Matrix metalloproteinase-9 activity and expression is reduced by melatonin during prevention of ethanol-induced gastric ulcer in mice

Matrix metalloproteinases (MMPs) play an important role in degradation of gastric extracellular matrix proteins. However, no reports are available on the relationship between the activity of MMPs and gastric ulceration induced by alcohol. Our objective was to investigate the effect of melatonin (N-acetyl-5-methoxytryptamine) on the regulation of MMP-9 and MMP-2 activities during prevention of ethanol-induced gastric ulcer. Biochemical and zymographic methods were used to analyze MMP-9 and -2 activities in gastric tissues of Balb/c mice following induction of gastric ulcer by ethanol. Our studies reveal that melatonin arrested cell injury, protein carbonyl formation, and lipid peroxidation in mice during gastroprotection. Melatonin dose-dependently reduced proMMP-9 activity that was induced ( approximately 25-fold) during ethanol-induced gastric ulceration. Severity of gastric ulcers were correlated proportionately with increased dose of ethanol and elevated activity of proMMP-9 and -2. The reduced activities of MMP-9 and -2 were associated with reduced expression of TNF-alpha and increased expression of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2). We conclude that melatonin's ability to prevent ethanol-induced gastric ulceration in mice is related to a reduction in proMMP-9 activity and expression.     

Effect of melatonin on secreted and induced matrix metalloproteinase-9 and -2 activity during prevention of indomethacin-induced gastric ulcer

Matrix metalloproteinases (MMPs) maintain the crucial role in physiological turnover of extracellular matrix (ECM) proteins in gastric tissues. However, a little is known about the relationship of MMPs with ECM degradation during gastric ulceration and ECM remodeling during healing. Our objective was to investigate the effect of melatonin (N-acetyl-5 methoxytryptamine) on the regulation of MMP-9 and MMP-2 activity during prevention of gastric ulcer. In the present study, biochemical and zymographic methods were used to analyze the mechanism of melatonin in indomethacin-induced gastric ulcer in a rat model. Our studies reveal that melatonin dose-dependently downregulates the expression and secretion of pro-MMP-9 which is induced (approximately 10-fold) during indomethacin-induced gastric ulceration. Furthermore, melatonin prevents gastric ulceration in a dose-dependent manner through upregulation (approximately two- to threefold) of both pro-MMP-2 and active MMP-2 at the level of induction as well as secretion. It also prevents gastric ulcers by blocking glutathione depletion and lipid peroxidation in cytosolic and microsomal fractions. The novel findings of this study are attributed to the attenuation of the pro-MMP-9 and increase of MMP-2 activity by pretreatment with melatonin. The finding defines one of the MMP-mediated pathways for melatonin's action in gastric ulcer.     

Melatonin reduces indomethacin‐induced gastric mucosal cell apoptosis by preventing mitochondrial oxidative stress and the activation of mitochondrial pathway of apoptosis

Abstract: Augmentation of gastric mucosal cell apoptosis due to development of oxidative stress is one of the main pathogenic events in the development of nonsteroidal anti‐inflammatory drug (NSAID)‐induced gastropathy. Identification of a nontoxic, anti‐apoptotic molecule is warranted for therapy against NSAID‐induced gastropathy. The objective of the present study was to define the mechanism of the anti‐apoptotic effect of melatonin, a nontoxic molecule which scavenges reactive oxygen species. Using an array of experimental approaches, we have shown that melatonin prevents the development of mitochondrial oxidative stress and activation of mitochondrial pathway of apoptosis induced by indomethacin (a NSAID) in the gastric mucosa. Melatonin inhibits the important steps of indomethacin‐induced activation of mitochondrial pathway of apoptosis such as upregulation of the expression of Bax and Bak, and the downregulation of Bcl‐2 and BclxL. Melatonin also prevents indomethacin‐induced mitochondrial translocation of Bax and prevents the collapse of mitochondrial membrane potential. Moreover, melatonin reduces indomethacin‐mediated activation of caspase‐9 and caspase‐3 by blocking the release of cytochrome c and finally rescues gastric mucosal cells from indomethacin‐induced apoptosis as measured by the TUNEL assay. Histologic studies of gastric mucosa further document that melatonin almost completely protects against gastric damage induced by indomethacin. Thus, melatonin has significant anti‐apoptotic effects to protect gastric mucosa from NSAID‐induced apoptosis and gastropathy, which makes its use as potential therapy against gastric damage during NSAID treatment.     

Induction of matrix metalloproteinase-9 and -3 in nonsteroidal anti-inflammatory drug-induced acute gastric ulcers in mice: regulation by melatonin

Abstract:  The pathogenesis of gastric ulcer is associated with remodeling of extracellular matrix (ECM) by various matrix metalloproteinases (MMPs). However, how MMPs are regulated during nonsteroidal anti-inflammatory drug (NSAID)-induced acute gastric ulceration is not well studied. In this study, different NSAIDs (80 mg/kg b.w.) were applied to generate acute gastric ulcer in the BALB/c mouse and the regulation of MMPs were investigated. NSAIDs caused dose-dependent induction in MMP-9 and -3 activities and expressions in ulcerated gastric tissues along with significant infiltration of inflammatory cells and disruption of gastric mucosal layer. In addition, an increase in tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-8 expression, excessive generation of hydroxyl radical (^•OH), and protein oxidation and lipid peroxidation were observed in acute ulcerated gastric tissues. In this study, the efficacy of melatonin on activities of MMP-9 and -3 during prevention of gastric ulcers was tested. Melatonin at a dose of 60 mg/kg b.w. downregulated MMP-9 and -3 both at the enzyme and protein levels in mouse gastric tissues during prevention as well as healing of acute gastric ulcers. It also blocked oxidative stress via inhibition of protein oxidation, lipid peroxidation, ^•OH generation and SOD-2 expression. Moreover, it suppressed myeloperoxidase activity and expressions of TNF-α, IL-1β and IL-8. This study documents for the first time that induction of MMP-9 and -3 activities accompany NSAID-induced inflammation and oxidative stress in gastric tissues and indicates that, melatonin may be a preventive or therapeutic remedy for gastric ulcers.     

