Melatonin reverses H2O2‐induced premature senescence in mesenchymal stem cells via the SIRT1‐dependent pathway

Mesenchymal stem cells (MSCs) represent an attractive source for stem cell‐based regenerative therapy, but they are vulnerable to oxidative stress‐induced premature senescence in pathological conditions. We previously reported antioxidant and antiarthritic effects of melatonin on MSCs against proinflammatory cytokines. In this study, we hypothesized that melatonin could protect MSCs from premature senescence induced by hydrogen peroxide (H₂O₂) via the silent information regulator type 1 (SIRT1)‐dependent pathway. In response to H₂O₂ at a sublethal concentration of 200 μm , human bone marrow‐derived MSCs (BM‐MSCs) underwent growth arrest and cellular senescence. Treatment with melatonin before H₂O₂ exposure cannot significantly prevent premature senescence; however, treatment with melatonin subsequent to H₂O₂ exposure successfully reversed the senescent phenotypes of BM‐MSCs in a dose‐dependent manner. This result was made evident by improved cell proliferation, decreased senescence‐associated β‐galactosidase activity, and the improved entry of proliferating cells into the S phase. In addition, treatment with 100 μm melatonin restored the osteogenic differentiation potential of BM‐MSCs that was inhibited by H₂O₂‐induced premature senescence. We also found that melatonin attenuated the H₂O₂‐stimulated phosphorylation of p38 mitogen‐activated protein kinase, decreased expression of the senescence‐associated protein p16^INK4α, and increased SIRT1. Further molecular experiments revealed that luzindole, a nonselective antagonist of melatonin receptors, blocked melatonin‐mediated antisenescence effects. Inhibition of SIRT1 by sirtinol counteracted the protective effects of melatonin, suggesting that melatonin reversed the senescence in cells through the SIRT1‐dependent pathway. Together, these findings lay new ground for understanding oxidative stress‐induced premature senescence and open perspectives for therapeutic applications of melatonin in stem cell‐based regenerative medicine.     

至真方调控M2巨噬细胞来源外泌体逆转大肠癌细胞耐药的机制研究

目的:探讨至真方通过调控M2巨噬细胞来源的外泌体逆转大肠癌细胞耐药的机制.方法:采取差异超速离心法分别提取M2巨噬细胞来源的外泌体以及100 μg/mL至真方醇提物预处理的M2型巨噬细胞来源的外泌体;用透射电子显微镜观察外泌体的形态,Western blot鉴定外泌体的标志蛋白;通过共培养体系将HCT116细胞分为3组...     

错配修复基因MLH1激活p53和线粒体凋亡通路参与雌激素诱导的结肠癌细胞凋亡

临床和流行病学研究显示雌激素替代治疗可降低绝经后妇女的结直肠癌发生风险。前期研究发现雌激素能上调结肠癌细胞的错配修复(MMR)基因MLH1表达,在MLH1基因缺失的结肠癌细胞中再表达MLH1能明显增强雌激素诱导的细胞凋亡。目的:探讨MLH1参与雌激素诱导结肠癌细胞凋亡所涉及的信号通路以及p53及其相关基因在此凋亡通路中的作用。方法:以含人野生型MLH1(hMLH1)全长cDNA的质粒转染MLH1基因缺失的人结肠癌细胞株HCT116。以转染空质粒的HCT1 16细胞作为对照,在有或无雌激素作用的条件下,采用电泳法检测凋亡DNA Ladder,蛋白质印迹法检测p53等凋亡相关蛋白表达。结果:转染hMLH1后,10~(-8)mol/L雌二醇(E_2)能明显诱导HCT116细胞凋亡。转染hMLH1并经E_2处理的HCT116细胞(D组)与经E_2处理但未转染hMLH1的HCT116细胞(B组)相比,其caspase-3、caspase-9、p53、Bax、胞质细胞色素C蛋白表达均显著增强,D组上述蛋白表达亦均高于转染hMLH1但未经E_2处理的HCT116细胞(C组)。结论:MMR基因MLH1主要通过激活p53和线粒体凋亡通路参与雌激素诱导的人结肠癌细胞株HCT116凋亡。     

