Melatonin mediates protective effects on inflammatory response induced by interleukin‐1 beta in human mesenchymal stem cells

Joint diseases like osteoarthritis usually are accompanied with inflammatory processes, in which pro‐inflammatory cytokines mediate the generation of intracellular reactive oxygen species (ROS) and compromise survival of subchondral osteoblasts. Melatonin is capable of manipulating bone formation and osteogenic differentiation of mesenchymal stem cells (MSCs). The aim of this work was to investigate the anti‐inflammatory effect of melatonin on MSC proliferation and osteogenic differentiation in the absence or presence of interleukin‐1 beta (IL‐1β), which was used to induce inflammation. Our data showed that melatonin improved cell viability and reduced ROS generation in MSCs in a dose‐dependent manner. When exposed to 10 ng/mL IL‐1β, various concentrations of melatonin resulted in significant reduction of ROS by 34.9% averagely. Luzindole as a melatonin receptor antagonist reversed the anti‐oxidant effect of melatonin in MSCs with co‐exposure to IL‐1β. Real‐time RT‐PCR data suggested that melatonin treatment up‐regulated the expression of CuZnSOD and MnSOD, while down‐regulated the expression of Bax. To investigate the effect of melatonin on osteogenesis, MSCs were cultured in osteogenic differentiation medium supplemented with IL‐1β, melatonin, or luzindole. After exposed to IL‐1β for 21 days, 1 μm melatonin treatment significantly increased the levels of type I collagen, ALP, and osteocalcin, and 100 μm melatonin treatment yielded the highest level of osteopontin. Our study demonstrated that melatonin maintained MSC survival and promoted osteogenic differentiation in inflammatory environment induced by IL‐1β, suggesting melatonin treatment could be a promising method for bone regenerative engineering in future studies.     

The protective effect of melatonin on methamphetamine‐induced calpain‐dependent death pathway in human neuroblastoma SH‐SY5Y cultured cells

Abstract: Methamphetamine (METH) is a potent psychostimulant drug that may cause neuronal cell degeneration. The underlying mechanisms of METH‐induced neuronal toxicity remains poorly understood. In this study, we investigated an important role of calpain‐dependent cascades in methamphetamine‐induced toxicity in human dopaminergic neuroblastoma SH‐SY5Y cultured cell lines. In addition, the protective effect of melatonin against METH‐induced calpain‐dependent death pathway was also investigated. The results of this study show that METH significantly decreased cell viability and tyrosine hydroxylase phosphorylation in SH‐SY5Y cultured cells. Melatonin reversed the toxic effect of METH by inducing cell viability. In addition, melatonin was able to restore the reduction in mitochondrial function and phosphorylation of tyrosine hydroxylase in SH‐SY5Y treated cells. An induction of calpain expression and activity but a reduction of calpain inhibitor (calpastatin) protein levels were observed in SH‐SY5Y cells treated with METH but these effects were diminished by melatonin. These results implicated calpain‐dependent death pathways in the processes of METH‐induced toxicity and also indicated that melatonin has the capacity to reverse this toxic effect in SH‐SY5Y cultured cells.     

The mechanism for the neuroprotective effect of melatonin against methamphetamine‐induced autophagy

Abstract: Methamphetamine (METH) is a common drug of abuse that induces toxicity in the central nervous system and is connected to neurological disorders such as Parkinson’s disease. METH neurotoxicity is induced by reactive oxygen species (ROS) production and apoptosis. Moreover, autophagy is an alternative to cell death and a means for eliminating dysfunctional organelles. In other cases, autophagy can end up in cell death. Nonetheless, it is not clear whether autophagy is also correlated with apoptotic signaling in drug‐induced neurotoxicity. Therefore, we hypothesized that METH‐generated toxicity associated with initiating the apoptotic signaling cascade can also increase the autophagic phenotype in neuronal cells. Using the SK–N–SH dopaminergic cell line as our model system, we found that METH‐induced autophagy by inhibiting dissociation of Bcl‐2/Beclin 1 complex and its upstream pathway that thereby led to cell death. We uncovered a novel function for the anti‐apoptotic protein Bcl‐2, as it played a role in negatively regulating autophagy by blocking an essential protein in the signaling pathway, Beclin 1. Furthermore, Bcl‐2 was activated by c‐Jun N‐terminal kinase 1 (JNK 1), which is upstream of Bcl‐2 phosphorylation, to induce Bcl‐2/Beclin 1 dissociation. Furthermore, we demonstrated a novel role for melatonin in protecting cells from autophagic cell death triggered by the Bcl‐2/Beclin 1 pathway by inhibiting the activation of the JNK 1, Bcl‐2 upstream pathway. This study provides information regarding the link between apoptosis and autophagy signaling, which could lead to the development of therapeutic strategies that exploit the neurotoxicity of drugs of abuse.     

