Inverse correlation between endogenous melatonin levels and oxidative damage in some tissues of SAM P8 mice

To assess whether oxidative damage in some tissues was related to their melatonin concentration, endogenous melatonin levels and the age-linked protein and lipid damage in spleen, thymus and liver in 5-month-old SAM P8 mice were examined. The results show that high levels of melatonin in spleen and thymus correlate with lower protein and lipid damage. The liver, which had much lower melatonin concentrations than the other two tissues, had much higher levels of oxidatively damaged protein, as measured by carbonyl values. These results add new evidence concerning the protective role of endogenous melatonin as an antioxidant agent, and suggest that a treatment with this molecule might help to reduce age-associated functional deficits in many organs, including those of the immune system.     

Melatonin treatment restores mitochondrial function in Alzheimer's mice: a mitochondrial protective role of melatonin membrane receptor signaling

Mitochondrial dysfunction is a hallmark of Alzheimer's disease (AD) and is observed in mutant amyloid precursor protein (APP) transgenic mouse models of familial AD. Melatonin is a potent antioxidant, can prevent toxic aggregation of Alzheimer's beta-amyloid (Aβ) peptide and, when taken long term, can protect against cognitive deficits in APP transgenic mice. To study the effects of melatonin on brain mitochondrial function in an AD model, APP/PS1 transgenic mice were treated for 1 month with melatonin. Analysis of isolated brain mitochondria from mice indicated that melatonin treatment decreased mitochondrial Aβ levels by two- to fourfold in different brain regions. This was accompanied by a near complete restoration of mitochondrial respiratory rates, membrane potential, and ATP levels in isolated mitochondria from the hippocampus, cortex, or striatum. When isolated mitochondria from untreated young mice were given melatonin, a slight increase in respiratory rate was observed. No such effect was observed in mitochondria from aged mice. In APP-expressing neuroblastoma cells in culture, mitochondrial function was restored by melatonin or by the structurally related compounds indole-3-propionic acid or N(1)-acetyl-N(2)-formyl-5-methoxykynuramine. This restoration was partially blocked by melatonin receptor antagonists indicating melatonin receptor signaling is required for the full effect. Therefore, treatments that stimulate melatonin receptor signaling may be beneficial for restoring mitochondrial function in AD, and preservation of mitochondrial function may an important mechanism by which long term melatonin treatment delays cognitive dysfunction in AD mice.     

Relationship between protective effects of rosiglitazone on endothelium and endogenous nitric oxide synthase inhibitor in streptozotocin-induced diabetic rats and cultured endothelial cells

BACKGROUND: Previous investigations have indicated that the level of asymmetric dimethylarginine (ADMA) is increased in diabetic patients and animals, and rosiglitazone has a protective effect on the endothelium. In the present study, we tested the relationship between protective effects of rosiglitazone and ADMA in streptozotocin (STZ)-induced diabetic rats and cultured endothelial cells. METHODS: Blood samples were collected from carotid artery. Vasodilator responses to acetylcholine (ACh) in the isolated aortic rings were measured, and serum concentrations of glucose, lipid, nitrite/nitrate, ADMA and tumour necrosis factor-alpha (TNF-alpha) were determined. Cultured endothelial cells were treated with ADMA, and the concentrations of intercellular adhesion molecule (ICAM-1), TNF-alpha, and the activity of nuclear factor-kappaB (NF-kappaB) were determined. RESULTS: Vasodilator responses to ACh were decreased markedly and the serum concentrations of TNF-alpha, nitrite/nitrate and ADMA were increased significantly in diabetic rats. Rosiglitazone (3, 10 or 30 mg/kg) produced a significant reduction of the inhibition of vasodilator responses to ACh, but had no effect on the serum concentrations of glucose, lipid, nitrite/nitrate and ADMA in diabetic rats. ADMA (30 microM) significantly increased the activity of NF-kappaB and elevated the levels of ICAM-1 and TNF-alpha, and pre-treatment with rosiglitazone (10 or 30 microM) markedly inhibited the increased activity of NF-kappaB and reduced the elevated levels of TNF-alpha and ICAM-1 induced by ADMA in cultured endothelial cells. CONCLUSIONS: Rosiglitazone improves endothelial function in diabetic rats, which is related to the reduction of the inflammatory response induced by ADMA.     