Fluid shear stress and melatonin in combination activate anabolic proteins in MC3T3‐E1 osteoblast cells

In this study, we investigated whether fluid shear stress and melatonin in combination stimulate the anabolic proteins through the phosphorylation of extracellular signal‐regulated kinase (p‐ERK) in MC3T3‐E1 osteoblast cells. First, we researched why fluid shear stress and melatonin in combination influence cell survival. Fluid shear stress (1 hr) and melatonin (1 mm ) in combination reduced autophagic marker LC3‐II compared with fluid shear stress (1 hr) and/or melatonin (0.1 mm ). Under the same conditions for fluid shear stress, markers of cell survival signaling pathway p‐ERK, phosphorylation of serine‐threonine protein kinase (p‐Akt), phosphorylation of mammalian target of rapamycin (p‐mTOR), and p85‐S6K were investigated. p‐Akt, p‐mTOR (Ser 2481) expressions increased with the addition of 1 mm melatonin prior to 0.1 mm melatonin treatment. However, p‐S6K expression did not change significantly. Next, mitochondria activity including Bcl‐2, Bax, catalase, and Mn‐superoxide dismutase (Mn‐SOD) were studied. Expressions of Bcl‐2, Bax, and catalase proteins were low under fluid shear stress plus 1 mm melatonin compared with only fluid shear stress alone, whereas Mn‐SOD expression was high compared with conditions of no fluid shear stress. Finally, the anabolic proteins of bone, osteoprotegerin, type I collagen (collagen I), and bone sialoprotein II (BSP II) were checked. These proteins increased with combined fluid shear stress (1, 4 hr) and melatonin (0.1, 1 mm ). Together, these results suggest that fluid shear stress and melatonin in combination may increase the expression of anabolic proteins through the p‐ERK in MC3T3‐E1 osteoblast cells. Therefore, fluid shear stress in combination with melatonin may promote the anabolic response of osteoblasts.     

Melatonin‐enhanced autophagy protects against neural apoptosis via a mitochondrial pathway in early brain injury following a subarachnoid hemorrhage

Melatonin is a strong antioxidant that has beneficial effects against early brain injury (EBI) following a subarachnoid hemorrhage (SAH) in rats; protection includes reduced mortality and brain water content. The molecular mechanisms underlying these clinical effects in the SAH model, however, have not been clearly identified. This study was undertaken to determine the influence of melatonin on neural apoptosis and the potential mechanism of these effects in EBI following SAH using the filament perforation model of SAH in male Sprague Dawley rats. Melatonin (150 mg/kg) or vehicle was given via an intraperitoneal injection 2 hr after SAH induction. Brain samples were extracted 24 hr after SAH. The results show that melatonin treatment markedly reduced caspase‐3 activity and the number of TUNEL‐positive cells, while the treatment increased the LC3‐II/LC3‐I, an autophagy marker, which indicated that melatonin‐enhanced autophagy ameliorated apoptotic cell death in rats subjected to SAH. To further identify the mechanism of autophagy protection, we demonstrated that melatonin administration reduced Bax translocation to the mitochondria and the release of cytochrome c into the cytosol. Taken together, this report demonstrates that melatonin improved the neurological outcome in rats by protecting against neural apoptosis after the induction of filament perforation SAH; moreover, the mechanism of these antiapoptosis effects was related to the enhancement of autophagy, which ameliorated cell apoptosis via a mitochondrial pathway.     