Melatonin: a well‐documented antioxidant with conditional pro‐oxidant actions

Melatonin (N‐acetyl‐5‐methoxytryptamine), an indoleamine produced in many organs including the pineal gland, was initially characterized as a hormone primarily involved in circadian regulation of physiological and neuroendocrine function. Subsequent studies found that melatonin and its metabolic derivatives possess strong free radical scavenging properties. These metabolites are potent antioxidants against both ROS (reactive oxygen species) and RNS (reactive nitrogen species). The mechanisms by which melatonin and its metabolites protect against free radicals and oxidative stress include direct scavenging of radicals and radical products, induction of the expression of antioxidant enzymes, reduction of the activation of pro‐oxidant enzymes, and maintenance of mitochondrial homeostasis. In both in vitro and in vivo studies, melatonin has been shown to reduce oxidative damage to lipids, proteins and DNA under a very wide set of conditions where toxic derivatives of oxygen are known to be produced. Although the vast majority of studies proved the antioxidant capacity of melatonin and its derivatives, a few studies using cultured cells found that melatonin promoted the generation of ROS at pharmacological concentrations (μm to mm range) in several tumor and nontumor cells; thus, melatonin functioned as a conditional pro‐oxidant. Mechanistically, melatonin may stimulate ROS production through its interaction with calmodulin. Also, melatonin may interact with mitochondrial complex III or mitochondrial transition pore to promote ROS production. Whether melatonin functions as a pro‐oxidant under in vivo conditions is not well documented; thus, whether the reported in vitro pro‐oxidant actions come into play in live organisms remains to be established.     

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 decreases muscular oxidative stress and inflammation induced by strenuous exercise and stimulates growth factor synthesis

Strenuous exercise is detrimental to athletes because of the overproduction of reactive oxygen species. Melatonin, a classic antioxidant, has been shown to exhibit beneficial effects regarding intense exercise and tissue repair. In this study, we evaluated the onset and resolution of inflammation in melatonin‐treated and nontreated rats subjected to a strenuous exercise session. We also analyzed the formation of thiobarbituric acid reactive substances (TBARS) and the activities of catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD). Control and treated rats were subjected to exhaustive exercise after a period of 10 days of melatonin treatment (20 mg/dL). Plasma and muscle levels of tumor necrosis factor‐alpha (TNF‐α), interleukin 1 beta (IL‐1β), interleukin 6 (IL‐6), cytokine‐induced neutrophil chemoattractant‐2‐alpha/beta (CINC‐2α/β), l‐selectin, macrophage inflammatory protein‐3‐alpha (MIP‐3α), and vascular endothelial growth factor (VEGF) were measured prior to, immediately after, and 2 hr after exercise. Our data revealed decreases in the muscle concentrations of IL‐1β (35%), TNF‐α (13%), IL‐6 (48%), and TBARS (40%) in the melatonin‐treated group compared with the control group. We also observed decreases in the plasma concentrations of IL‐1β (17%) in the melatonin‐treated group. VEGF‐α concentrations and SOD activity increased by 179% and 22%, respectively, in the melatonin‐treated group compared with the control group. We concluded that muscle inflammation and oxidative stress resulting from exhaustive exercise were less severe in the muscles of melatonin‐treated animals than in the muscles of control animals. Thus, melatonin treatment may reverse exercise‐induced skeletal muscle inflammation and stimulate growth factor synthesis.     