Melatonin induces neural SOD2 expression independent of the NF-kappaB pathway and improves the mitochondrial population and function in old mice

Aging is commonly defined as a physiological phenomenon associated with morphological and functional deleterious changes in which oxidative stress has a fundamental impact; therefore, readjusting the oxidative balance should have beneficial effects. In our study, we tested the antioxidant melatonin in old mouse brains and showed positive effects at the cellular and mitochondrial levels. Melatonin attenuated β-amyloid protein expression and α-synuclein deposits in the brain compared to aged group. Furthermore, oxidative stress was increased by aging and induced the nuclear translocation of nuclear factor-kappa B (NF-κB), which was suppressed by melatonin treatment. The antioxidant mitochondrial expression, superoxide dismutase 2 (SOD2), was increased in both control and melatonin-treated old mice, despite the different activation states of the NF-κB pathway. The NF-κB pathway was activated in the old mice, which may be explained by this group's response to the increased oxidative insult; this insult was inhibited in melatonin-treated animals, showing this group an increase in active mitochondria population that was not observed in old group. We also report that melatonin is capable of restoring the mitochondrial potential of age-damaged neurons. In conclusion, melatonin's beneficial effects on brain aging are linked to the increase in mitochondrial membrane potential and SOD2 expression, which probably reduces the mitochondrial contribution to the oxidative stress imbalance.     

Melatonin suppresses activation of hepatic stellate cells through RORα‐mediated inhibition of 5‐lipoxygenase

Liver fibrosis is scar tissue resulting from an uncontrolled wound‐healing process in response to chronic liver injury. Liver damage generates an inflammatory reaction that activates hepatic stellate cells (HSC) that transdifferentiate from quiescent cells that control retinol metabolism to proliferative and migratory myofibroblasts that produce excessive amounts of extracellular matrix proteins, in particular collagen 1a1 (COL1A1). Although liver fibrosis is reversible, no effective drug therapy is available to prevent or reverse HSC activation. Melatonin has potent hepatoprotective properties in a variety of acute and chronic liver injury models and suppresses liver fibrosis. However, it remains unclear whether melatonin acts indirectly or directly on HSC to prevent liver fibrosis. Here, we studied the effect of melatonin on culture‐activated rat HSC. Melatonin dose‐dependently suppressed the expression of HSC activation markers Col1a1 and alpha‐smooth muscle actin (αSMA, Acta2), as well as HSC proliferation and loss of lipid droplets. The nuclear melatonin sensor retinoic acid receptor‐related orphan receptor‐alpha (RORα/Nr1f1) was expressed in quiescent and activated HSC, while the membranous melatonin receptors (Mtrn1a and Mtrn1b) were not. The synthetic RORα agonist SR1078 more potently suppressed Col1a1 and αSma expression, HSC proliferation, and lipid droplet loss, while the RORα antagonist SR1001 blocked the antifibrotic features of melatonin. Melatonin and SR1078 inhibited the expression of Alox5, encoding 5‐lipoxygenase (5‐LO). The pharmacological 5‐LO inhibitor AA861 reduced Acta2 and Col1a1 expression in activated HSC. We conclude that melatonin directly suppresses HSC activation via RORα‐mediated inhibition of Alox5 expression, which provides novel drug targets to treat liver fibrosis.     

Alpha-interferon 2a and 1 3-cis-retinoic acid for the treatment of metastatic renal cell carcinoma

Background: It is suggested that immunotherapy may have a better role than cytotoxic chemotherapy in the treatment of metastatic renal cell carcinoma. Aims: A phase II study of α‐interferon 2a (IFN2a) and 13‐cis‐retinoic acid (CRA) in the treatment of metastatic renal cell carcinoma. Methods: Twenty‐two patients with no previous systemic therapy were treated with IFN2a daily at 3 million units (MU) and escalated to 6 and 9 MU if tolerated, together with CRA given orally at 1 mg/kg per day in two divided doses. Changes in quality of life were also assessed. Results: Twenty patients were available for assessment. Three patients (14%) achieved a partial response and five patients (23%) had stable disease. No patient achieved a complete response. A durable response was observed in partial responders with median length of response of 44 weeks (range 32?59 weeks). Therapy was stopped in seven (35%) patients due to treatment‐related toxicities, and quality of life was worsened in the majority of patients. Conclusion: IFN2a and CRA has a low response rate and significant toxicity, and the combination as standard treatment of metastatic renal cell carcinoma is not recommended, despite the suggestion that CRA may lengthen the response to IFN2a. (Intern Med J 2002; 32: 158?162)     