Melatonin suppresses doxorubicin‐induced premature senescence of A549 lung cancer cells by ameliorating mitochondrial dysfunction

Abstract: Melatonin is an indolamine that is synthesized in the pineal gland and shows a wide range of physiological functions. Although the anti‐aging properties of melatonin have been reported in a senescence‐accelerated mouse model, whether melatonin modulates cellular senescence has not been determined. In this study, we examined the effect of melatonin on anticancer drug‐induced cellular premature senescence. We found that the doxorubicin (DOX)‐induced senescence of A549 human lung cancer cells and IMR90 normal lung cells was substantially inhibited by cotreatment with melatonin in a dose‐dependent manner. Mechanistically, the DOX‐induced G2/M phase cell cycle arrest and the decrease in cyclinB and cdc2 expression were not affected by melatonin. However, the DOX‐induced increase in intracellular levels of ROS, which is necessary for premature senescence, was completely abolished upon melatonin cotreatment. In addition, the reduction in mitochondrial membrane potential that occurs upon DOX treatment was inhibited by melatonin. An aberrant increase in mitochondrial respiration was also significantly suppressed by melatonin, indicating that melatonin ameliorates the mitochondrial dysfunction induced by DOX treatment. The treatment of A549 cells with luzindole, a potent inhibitor of melatonin receptors, failed to prevent the effects of melatonin treatment on mitochondrial functions and premature senescence in cells also treated with DOX; this suggests that melatonin suppresses DOX‐induced senescence in a melatonin receptor‐independent manner. Together, these results reveal that melatonin has an inhibitory effect of melatonin on premature senescence at the cellular level and that melatonin protects A549 cells from DOX‐induced senescence. Thus, melatonin might have the therapeutic potential to prevent the side effects of anticancer drug therapy.     

Melatonin attenuates methamphetamine‐induced inhibition of proliferation of adult rat hippocampal progenitor cells in vitro

Methamphetamine (METH) is an extremely addictive stimulatory drug. A recent study suggested that METH may cause an impairment in the proliferation of hippocampal neural progenitor cells, but the underlying mechanism of this effect remains unknown. Blood and cerebrospinal levels of melatonin derive primarily from the pineal gland, and that performs many biological functions. Our previous study demonstrated that melatonin promotes the proliferation of progenitor cells originating from the hippocampus. In this study, hippocampal progenitor cells from adult Wistar rats were used to determine the effects of METH on cell proliferation and the mechanisms underlying these effects. We investigated the effects of melatonin on the METH‐induced alteration in cell proliferation. The results demonstrated that 500 μm METH induced a decrease (63.0%) in neurosphere cell proliferation and altered the expression of neuronal phenotype markers in the neurosphere cell population. Moreover, METH induced an increase in the protein expression of the tumor suppressor p53 (124.4%) and the cell cycle inhibitor p21^CIP1 (p21) (128.1%), resulting in the accumulation of p21 in the nucleus. We also found that METH altered the expression of the N‐methyl‐d ‐aspartate (NMDA) receptor subunits NR2A (79.6%) and NR2B (126.7%) and Ca²⁺/calmodulin‐dependent protein kinase II (CAMKII) (74.0%). In addition, pretreatment with 1 μm melatonin attenuated the effects induced by METH treatment. According to these results, we concluded that METH induces a reduction in cell proliferation by upregulating the cell cycle regulators p53/p21 and promoting the accumulation of p21 in the nucleus and that melatonin ameliorates these negative effects of METH.     

Additional benefit of combined therapy with melatonin and apoptotic adipose‐derived mesenchymal stem cell against sepsis‐induced kidney injury