Melatonin attenuates TNF‐α and IL‐1β expression in synovial fibroblasts and diminishes cartilage degradation: Implications for the treatment of rheumatoid arthritis

The hormone melatonin has many properties, including antioxidant, anti‐inflammatory, and immunomodulatory effects. Melatonin has been demonstrated to be beneficial in several inflammatory autoimmune diseases, but its effects in rheumatoid arthritis (RA) remain controversial. We sought to determine how melatonin regulates inflammation in RA. We found that melatonin dose‐dependently inhibits tumor necrosis factor‐α (TNF‐α) and interleukin (IL)‐1β expression through the PI3K/AKT, ERK, and NF‐κB signaling pathways. We also identified that melatonin inhibits TNF‐α and IL‐1β production by upregulating miR‐3150a‐3p expression. Synovial tissue specimens from RA patients and culture of human rheumatoid fibroblast‐like synoviocytes confirmed that the MT1 receptor is needed for the anti‐inflammatory activities of melatonin. Importantly, melatonin also significantly reduced paw swelling, cartilage degradation, and bone erosion in the collagen‐induced arthritis mouse model. Our results indicate that melatonin ameliorates RA by inhibiting TNF‐α and IL‐1β production through downregulation of the PI3K/AKT, ERK, NF‐κB signaling pathways, as well as miR‐3150a‐3p overexpression. The role of melatonin as an adjuvant treatment in patients with RA deserves further clinical studies.     

Enhancement of high glucose‐induced PINK1 expression by melatonin stimulates neuronal cell survival: Involvement of MT2/Akt/NF‐κB pathway

Hyperglycemia is a representative hallmark and risk factor for diabetes mellitus (DM) and is closely linked to DM‐associated neuronal cell death. Previous investigators reported on a genome‐wide association study and showed relationships between DM and melatonin receptor (MT), highlighting the role of MT signaling by assessing melatonin in DM. However, the role of MT signaling in DM pathogenesis is unclear. Therefore, we investigated the role of mitophagy regulators in high glucose‐induced neuronal cell death and the effect of melatonin against high glucose‐induced mitophagy regulators in neuronal cells. In our results, high glucose significantly increased PTEN‐induced putative kinase 1 (PINK1) and LC‐3B expressions; as well it decreased cytochrome c oxidase subunit 4 expression and Mitotracker? fluorescence intensity. Silencing of PINK1 induced mitochondrial reactive oxygen species (ROS) accumulation and mitochondrial membrane potential impairment, increased expressions of cleaved caspases, and increased the number of annexin V‐positive cells. In addition, high glucose‐stimulated melatonin receptor 1B (MTNR1B) mRNA and PINK1 expressions were reversed by ROS scavenger N‐acetyl cysteine pretreatment. Upregulation of PINK1 expression in neuronal cells is suppressed by pretreatment with MT₂ receptor‐specific inhibitor 4‐P‐PDOT. We further showed melatonin stimulated Akt phosphorylation, which was followed by nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) phosphorylation and nuclear translocation. Silencing of PINK1 expression abolished melatonin‐regulated mitochondrial ROS production, cleaved caspase‐3 and caspase‐9 expressions, and the number of annexin V‐positive cells. In conclusion, we have demonstrated the melatonin stimulates PINK1 expression via an MT₂/Akt/NF‐κB pathway, and such stimulation is important for the prevention of neuronal cell apoptosis under high glucose conditions.     

Insulin and IGF1 enhance IL‐17‐induced chemokine expression through a GSK3B‐dependent mechanism: a new target for melatonin's anti‐inflammatory action

Obesity is a chronic inflammation with increased serum levels of insulin, insulin‐like growth factor 1 (IGF1), and interleukin‐17 (IL‐17). The objective of this study was to test a hypothesis that insulin and IGF1 enhance IL‐17‐induced expression of inflammatory chemokines/cytokines through a glycogen synthase kinase 3β (GSK3B)‐dependent mechanism, which can be inhibited by melatonin. We found that insulin/IGF1 and lithium chloride enhanced IL‐17‐induced expression of C‐X‐C motif ligand 1 (Cxcl1) and C‐C motif ligand 20 (Ccl20) in the Gsk3b^+/+, but not in Gsk3b^?/? mouse embryonic fibroblast (MEF) cells. IL‐17 induced higher levels of Cxcl1 and Ccl20 in the Gsk3b^?/? MEF cells, compared with the Gsk3b^+/+ MEF cells. Insulin and IGF1 activated Akt to phosphorylate GSK3B at serine 9, thus inhibiting GSK3B activity. Melatonin inhibited Akt activation, thus decreasing P‐GSK3B at serine 9 (i.e., increasing GSK3B activity) and subsequently inhibiting expression of Cxcl1 and Ccl20 that was induced either by IL‐17 alone or by a combination of insulin and IL‐17. Melatonin's inhibitory effects were only observed in the Gsk3b^+/+, but in not Gsk3b^?/? MEF cells. Melatonin also inhibited expression of Cxcl1, Ccl20, and Il‐6 that was induced by a combination of insulin and IL‐17 in the mouse prostatic tissues. Further, nighttime human blood, which contained high physiologic levels of melatonin, decreased expression of Cxcl1, Ccl20, and Il‐6 in the PC3 human prostate cancer xenograft tumors. Our data support our hypothesis and suggest that melatonin may be used to dampen IL‐17‐mediated inflammation that is enhanced by the increased levels of insulin and IGF1 in obesity.     