Melatonin inhibits matrix metalloproteinase-9 (MMP-9) activation in the lipopolysaccharide (LPS)-stimulated RAW 264.7 and BV2 cells and a mouse model of meningitis

Abstract: We explored anti‐inflammatory potential of melatonin against the lipopolysaccharide (LPS)‐induced inflammation in vivo and in vitro. RAW 264.7 and BV2 cells were stimulated by LPS, followed by the treatment with melatonin or vehicle at various time intervals. In a mouse model of meningitis induced by LPS, melatonin (5 mg/kg) or vehicle was intravenously injected at 30 min postinsult. The activity of matrix metalloproteinase‐2 (MMP‐2) and metalloproteinase‐9 (MMP‐9) was determined by gelatin zymography. Nuclear factor‐kappa B (NFκB) translocation and binding activity were determined by immunocytochemistry and electrophoretic mobility shift assay (EMSA). Our results showed that either pretreatment or cotreatment with melatonin at 50–500 μm effectively inhibited the LPS‐induced proMMP‐9 activation in the RAW 264.7 and BV2 cells, respectively (P < 0.05). This melatonin‐induced proMMP‐9 inhibition remained effective when treatment was delayed up to 2 and 6 hr postinsult for RAW 264.7 and BV2 cells, respectively (P < 0.05 for both groups). Additionally, melatonin significantly attenuated the rises of circulatory and cerebral MMP‐9 activity, respectively (P < 0.05) and reduced the loss of body weight (P < 0.05) in mice with meningitis. Moreover, melatonin (50 μm ) effectively inhibited nuclear factor‐kappa B (NFκB) translocation and binding activity in the LPS‐treated RAW 264.7 and BV2 cells, respectively (P < 0.05). These results demonstrate direct inhibitory actions of melatonin against postinflammatory NFκB translocation and MMP‐9 activation and highlight its ability to inhibit systemic and cerebral MMP‐9 activation following brain inflammation.     

Similar effects of recombinant interferon-alpha-2b and natural interferon-alpha when combined with intra-arterial 5-fluorouracil for the treatment of advanced hepatocellular carcinoma.

AIM: Intra-arterial 5-fluorouracil (5-FU) plus interferon (IFN) combination therapy is effective against advanced hepatocellular carcinoma (HCC) with portal vein tumour thrombosis. In this study, we compared the efficiency and safety of recombinant IFN-alpha-2b with natural IFN-alpha as components of the combination therapy. METHODS: Consecutive HCC patients (n=31) with portal vein tumour thrombosis were enrolled in this prospective study. They received combination therapy of 5-FU and either recombinant IFN-alpha-2b (R group, n=15) or natural IFN-alpha (N group, n=16). We compared the two groups for the early response rate, adverse reactions, time to progression (TTP) and survival rates. In addition, we assessed the cost-effectiveness of each protocol. RESULTS: The early response rate (R: 26.7%, N: 31.2%), median TTP (R: 5.8 months, N: 5.6 months) and median survival time (R: 7.5 months, N: 6.5 months) were not significantly different between the R and N groups. There were no differences in adverse reactions between the two groups. The estimated cost-effectiveness ratio of recombinant IFN-alpha-2b was better than natural IFN-alpha. CONCLUSIONS: In our protocol of combination therapy, there were no significant differences between recombinant IFN-alpha-2b and natural IFN-alpha with regard to early response to therapy, adverse effects, TTP and survival rates. 5-FU could be combined with either recombinant IFN-alpha-2b or natural IFN-alpha, although the cost-effectiveness of the former warrants its use clinically.     

In vitro effect of granulocyte-colony stimulating factor and all-trans retinoic acid on the expression of inflammatory cytokines and adhesion molecules in acute promyelocytic leukemic cells.