This study tested whether combined therapy with melatonin and apoptotic adipose‐derived mesenchymal stem cells (A‐ADMSCs) offered additional benefit in ameliorating sepsis‐induced acute kidney injury. Adult male Sprague–Dawley rats (n = 65) were randomized equally into five groups: Sham controls (SC), sepsis induced by cecal‐ligation and puncture (CLP), CLP‐melatonin, CLP‐A‐ADMSC, and CLP‐melatonin‐A‐ADMSC. Circulating TNF‐α level at post‐CLP 6 hr was highest in CLP and lowest in SC groups, higher in CLP‐melatonin than in CLP‐A‐ADMSC and CLP‐melatonin‐A‐ADMSC groups (all P < 0.001). Immune reactivity as reflected in the number of splenic helper‐, cytoxic‐, and regulatory‐T cells at post‐CLP 72 hr exhibited the same pattern as that of circulating TNF‐α among all groups (P < 0.001). The histological scoring of kidney injury and the number of F4/80+ and CD14+ cells in kidney were highest in CLP and lowest in SC groups, higher in CLP‐melatonin than in CLP‐A‐ADMSC and CLP‐melatonin‐A‐ADMSC groups, and higher in CLP‐A‐ADMSC than in CLP‐melatonin‐A‐ADMSC groups (all P < 0.001). Changes in protein expressions of inflammatory (RANTES, TNF‐1α, NF‐κB, MMP‐9, MIP‐1, IL‐1β), apoptotic (cleaved caspase 3 and PARP, mitochondrial Bax), fibrotic (Smad3, TGF‐β) markers, reactive‐oxygen‐species (NOX‐1, NOX‐2), and oxidative stress displayed a pattern identical to that of kidney injury score among the five groups (all P < 0.001). Expressions of antioxidants (GR+, GPx+, HO‐1, NQO‐1+) were lowest in SC group and highest in CLP‐melatonin‐A‐ADMSC group, lower in CLP than in CLP‐melatonin and CLP‐A‐ADMSC groups, and lower in CLP‐melatonin‐ than in CLP‐A‐ADMSC‐tretaed animals (all P < 0.001). In conclusion, combined treatment with melatonin and A‐ADMSC was superior to A‐ADMSC alone in protecting the kidneys from sepsis‐induced injury.     

Melatonin limits paclitaxel‐induced mitochondrial dysfunction in vitro and protects against paclitaxel‐induced neuropathic pain in the rat

Chemotherapy‐induced neuropathic pain is a debilitating and common side effect of cancer treatment. Mitochondrial dysfunction associated with oxidative stress in peripheral nerves has been implicated in the underlying mechanism. We investigated the potential of melatonin, a potent antioxidant that preferentially acts within mitochondria, to reduce mitochondrial damage and neuropathic pain resulting from the chemotherapeutic drug paclitaxel. In vitro, paclitaxel caused a 50% reduction in mitochondrial membrane potential and metabolic rate, independent of concentration (20‐100 μmol/L). Mitochondrial volume was increased dose‐dependently by paclitaxel (200% increase at 100 μmol/L). These effects were prevented by co‐treatment with 1 μmol/L melatonin. Paclitaxel cytotoxicity against cancer cells was not affected by co‐exposure to 1 μmol/L melatonin of either the breast cancer cell line MCF‐7 or the ovarian carcinoma cell line A2780. In a rat model of paclitaxel‐induced painful peripheral neuropathy, pretreatment with oral melatonin (5/10/50 mg/kg), given as a daily bolus dose, was protective, dose‐dependently limiting development of mechanical hypersensitivity (19/43/47% difference from paclitaxel control, respectively). Melatonin (10 mg/kg/day) was similarly effective when administered continuously in drinking water (39% difference). Melatonin also reduced paclitaxel‐induced elevated 8‐isoprostane F₂α levels in peripheral nerves (by 22% in sciatic; 41% in saphenous) and limited paclitaxel‐induced reduction in C‐fibre activity‐dependent slowing (by 64%). Notably, melatonin limited the development of mechanical hypersensitivity in both male and female animals (by 50/41%, respectively), and an additive effect was found when melatonin was given with the current treatment, duloxetine (75/62% difference, respectively). Melatonin is therefore a potential treatment to limit the development of painful neuropathy resulting from chemotherapy treatment.     

Melatonin enhances hyperthermia‐induced apoptotic cell death in human leukemia cells