Melatonin ameliorates cerulein‐induced pancreatitis by the modulation of nuclear erythroid 2‐related factor 2 and nuclear factor‐kappaB in rats

Abstract: Melatonin exhibits a wide variety of biological effects, including antioxidant and anti‐inflammatory functions. Its antioxidant role impedes the etiopathogenesis of pancreatitis, but little is known about the signaling pathway of melatonin in the induction of antioxidant enzymes in acute pancreatitis (AP). The aim of this study was to determine whether melatonin could prevent cerulein‐induced AP through nuclear factor erythroid 2‐related factor 2 (Nrf2) and curtail inflammation by inhibition of NF‐κB. AP was induced by two intraperitoneal (i.p.) injections of cerulein at 2 h intervals (50 μg/kg) in Sprague‐Dawley rats. Melatonin (10 or 50 mg/kg/daily, i.p.) was administered 24 h before each injection of cerulein. The rats were killed 12 h after the last injection. Acinar cell degeneration, pancreatic edema, and inflammatory infiltration were significantly different in cerulein‐ and melatonin‐treated rats. Melatonin significantly reduced amylase, lipase, MPO, and MDA levels, and increased antioxidant enzyme activities including SOD and GPx, which were decreased in AP (P < 0.05). Melatonin increased the expression of NQO1, HO‐1, and SOD2 when compared with the cerulein‐induced AP group (P < 0.05). In addition, melatonin increased Nrf2 expression, and reduced expressions of tumor necrosis factor‐alpha, IL‐1β, IL‐6, IL‐8, and iNOS. The elevated nuclear binding of NF‐κB in the cerulein‐induced pancreatitis group was inhibited by melatonin. These results show that melatonin increases antioxidant enzymes and Nrf2 expression, and limits inflammatory mediators in cerulein‐induced AP. It is proposed that melatonin may play an important role in oxidative stress via the Nrf2 pathway in parallel with reduction of inflammation by NF‐κB inhibition.     

Melatonin modulates TLR4‐mediated inflammatory genes through MyD88‐ and TRIF‐dependent signaling pathways in lipopolysaccharide‐stimulated RAW264.7 cells

Abstract: Increasing evidence demonstrates that melatonin has an anti‐inflammatory effect. Nevertheless, the molecular mechanisms remain obscure. In this study, we investigated the effect of melatonin on toll‐like receptor 4 (TLR4)‐mediated molecule myeloid differentiation factor 88 (MyD88)‐dependent and TRIF‐dependent signaling pathways in lipopolysaccharide (LPS)‐stimulated macrophages. RAW264.7 cells were incubated with LPS (2.0 μg/mL) in the absence or presence of melatonin (10, 100, 1000 μm ). As expected, melatonin inhibited TLR4‐mediated tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐1β, IL‐6, IL‐8, and IL‐10 in LPS‐stimulated macrophages. In addition, melatonin significantly attenuated LPS‐induced upregulation of cyclooxygenase (COX)‐2 and inducible nitric oxide synthase (iNOS) in macrophages. Further analysis showed that melatonin inhibited the expression of MyD88 in LPS‐stimulated macrophages. Although it had no effect on TLR4‐mediated phosphorylation of c‐Jun N‐terminal kinase (JNK), p38, and extracellular regulated protein kinase (ERK), melatonin significantly attenuated the activation of nuclear factor kappa B (NF‐κB) in LPS‐stimulated macrophages. In addition, melatonin inhibited TLR4‐mediated Akt phosphorylation in LPS‐stimulated macrophages. Moreover, melatonin significantly attenuated the elevation of interferon (IFN)‐regulated factor‐3 (IRF3), which was involved in TLR4‐mediated TRIF‐dependent signaling pathway, in LPS‐stimulated macrophages. Correspondingly, melatonin significantly alleviated LPS‐induced IFN‐β in macrophages. In conclusion, melatonin modulates TLR4‐mediated inflammatory genes through MyD88‐dependent and TRIF‐dependent signaling pathways.     