Differentiation therapy with all-trans retinoic acid (ATRA) represents a landmark approach in the treatment of acute promyelocytic leukemia (APL). However, a potentially fatal complication of retinoic acid (RA) syndrome occurs in about a quarter of patients and its pathophysiology is still unclear. In order to investigate whether or not the treatment with ATRA leads to increased elaboration of inflammatory cytokines and adhesion molecules by the APL cells, the expression of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-8, L-selectin and intercellular adhesion molecule-1 (ICAM-1) was examined in the APL cells after induction of differentiation with ATRA in the presence or absence of granulocyte-colony stimulating factor (G-CSF) or IL-3 in the present study. Cytokine elaboration by the treated cells was detected using both Northern blotting and enzyme-linked immunosorbent assay. Our results have shown that ATRA induces an increased expression of IL-8, IL-1beta, TNF-alpha and ICAM-1 in APL cells, which can be amplified by the addition of G-CSF. These data imply that the induction of inflammatory cytokines in APL cells may play an important role in the pathogenesis of RA syndrome. Furthermore, G-CSF, through its potent differentiating activity, may increase the risk of such complications during ATRA treatment.     

Effects of low glucose degradation products peritoneal dialysis fluid on the peritoneal fibrosis and vascularization in a chronic rat model

In the present study, we examined the effects of a new peritoneal dialysis fluid (PDF) with a low level of low glucose degradation products (GDP) on the functional and structural stability of the peritoneal membrane (PM). Male Sprague-Dawley rats were divided into three groups: group C (n = 8), without dialysate infusion; group P (n = 12), infused with low-level GDP solution (4.25% Physioneal, pH 7.0-7.4); and group D (n = 12), infused with conventional solution (4.25% Dianeal, pH 5.2, adjusted to pH 7.0). In groups D and P, animals were infused through a permanent catheter with 25 mL of PDF, twice daily for 8 weeks. Lipopolysaccharide was added into the PDF immediately before infusion on days 8, 9 and 10 in the two dialysis groups. When compared with group P, group D showed a higher glucose mass transfer at weeks 6 and 8, D/P urea at week 8, TGF-beta1 at weeks 4 and 8, and VEGF level at week 8. The submesothelial matrix layer of the parietal peritoneum was significantly thickened in group D and the lectin-stained blood vessels in this layer were well-visualized in group D compared with group P. There were significantly more peritoneal blood vessels in group D than group P. The transforming growth factor-beta induced gene-h3 (betaig-h3) and TGF-beta1 levels in the peritoneal effluent correlated with the submesothelial thickness, which correlated with the dialysate-to-plasma ratio (D/P) of protein and, inversely, with the rate of glucose transport (D/D(0) glucose, where D is glucose concentration in the dialysate and D(0) is glucose concentration in the dialysis solution before it is infused into the peritoneal cavity). The present study showed that low-GDP PDF effectively attenuated the peritoneal vascularization and fibrosis related to conventional solution.     

Protective effects of melatonin against oxidative stress in Fmr1 knockout mice: a therapeutic research model for the fragile X syndrome

Abstract:  Fragile X syndrome is the most common form of inherited mental retardation. It is typically caused by a mutation of the Fragile X mental-retardation 1 (Fmr1) gene. To better understand the role of the Fmr1 gene and its gene product, the fragile X mental-retardation protein in central nervous system functions, an fmr1 knockout mouse that is deficient in the fragile X mental-retardation protein was bred. In the present study, fragile X mental retardation 1-knockout and wild-type mice are used to determine behaviour and oxidative stress alterations, including reduced glutathione, oxidized glutathione and thiobarbituric acid-reactive substances, before and after chronic treatment with melatonin or tianeptine. Reduced glutathione levels were reduced in the brain of fmr1-knockout mice and chronic melatonin treatment normalized the glutathione levels compared with the control group. Lipid peroxidation was elevated in brain and testes of fmr1-knockout mice and chronic melatonin treatment prevents lipid peroxidation in both tissues. Interestingly, chronic treatment with melatonin alleviated the altered parameters in the fmr1-knockout mice, including abnormal context-dependent exploratory and anxiety behaviours and learning abnormalities. Chronic treatment with tianeptine (a serotonin reuptake enhancer) did not normalize the behaviour in fmr1-knockout mice. The prevention of oxidative stress in the fragile X mouse model, by an antioxidant compound such as melatonin, emerges as a new and promising approach for further investigation on treatment trials for the disease.     