Melatonin is an endogenous indoleamine with a wide range of biological functions. In addition to modulating circadian rhythms, it plays important roles in the health as an antioxidant. Melatonin has also the ability to induce apoptosis in cancer cells and to enhance the antitumoral activity of chemotherapeutic agents. In this study, the effect of melatonin on hyperthermia‐induced apoptosis was explored using human leukemia cells. The results demonstrate that melatonin greatly improved the cytotoxicity of hyperthermia in U937 cells. The potentiation of cell death was achieved with 1 mmol/L concentrations of the indoleamine but not with concentrations close to physiological levels in blood (1 nmol/L). This effect was associated to an enhancement of the apoptotic response, revealed by an increase in cells with hypodiploid DNA content and activation of multiple caspases (caspase‐2, caspase‐3, caspase‐8, and caspase‐9). Melatonin also increased hyperthermia‐induced Bid activation as well as translocation of Bax from the cytosol to mitochondria and cytochrome c release. Hyperthermia‐provoked apoptosis and potentiation by melatonin were abrogated by a broad‐spectrum caspase inhibitor (z‐VAD‐fmk) as well as by specific inhibitors against caspase‐8 or caspase‐3. In contrast, blocking of the mitochondrial pathway of apoptosis either with a caspase‐9 inhibitor or overexpressing the anti‐apoptotic protein Bcl‐2 (U937/Bcl‐2) reduced the number of apoptotic cells in response to hyperthermia but it was unable to suppress melatonin enhancement. Melatonin appears to modulate the apoptotic response triggered by hyperthermia in a cell type‐specific manner as similar results were observed in HL‐60 but not in K562 or MOLT‐3 cells.     

Melatonin enhances L‐DOPA therapeutic effects,helps to reduce its dose,and protects dopaminergic neurons in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐induced parkinsonism in mice

L‐3,4‐dihydroxyphenylalanine (L‐DOPA) reduces symptoms of Parkinson's disease (PD), but suffers from serious side effects on long‐term use. Melatonin (10–30 mg/kg, 6 doses at 10 hr intervals) was investigated to potentiate L‐DOPA therapeutic effects in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced parkinsonism in mice. Striatal tyrosine hydroxylase (TH) immunoreactivity, TH, and phosphorylated ser 40 TH (p‐TH) protein levels were assayed on 7th day. Nigral TH‐positive neurons stereology was conducted on serial sections 2.8 mm from bregma rostrally to 3.74 mm caudally. MPTP caused 39% and 58% decrease, respectively, in striatal fibers and TH protein levels, but 2.5‐fold increase in p‐TH levels. About 35% TH neurons were lost between 360 and 600 μm from 940 μm of the entire nigra analyzed, but no neurons were lost between 250 μm rostrally and 220 μm caudally. When L‐DOPA in small doses (5–8 mg/kg) failed to affect MPTP‐induced akinesia or catalepsy, co‐administration of melatonin with L‐DOPA attenuated these behaviors. Melatonin administration significantly attenuated MPTP‐induced loss in striatal TH fibers (82%), TH (62%) and p‐TH protein (100%) levels, and nigral neurons (87–100%). Melatonin failed to attenuate MPTP‐induced striatal dopamine depletion. L‐DOPA administration (5 mg/kg, once 40 min prior to sacrifice, p.o.) in MPTP‐ and melatonin‐treated mice caused significant increase in striatal dopamine (31%), as compared to L‐DOPA and MPTP‐treated mice. This was equivalent to 8 mg/kg L‐DOPA administration in parkinsonian mouse. Therefore, prolonged, effective use of L‐DOPA in PD with lesser side effects could be achieved by treating with 60% lower doses of L‐DOPA along with melatonin.     

Neuroprotective effect of melatonin against hypoxia‐induced retinal ganglion cell death in neonatal rats

