Melatonin improves neuroplasticity by upregulating the growth‐associated protein‐43 (GAP‐43) and NMDAR postsynaptic density‐95 (PSD‐95) proteins in cultured neurons exposed to glutamate excitotoxicity and in rats subjected to transient focal cerebral ischemia even during a long‐term recovery period

Recent evidence shows that the NMDAR postsynaptic density‐95 (PSD‐95), growth‐associated protein‐43 (GAP‐43), and matrix metalloproteinase‐9 (MMP‐9) protein enhance neuroplasticity at the subacute stage of stroke. Here, we evaluated whether melatonin would modulate the PSD‐95, GAP‐43, and MMP‐9 proteins in cultured neurons exposed to glutamate excitotoxicity and in rats subjected to experimental stroke. Adult male Sprague–Dawley rats were treated with melatonin (5 mg/kg) or vehicle at reperfusion onset after transient occlusion of the right middle cerebral artery (tMCAO) for 90 min. Animals were euthanized for Western immunoblot analyses for the PSD‐95 and GAP‐43 proteins and gelatin zymography for the MMP‐9 activity at 7 days postinsult. Another set of animals was sacrificed for histologic and Golgi–Cox‐impregnated sections at 28 days postinsult. In cultured neurons exposed to glutamate excitotoxicity, melatonin significantly upregulated the GAP‐43 and PSD‐95 expressions and improved dendritic aborizations (P < 0.05, respectively). Relative to controls, melatonin‐treated stroke animals caused a significant improvement in GAP‐43 and PSD‐95 expressions as well as the MMP‐9 activity in the ischemic brain (P < 0.05). Consequently, melatonin also significantly promoted the dendritic spine density and reduced infarction in the ischemic brain, and improved neurobehaviors as well at 28 days postinsult (P < 0.05, respectively). Together, melatonin upregulates GAP‐43, PSD‐95, and MMP‐9 proteins, which likely accounts for its actions to improve neuroplasticity in cultured neurons exposed to glutamate excitotoxicity and to enhance long‐term neuroprotection, neuroplasticity, and brain remodeling in stroke rats.     

Phosphorylation of extracellular signal‐regulated kinase 1/2, p38 mitogen‐activated protein kinase and nuclear translocation of nuclear factor‐κB are involved in upregulation of matrix metalloproteinase‐9 by tumour necrosis factor‐α

Background: Upregulation of matrix metalloproteinase‐9 (MMP‐9) induced by tumour necrosis factor‐α (TNF‐α) is reportedly involved in a variety of non‐neoplastic and neoplastic diseases. In this study, we examined which signalling pathways are involved in TNF‐α‐induced MMP‐9 upregulation in cholangiocarcinoma (CC). Methods: We used two CC cell lines: HuCCT‐1 and CCKS‐1. Results: In an ex vivo study using HuCCT‐1 and CCKS‐1 cells, TNF‐α treatment induced MMP‐9 production and activation via interaction with TNF receptor‐1 (TNF‐R1) but not with TNF receptor‐2 (TNF‐R2), shown by zymography, and increased MMP‐9 promoter activity (luciferase assay). As for the signalling pathway, TNF‐α stimulation led to the phosphorylation of extracellular signal‐regulated kinase 1/2 (Erk1/2) and p38 mitogen‐activated protein kinase (p38MAPK) and translocation of nuclear factor κB (NF‐κB) (p65) into the nuclei. Inhibition studies using SB203580 (inhibitor of p38MAPK), U0126 (inhibitor of mitogen‐activated or extracellular signal‐regulated protein kinase 1/2) and MG132 (inhibitor of NF‐κB) showed that the phosphorylation of Erk1/2 and p38MAPK with activation of NF‐κB was closely related to MMP‐9 upregulation in both cell lines. Conclusion: These data suggest that TNF‐α/TNF‐R1 interaction leads to the phosphorylation of Erk1/2 and p38MAPK and nuclear translocation of NF‐κB, which is closely associated with the production and activation of MMP‐9 in cultured CC cells of HuCTT‐1 and CCKS‐1. Upregulation of MMP‐9 with NF‐κB activation may be involved in the tumour invasion of CC.     