Biological effects of melatonin on osteoblast/osteoclast cocultures,bone, and quality of life: Implications of a role for MT2 melatonin receptors,MEK1/2, and MEK5 in melatonin‐mediated osteoblastogenesis

The Melatonin Osteoporosis Prevention Study (MOPS) demonstrated that nightly melatonin resulted in a time‐dependent decrease in equilibrium ratios of serum osteoclasts and osteoblasts in perimenopausal women. This study examines mechanisms related to the ratios of osteoblasts and osteoclasts using coculture models (transwell or layered) of human mesenchymal stem cell (MSC) and human peripheral blood monocytes (PBMCs). Human MSC/PBMC cocultures exposed to melatonin in osteogenic (OS+) medium for 21 days induced osteoblast differentiation and mineralization; however, only in layered cocultures did melatonin inhibit osteoclastogenesis. Melatonin effects were mediated through MT2 melatonin receptors, MEK1/2, and MEK5. In layered but not transwell cocultures, melatonin increased OPG:RANKL ratios by inhibiting RANKL, suggesting that contact with osteoclasts during osteoblastogenesis inhibits RANKL secretion. Melatonin modulated expression of ERK1/2, ERK5, β1 integrin, GLUT4, and IRβ that was dependent upon the type of coculture; however, in both cultures, melatonin increased RUNX2 and decreased PPARγ expression, indicating a role for metabolic processes that control osteogenic vs adipogenic cell fates of MSCs. Furthermore, melatonin also has osteoblast‐inducing effects on human adipose‐derived MSCs. In vivo, one‐year nightly melatonin (15 mg/L) given to neu female mice in their drinking water increased pErk1/2, pErk5, Runx2, and Opg and Rankl levels in bone consistent with melatonin's already reported bone‐enhancing effects. Finally, analysis of daily logs from the MOPS demonstrated a significant improvement in mood and perhaps sleep quality in women receiving melatonin vs placebo. The osteoblast‐inducing, bone‐enhancing effects of melatonin and improvement in quality of life suggest that melatonin is a safe and effective bone loss therapy.     

Low serum level of high‐sensitivity C‐reactive protein in a Japanese patient with maturity‐onset diabetes of the young type 3 (MODY3)

High‐sensitivity C‐reactive protein (hs‐CRP) levels in European populations are lower in patients with maturity‐onset diabetes of the young type 3 (MODY3) than in those with type 2 diabetes. hs‐CRP levels have been suggested to be useful for discriminating MODY3 from type 2 diabetes. As hs‐CRP levels are influenced by various factors including race and body mass index, it is worthwhile to examine whether hs‐CRP can serve as a biomarker for MODY3 in Japanese. Here we describe the case of a Japanese MODY3 patient with a nonsense mutation in the HNF1A gene. Two measurements showed consistently lower hs‐CRP levels (<0.05 and 0.09 mg/L) than in Japanese patients with type 1 and type 2 diabetes. Hepatic expression of Crp messenger ribonucleic acid was significantly decreased in Hnf1a knockout mice. The hs‐CRP level might be a useful biomarker for MODY3 in both Japanese and European populations.     

Multiple osteolytic bone lesions with high serum levels of interleukin-6 and CCL chemokines in a patient with adult T cell leukemia

A 37-year-old woman was diagnosed as having chronic adult T-cell leukemia (ATL) of the skin by a skin biopsy and human T-cell leukemia virus type-1 serology at our hospital in August 1992. The skin lesions of ATL were improved by treatment with psoralen ultraviolet ray A. She complained of severe pain in her bilateral forearms, hands and ankles, and X-ray examination in July 1999 revealed multiple punched-out lesions of the extremities. Serum levels of parathyroid hormone-related peptide, interleukin-1beta (IL-1beta), tumor necrosis factor-alpha and total serum receptor activator of nuclear factor kappaB ligand were not elevated. However, serum levels of IL-6, CCL2 monocyte chemoattractant protein-1 (MCP-1), CCL3 [macrophage inflammatory protein-1alpha (MIP-1alpha)] and CCL4 (MIP-1beta) were markedly elevated. Here, we have discussed the possible mechanism underlying the onset of the osteolytic lesions.