The purpose of this study was to determine whether melatonin treatment would mitigate retinal ganglion cell (RGC) death in the developing retina following a hypoxic insult. Lipid peroxidation (LPO), glutathione (GSH), tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) concentrations, expression of vascular endothelial growth factor receptors, Flt‐1 and Flk‐1, release of cytochrome c from mitochondria, and caspase‐3 expression were examined in the retinas of 1‐day‐old rats at 3 hr to 14 days after a hypoxic exposure. The mRNA and protein expression of Flt‐1 and Flk‐1 and the tissue concentration of LPO, TNF‐α, and IL‐1β were upregulated significantly after the hypoxic exposure, whereas the content of GSH was decreased significantly. RGC cultures also showed increased LPO and decreased GSH levels after hypoxic exposure but these effects were reversed in cells treated with melatonin. TNF‐α and IL‐1β expression was specifically located on microglial cells, whereas Flt‐1 and Flk‐1 was limited to RGCs as confirmed by double immunofluorescence labeling. Cultures of hypoxic microglial cells treated with melatonin showed a significant reduction in the release of these cytokines as compared to untreated hypoxic cells. Hypoxia induced increase in the cytosolic cytochrome c and caspase‐3 in RGCs was attenuated with melatonin treatment. The results suggest that, in hypoxic injuries, melatonin is neuroprotective to RGCs in the developing retina through its antioxidative, anti‐inflammatory, and anti‐apoptotic effects. Melatonin suppressed Flt‐1 and Flk‐1 expression in retinal blood vessels, which may result in reduced retinal vascular permeability and it also preserved mitochondrial function as shown by a reduction in cytochrome c leakage into the cytosol. The results may have therapeutic implications for the management of retinopathy of prematurity.     

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

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

Melatonin synergizes with low doses of L‐DOPA to improve dendritic spine density in the mouse striatum in experimental Parkinsonism

The dopamine precursor, L‐3,4‐dihydroxyphenylalanine (L‐DOPA), is the preferred drug for Parkinson's disease, but long‐term treatment results in the drug‐induced dyskinesias and other side effects. This study was undertaken to examine whether melatonin could potentiate low dose L‐DOPA effects in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced experimental parkinsonism. Mice were treated with the parkinsonian neurotoxin, MPTP, and different doses of melatonin and low doses of L‐DOPA. Behavior, striatal histology, and dopamine metabolism were evaluated on the 7th day. MPTP‐induced striatal dopamine loss was not modified by melatonin administration (10–30 mg/kg; i.p. at 10‐hr intervals, 6 times; or at 2‐hr intervals, by day). However, low doses of L‐DOPA (5 mg/kg, by oral gavage) administered alone or along with melatonin (10 mg/kg, i.p.) twice everyday for 2 days, 10 hr apart, after two doses of MPTP significantly attenuated striatal dopamine loss and provided improvements in both catalepsy and akinesia. Additionally, Golgi‐impregnated striatal sections showed preservation of the medium spiny neurons, which have been damaged in MPTP‐treated mouse. The results demonstrated that melatonin, but not L‐DOPA, restored spine density and spine morphology of medium spiny neurons in the striatum and suggest that melatonin could be an ideal adjuvant to L‐DOPA therapy in Parkinson's disease, and by the use of this neurohormone, it is possible to bring down the therapeutic doses of L‐DOPA.     

Neuroprotective effect of melatonin against ischemia is partially mediated by alpha‐7 nicotinic receptor modulation and HO‐1 overexpression

Melatonin has been widely studied as a protective agent against oxidative stress. However, the molecular mechanisms underlying neuroprotection in neurodegeneration and ischemic stroke are not yet well understood. In this study, we evaluated the neuroprotective/antioxidant mechanism of action of melatonin in organotypic hippocampal cultures (OHCs) as well as in photothrombotic stroke model in vivo. Melatonin (0.1, 1, and 10 μm ) incubated postoxygen and glucose deprivation (OGD) showed a concentration‐dependent protection; maximum protection was achieved at 10 μm (90% protection). Next, OHCs were exposed to 10 μm melatonin at different post‐OGD times; the protective effect of melatonin was maintained at 0, 1, and 2 hr post‐OGD treatment, but it was lost at 6 hr post‐OGD. The protective effect of melatonin and the reduction in OGD‐induced ROS were prevented by luzindole (melatonin antagonist) and α‐bungarotoxin (α‐Bgt, a selective α7 nAChR antagonist). In Nrf2 knockout mice, the protective effect of melatonin was reduced by 40% compared with controls. Melatonin, incubated 0, 1, and 2 hr post‐OGD, increased the expression of heme oxygenase‐1 (HO‐1), and this overexpression was prevented by luzindole and α‐bungarotoxin. Finally, administration of 15 mg/kg melatonin following the induction of photothrombotic stroke in vivo, reduced infarct size (50%), and improved motor skills; this effect was partially lost in 0.1 mg/kg methyllycaconitine (MLA, selective α7 nAChR antagonist)‐treated mice. Taken together, these results demonstrate that postincubation of melatonin provides a protective effect that, at least in part, depends on nicotinic receptor activation and overexpression of HO‐1.     