Melatonin overcomes gemcitabine resistance in pancreatic ductal adenocarcinoma by abrogating nuclear factor‐κB activation

Constitutive activation and gemcitabine induction of nuclear factor‐κB (NF‐κB) contribute to the aggressive behavior and chemotherapeutic resistance of pancreatic ductal adenocarcinoma (PDAC). Thus, targeting the NF‐κB pathway has proven an insurmountable challenge for PDAC therapy. In this study, we investigated whether the inhibition of NF‐κB signaling pathway by melatonin might lead to tumor suppression and overcome gemcitabine resistance in pancreatic tumors. Our results showed that melatonin inhibited activities of NF‐κB by suppressing IκBα phosphorylation and decreased the expression of NF‐κB response genes in MiaPaCa‐2, AsPc‐1, Panc‐28 cells and gemcitabine resistance MiaPaCa‐2/GR cells. Moreover, melatonin not only inhibited cell proliferation and invasion in a receptor‐independent manner, but also enhanced gemcitabine cytotoxicity at pharmacologic concentrations in these PDAC cells. In vivo, the mice treated with both agents experienced a larger reduction in tumor burden than the single drug‐treated groups in an orthotopic xenograft mouse model. Taken together, these results indicate that melatonin inhibits proliferation and invasion of PDAC cells and overcomes gemcitabine resistance of pancreatic tumors through NF‐κB inhibition. Our findings therefore provide novel preclinical knowledge about melatonin inhibition of NF‐κB in PDAC and suggest that melatonin should be investigated clinically, alone or in combination with gemcitabine for PDAC treatment.     

Melatonin potentiates the antiproliferative and pro‐apoptotic effects of ursolic acid in colon cancer cells by modulating multiple signaling pathways

Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid, is largely distributed in medical herbs and edible plants. Melatonin is an indoleamine compound produced in the pineal gland and also a plant‐derived product. Both UA and melatonin have been shown to inhibit cancer cell growth in numerous studies, but they have never been combined altogether as an anticolon cancer treatment. In this study, we investigated whether the association between UA and melatonin leads to an enhanced antiproliferative and pro‐apoptotic activities in colon cancer SW480 and LoVo cells. We found that combined treatment with UA and melatonin significantly enhanced inhibition of cell viability and migration, promoted changes in cell morphology and spreading, and increased induction of apoptosis, thereby potentiating the effects of UA alone in colon cancer cells. Moreover, we found that the enhanced effects of UA and melatonin combination are mediated through simultaneous modulation of cytochrome c/caspase, MMP9/COX‐2, and p300/NF‐κB signaling pathways. Combined treatment with UA and melatonin triggered the release of cytochrome c from the mitochondrial intermembrane space into the cytosol, induced cleavage of caspase and PARP proteins, enhanced inhibition of MMP9 and COX‐2 expression, promoted p300 and NF‐κB translocation from cell nuclei to cytoplasm, and abrogated NF‐κB binding and p300 recruitment to COX‐2 promoter in colon cancer cells. These results, therefore, demonstrated that melatonin potentiated the antiproliferative and pro‐apoptotic effects of UA in colon cancer cells by modulating multiple signaling pathways and suggest that such a combinational treatment might potentially become an effective way in colon cancer therapy.     

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.     

Melatonin inhibits the sphingosine kinase 1/sphingosine‐1‐phosphate signaling pathway in rabbits with fulminant hepatitis of viral origin

The sphingosine kinase (SphK)1/sphingosine‐1‐phosphate (S1P) pathway is involved in multiple biological processes, including liver diseases. This study investigate whether modulation of the SphK1/S1P system associates to the beneficial effects of melatonin in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2 × 10⁴ hemagglutination units of a RHDV isolate and received 20 mg/kg of melatonin at 0, 12, and 24 hr postinfection. Liver mRNA levels, protein concentration, and immunohistochemical labeling for SphK1 increased in RHDV‐infected rabbits. S1P production and protein expression of the S1PR1 receptor were significantly elevated following RHDV infection. These effects were significantly reduced by melatonin. Rabbits also exhibited increased expression of toll‐like receptor (TLR)4, tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐6, nuclear factor‐kappa B (NF‐κB) p50 and p65 subunits, and phosphorylated inhibitor of kappa B (IκB)α. Melatonin administration significantly inhibited those changes and induced a decreased immunoreactivity for RHDV viral VP60 antigen in the liver. Results obtained indicate that the SphK1/S1P system activates in parallel to viral replication and the inflammatory process induced by the virus. Inhibition of the lipid signaling pathway by the indole reveals novel molecular pathways that may account for the protective effect of melatonin in this animal model of ALF, and supports the potential of melatonin as an antiviral agent.     