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 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.     

Melatonin attenuates smoking‐induced hyperglycemia via preserving insulin secretion and hepatic glycogen synthesis in rats

Epidemiology survey indicated that cigarette smoking is a risk factor of diabetes. However, the precise mechanisms remain to be clarified. In this study, we found that smoking caused metabolic malfunctions on pancreas and liver in experimental animal model. These were indicated by hyperglycemia, increased serum hemoglobin A1c level and decreased insulin secretion, inhibition of liver glycogen synthase (LGS), and hepatic glycogen synthesis. Mechanistic studies revealed that all these alterations were caused by the inflammatory reaction and reactive oxygen species (ROS) induced by the smoking. Melatonin treatment significantly preserved the functions of both pancreas and liver by reducing β cell apoptosis, CD68‐cell infiltration, ROS production, and caspase‐3 expression. The siRNA‐knockdown model identified that the protective effects of melatonin were mediated by melatonin receptor‐2 (MT2). This study uncovered potentially underlying mechanisms related to the association between smoking and diabetes. In addition, it is, for first time, to report that melatonin effectively protects against smoking‐induced glucose metabolic alterations and the signal transduction pathway of melatonin is mainly mediated by its MT2 receptor. These observations provide solid evidence for the clinically use of melatonin to reduce smoking‐related diabetes, and the therapeutic regimens are absent currently.     

The role of mitochondrial complex III in melatonin-induced ROS production in cultured mesangial cells

Melatonin is a potent scavenger of reactive oxygen (ROS) and reactive nitrogen species (RNS). At pharmacological concentrations, however, melatonin is documented to cause ROS/RNS production, especially in cultured cancerous cells. Currently, the mechanism responsible for melatonin-induced ROS generation remains elusive. In this study, we provided evidence that melatonin, at micromolar concentrations, induced rapid ROS generation by a mitochondrial-dependent mechanism in primary human mesangial (HM) cells. The melatonin-induced ROS production occurred independent of changes in Ca(2+) concentrations in the cytosol and/or in mitochondria. In mitochondria isolated from HM cells and mice kidney tissues, melatonin caused ROS production; this melatonin response was completely blocked by the complex III inhibitor antimycin A. In contrast, both the mitochondrial complex I inhibitor, rotenone, and another complex III inhibitor, myxothiazol, which interacts with complex III at a distinct site, had no significant inhibitory effect on melatonin-induced ROS generation. These results demonstrate that melatonin induced rapid ROS generation via the antimycin A-sensitive site of mitochondrial complex III.     

Curcumin attenuates high glucose‐induced inflammatory injury through the reactive oxygen species–phosphoinositide 3‐kinase/protein kinase B–nuclear factor‐κB signaling pathway in rat thoracic aorta endothelial cells

Aims/Introduction

Endothelial cell inflammatory injury is likely required for barrier dysfunction under hyperglycemic conditions. Curcumin (CUR) is well known for its anti‐inflammatory effect. However, there have been few reports about the anti‐inflammatory effect of CUR induced by high glucose in endothelial cells. The aim of the present study was to investigate the inflammatory effect of high glucose and the anti‐inflammatory effect of CUR induced by high glucose in rat thoracic aorta endothelial cells (TAECs).

Materials and Methods

Well characterized TAECs were established and cell viability was assayed by the cell counting kit‐8 method, messenger ribonucleic acid and protein expression were identified by real‐time polymerase chain reaction, western blot or enzyme‐linked immunosorbent assay, respectively. The production of reactive oxygen species was observed by a fluorescence microscope.