Protective effect of melatonin on TNF‐α‐induced muscle atrophy in L6 myotubes

Muscle atrophy, characterized by decreased cell number and size, is a serious concern for patients afflicted with inflammatory diseases. Mounting evidence indicates that tumor necrosis factor alpha (TNF‐α) plays a critical role in muscle atrophy in a number of clinical settings. We hypothesize that reactive oxygen species (ROS) mediate TNF‐α‐induced muscle cell death and hypotrophy. Recently, melatonin has attracted attention because of its free‐radical scavenging and antioxidant properties. The aim of the current study was to evaluate the possible protective role of melatonin in TNF‐α‐induced muscle cell death and hypotrophy in rat L6 myotubes. To examine this possible role, L6 myotubes were exposed to various concentrations of recombinant TNF‐α for 24 hr. We found that TNF‐α at a concentration of 100 ng/mL induced ROS generation and decreased cell viability. Further analysis revealed that apoptosis, but not autophagy, may be important for TNF‐α‐induced cell death. Melatonin significantly attenuated TNF‐α‐induced ROS generation and apoptosis. In addition, decreased muscle fiber diameter and increased muscle cell proteolysis by TNF‐α was highly attenuated by treatment with melatonin. The effects of melatonin were mediated neither through its plasmalemmal receptors nor by modulating the nuclear factor kappa B pathway activated by TNF‐α. Taken together, these results suggest that TNF‐α may mediate ROS‐induced muscle cell death and hypotrophy and that melatonin may be a useful tool for protecting against muscle atrophy stemming from inflammatory diseases.     

Melatonin promotes seed germination under high salinity by regulating antioxidant systems,ABA and GA4 interaction in cucumber (Cucumis sativus L.)

Although previous studies have found that melatonin can promote seed germination, the mechanisms involved in perceiving and signaling melatonin remain poorly understood. In this study, it was found that melatonin was synthesized during cucumber seed germination with a peak in melatonin levels occurring 14 hr into germination. This is indicative of a correlation between melatonin synthesis and seed germination. Meanwhile, seeds pretreated with exogenous melatonin (1 μm ) showed enhanced germination rates under 150 mm NaCl stress compared to water‐pretreated seeds under salinity stress. There are two apparent mechanisms by which melatonin alleviated salinity‐induced inhibition of seed germination. Exogenous melatonin decreased oxidative damage induced by NaCl stress by enhancing gene expression of antioxidants. Under NaCl stress, compared to untreated control, the activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were significantly increased by approximately 1.3–5.0‐fold, with a concomitant 1.4–2.0‐fold increase of CsCu‐ZnSOD, CsFe‐ZnSOD, CsCAT, and CsPOD in melatonin‐pretreated seeds. Melatonin also alleviated salinity stress by affecting abscisic acid (ABA) and gibberellin acid (GA) biosynthesis and catabolism during seed germination. Compared to NaCl treatment, melatonin significantly up‐regulated ABA catabolism genes (e.g., CsCYP707A1 and CsCYP707A2, 3.5 and 105‐fold higher than NaCl treatment at 16 hr, respectively) and down‐regulated ABA biosynthesis genes (e.g., CsNECD2, 0.29‐fold of CK2 at 16 hr), resulting in a rapid decrease of ABA content during the early stage of germination. At the same time, melatonin positively up‐regulated GA biosynthesis genes (e.g., GA20ox and GA3ox, 2.3 and 3.9‐fold higher than NaCl treatment at 0 and 12 hr, respectively), contributing to a significant increase of GA (especially GA₄) content. In this study, we provide new evidence suggesting that melatonin alleviates the inhibitory effects of NaCl stress on germination mainly by regulating the biosynthesis and catabolism of ABA and GA₄.