(Z)2-(5-(4-methoxybenzylidene)-2, 4-dioxothiazolidin-3-yl) acetic acid protects rats from CCl(4) -induced liver injury

Background and Aim: (Z)2‐(5‐(4‐methoxybenzylidene)‐2, 4‐dioxothiazolidin‐3‐yl) acetic acid (MDA) is an aldose reductase (AR) inhibitor. Recent studies suggest that AR contributes to the pathogenesis of inflammation by affecting the nuclear factor κB (NF‐κB)‐dependent expression of cytokines and chemokines and therefore could be a novel therapeutic target for inflammatory pathology. The current study evaluated the in vivo role of MDA in protecting the liver against injury and fibrogenesis caused by CCl₄ in rats, and the underlying mechanisms. Methods: A single injection of CCl₄ induced acute hepatitis, and repeated injections were used to induce hepatic fibrosis in rats. Therapeutic efficacy was assessed by comparison of the severity of hepatic injury and fibrosis in MDA ‐ treated rats versus untreated controls. Results: MDA significantly protected the liver from injury by reducing the activity of serum alanine aminotransferase, and improving the histological architecture of the liver. MDA modulated NF‐κB‐dependent activation of inflammatory cytokines by reducing hepatic mRNA levels of tumor necrosis factor‐α, interleukin‐1β, inducible nitric oxide (NO) synthase and transforming growth factor‐β. In addition, MDA attenuated oxidative stress by increasing the content of hepatic glutathione. These favorable changes were associated with suppressed hepatic NF‐κB activation by MDA. MDA treatment improved liver fibrosis in rats that received repeated CCl₄ injections. In vitro, MDA attenuated phosphorylation of IκB and activation of NF‐κB, and thus prevented biosynthesis of NO in lipopolysaccharide‐activated RAW264.7 cells. Conclusions: The present study suggests that AR is a novel therapeutic anti‐inflammatory target for the treatment of hepatitis and liver fibrosis.     

Protective effect of melatonin against Opisthorchis viverrini‐induced oxidative and nitrosative DNA damage and liver injury in hamsters

Abstract: The liver fluke, Opisthorchis viverrini, is the risk factor of cholangiocarcinoma, which is a major health problem in northeastern Thailand. Production of reactive oxygen and nitrogen species during the host’s response leads to oxidative and nitrosative stress contributing to carcinogenesis. We investigated the protective effect of melatonin against O. viverrini‐induced oxidative and nitrosative stress and liver injury. Hamsters were infected with O. viverrini followed by oral administration of various doses of melatonin (5, 10, and 20 mg/kg body weight) for 30 days. Uninfected hamsters served as controls. Compared to the levels in O. viverrini‐infected hamsters without melatonin treatment, the indoleamine decreased the formation of oxidative and nitrosative DNA lesions, 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine and 8‐nitroguanine, in the nucleus of bile duct epithelium and inflammatory cells, in parallel with a reduction in 3‐nitrotyrosine. Melatonin also reduced the expression of heme oxygenase‐1 and cytokeratin 19, nitrate/nitrite levels, and bile duct proliferation in the liver. Alanine transaminase activity and the levels of 8‐isoprostane and vitamin E were also dose dependently decreased in the plasma of melatonin‐treated hamsters. Melatonin reduced the mRNA expression of oxidant‐generating genes [inducible nitric oxide synthase, nuclear factor‐kappa B (NF‐κB), and cyclooxygenase‐2] and proinflammatory cytokines (TNF‐α and IL‐1β), accompanied by an increase in the expression of antioxidant genes [nuclear erythroid 2‐related factor 2 (Nrf2) and manganese superoxide dismutase]. Thus, melatonin may be an effective chemopreventive agent against O. viverrini‐induced cholangiocarcinoma by reducing oxidative and nitrosative DNA damage via induction of Nrf2 and inhibition of NF‐κB‐mediated pathways.     

Melatonin inhibits IL1β-induced MMP9 expression and activity in human umbilical vein endothelial cells by suppressing NF-κB activation

Matrix metalloproteinases (MMPs) have been involved in inflammatory and degradative processes in pathologic conditions. The purpose of this study was to investigate the protective effect of melatonin in human umbilical vein endothelial cell (HUVEC) monolayer permeability and the regulation of MMP9 induced by interleukin 1β (IL1β (IL1B)) in HUVECs. Protection studies were carried out with melatonin, a well-known antioxidant and antiinflammatory molecule. MMP9 expression was increased with IL1β induction in HUVECs. Melatonin showed a barrier-protective role by downregulation of MMP9 and upregulation of tissue inhibitor of metalloproteinase-1 expression in HUVECs. Meanwhile, melatonin also decreased sodium fluorescein permeability and counteracted the downregulation of vascular endothelial cadherin and occludin expression in HUVECs. During inflammatory stimulus, nuclear factor-κB (NF-κB) plays a significant role in regulating MMP genes expression, thus the function of NF-κB in HUVECs' barrier disruption was investigated. IL1β induced nuclear translocation of NF-κB in HUVECs and regulated MMP9 expression. However, NF-κB translocation into the nucleus was inhibited significantly by melatonin. Our results show that melatonin decreases the permeability of monolayer endothelial cell induced by IL1β. At the same time, melatonin decreased the expression and activity of MMP9 by a NF-κB-dependent pathway in HUVECs induced by IL1β.     

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.