Results

High glucose (30 mmol/L) significantly decreased the cell viability of TAECs after being co‐cultivated for 12 h and showed a time‐dependent manner, and increased interleukin (IL)‐1β, IL‐6 and tumor necrosis factor‐α secretion in TAECs. The injury effect of high glucose was involved in the reactive oxygen species–phosphoinositide 3‐kinase (PI3K)/protein kinase B (AKT)–nuclear factor (NF)‐κB signaling pathway. Anti‐oxidant N‐acetylcysteine, PI3K and NF‐κB‐specific pathway inhibitors can abolish the secretion of these inflammatory factors; pretreatment with anti‐oxidant N‐acetylcysteine significantly decreased PI3K expression, the level of phosphorylated AKT and nuclear NF‐κB; pretreatment of LY294002 can significantly decrease the NF‐κB level in nuclei. After treatment with CUR for 12 h, IL‐1β, IL‐6 and tumor necrosis factor‐α secretion were markedly decreased, and PI3K expression, the phosphorylation of AKT and nuclear NF‐κB level were also decreased.

Conclusion

Curcumin attenuates high glucose‐induced inflammatory injury through the reactive oxygen species–PI3K/AKT–NF‐κB signaling pathway in rat thoracic aorta endothelial cells.     

Melatonin sensitizes human cervical cancer HeLa cells to cisplatin‐induced cytotoxicity and apoptosis: effects on oxidative stress and DNA fragmentation

Melatonin has antitumor activity via several mechanisms including its antiproliferative and pro‐apoptotic effects as well as its potent antioxidant actions, although recent evidence has indicated that melatonin may perform pro‐oxidant actions in tumor cells. Therefore, melatonin may be useful in the treatment of tumors in association with chemotherapy drugs. This study was intended to evaluate the in vitro effect of melatonin on the cytotoxic and pro‐apoptotic actions of various chemotherapeutic agents in cervical cancer HeLa cells. Herein, we found that both melatonin and three of the chemotherapeutic drugs tested, namely cisplatin (CIS), 5‐fluorouracil (5‐FU), and doxorubicin, induced a decrease in HeLa cell viability. Furthermore, melatonin significantly increased the cytotoxic effect of such chemotherapeutic agents. Consistently, costimulation of HeLa cells with any chemotherapeutic agent in the presence of melatonin further increased caspase‐3 activation, particularly in CIS‐ and 5‐FU‐challenged cells. Likewise, concomitant treatments with melatonin and CIS significantly enhanced the ratio of cells entering mitochondrial apoptosis due to reactive oxygen species (ROS) overproduction, substantially augmented the population of apoptotic cells, and markedly enlarged DNA fragmentation compared to the treatments with CIS alone. Nonetheless, melatonin only displayed moderate chemosensitizing effects in 5‐FU‐stimulated HeLa cells, as suggested by slight increments in the percentage of cells stimulated for ROS production and in the proportion of early apoptotic cells compared to the treatments with 5‐FU alone. In summary, our findings provided evidence that in vitro melatonin strongly enhances CIS‐induced cytotoxicity and apoptosis in HeLa cells and, hence, the indoleamine could be potentially applied to cervical cancer treatment as a powerful synergistic agent.     

Melatonin attenuates traumatic brain injury‐induced inflammation: a possible role for mitophagy

Melatonin functions as a crucial mediator of sterile neuroinflammation; however, the underlying mechanisms remain poorly understood. Dysfunctional mitochondria, a main source of reactive oxygen species, are impacted in inflammation activation. This study aimed to examine the effect of melatonin on inflammation via elimination of damaged mitochondria after controlled cortical impact, an in vivo model of traumatic brain injury (TBI). Here, we demonstrated that inhibition of mitophagy, the selective degradation of damaged mitochondria by autophagy, markedly enhanced inflammation induced by TBI. Melatonin treatment activated mitophagy through the mTOR pathway, then to attenuate TBI‐induced inflammation. Furthermore, treatment with melatonin significantly ameliorated neuronal death and behavioral deficits after TBI, while 3‐methyladenine reversed this effect by inhibiting mitophagy. Taken together, these results highlight a role for melatonin in protecting against TBI‐triggered immunopathology, which is accomplished by negatively regulating inflammation activation and IL‐1β secretion via the autophagy of damaged mitochondria.