over-training groups were fed with normal saline (15 mg/kg) once a day and 5 times per week, while rats in the melatonin intervention group were administrated with melatonin, perfusion in the intervention group (15 mg/kg). Excessive training group and melatonin intervention group rats were subjected to excessive training at 5 times a week for 6 weeks. After 6 weeks, the gastric emptying rate, small intestinal propulsion ratio and levels of plasma motilin (MTL) and calcitonin gene-related peptide (CGRP), cortisol (CORT) and catecholamines (CA) were observed in all groups.

RESULTSCompared with the control group, the gastric emptying rate, small intestinal propulsion ratio and levels of plasma MTL, CORT and CA were increased significantly (P < 0.01) while the content of CGRP was reduced (P < 0.01) in over-training group. After treated with melatonin, this trend was reversed, that was, the gastric emptying rate, small intestinal propulsion ratio and levels of plasma MTL, CORT and CA were surpressed significantly (P < 0.01) while the content of CGRP was improved obviously (P < 0.01) in over-training group.

CONCLUSIONMelatonin plays an important role in protecting gastrointestinal tract from dysfunction, in which MTL, CGRP, CORT and CA are all involved.

Animals ; Calcitonin Gene-Related Peptide ; blood ; Catecholamines ; blood ; Fatigue ; Gastrointestinal Motility ; Hydrocortisone ; blood ; Melatonin ; pharmacology ; Motilin ; blood ; Physical Conditioning, Animal ; Rats ; Rats, Sprague-Dawley ; Stress, Physiological