Melatonin promotes angiogenesis during protection and healing of indomethacin‐induced gastric ulcer: role of matrix metaloproteinase‐2

Abstract: Matrix metalloproteinase (MMP)‐2 is considered as a crucial regulator of angiogenesis, a process of new blood vessel formation. We reported previously that melatonin (N‐acetyl‐5‐methoxy tryptamine), an antioxidant and anti‐inflammatory agent, prevents indomethacin‐induced gastric ulcers. Herein, we investigated the effect of melatonin on MMP‐2‐mediated angiogenesis during gastroprotection. Angiogenic properties of melatonin were tested in both rat corneal micropocket assay and in mouse model of indomethacin‐induced gastric lesions. Melatonin augmented angiogenesis that was associated with amelioration of MMP‐2 expression and activity and, upregulation of vascular endothelial growth factor (VEGF) in rat cornea. Melatonin prevented gastric lesions by promoting angiogenesis via upregulation of VEGF followed by over‐expression of MMP‐2. Similarly, healing of gastric lesions was associated with early expression of VEGF followed by MMP‐2. In addition, upregulation of MMP‐2 was parallel to MMP‐14 and inverse to tissue inhibitor of metalloprotease (TIMP)‐2 expression during gastroprotection. Our data demonstrated that melatonin exerts angiogenesis through MMP‐2 and VEGF over‐expression during protection and healing of gastric ulcers. This study highlights for the first time a phase‐associated regulation of MMP‐2 activity in gastric mucosa and an angiogenic action of melatonin to rescue indomethacin‐induced gastropathy.     

Reduced silent information regulator 1 signaling exacerbates myocardial ischemia–reperfusion injury in type 2 diabetic rats and the protective effect of melatonin

Diabetes mellitus (DM) increases myocardial oxidative stress and endoplasmic reticulum (ER) stress. Melatonin confers cardioprotective effect by suppressing oxidative damage. However, the effect and mechanism of melatonin on myocardial ischemia–reperfusion (MI/R) injury in type 2 diabetic state are still unknown. In this study, we developed high‐fat diet‐fed streptozotocin (HFD‐STZ) rat, a well‐known type 2 diabetic model, to evaluate the effect of melatonin on MI/R injury with a focus on silent information regulator 1 (SIRT1) signaling, oxidative stress, and PERK/eIF2α/ATF4‐mediated ER stress. HFD‐STZ treated rats were exposed to melatonin treatment in the presence or the absence of sirtinol (a SIRT1 inhibitor) and subjected to MI/R surgery. Compared with nondiabetic animals, type 2 diabetic rats exhibited significantly decreased myocardial SIRT1 signaling, increased apoptosis, enhanced oxidative stress, and ER stress. Additionally, further reduced SIRT1 signaling, aggravated oxidative damage, and ER stress were found in diabetic animals subjected to MI/R surgery. Melatonin markedly reduced MI/R injury by improving cardiac functional recovery and decreasing myocardial apoptosis in type 2 diabetic animals. Melatonin treatment up‐regulated SIRT1 expression, reduced oxidative damage, and suppressed PERK/eIF2α/ATF4 signaling. However, these effects were all attenuated by SIRT1 inhibition. Melatonin also protected high glucose/high fat cultured H9C2 cardiomyocytes against simulated ischemia–reperfusion injury‐induced ER stress by activating SIRT1 signaling while SIRT1 siRNA blunted this action. Taken together, our study demonstrates that reduced cardiac SIRT1 signaling in type 2 diabetic state aggravates MI/R injury. Melatonin ameliorates reperfusion‐induced oxidative stress and ER stress via activation of SIRT1 signaling, thus reducing MI/R damage and improving cardiac function.     

Matrix metalloproteinase-9 and metalloproteinase-2 activity and expression is reduced by melatonin during experimental colitis

Abstract:  Matrix metalloproteinases (MMPs) are associated with matrix turnover in both physiological and pathological conditions. Several data indicate that MMPs play an important role in the pathogenesis of colitis. Various evidence has documented that the pineal secretory product melatonin exerts an important anti-inflammatory effect in different experimental models including colitis. However, no reports are available on the relationship between the activity and expression of MMPs and anti-inflammatory effect of melatonin. The aim of the present study was to evaluate whether melatonin prevents the experimental colitis in rats by regulating MMP-9 and MMP-2 activity and expression. Colitis was induced by intracolonic instillation of dinitrobenzene sulphonic acid (DNBS). Four days after DNBS administration, colon TNF- α production was associated with colon damage. Biochemical methods and zymography were used to analyse MMP-9 and MMP-2 activities in colon tissues from DNBS-injured rats. Our studies reveal that melatonin prevented colon injury and lipid peroxidation in rats at 4 days after DNBS-induced colitis. Melatonin also reduced proMMP-9 and MMP-2 activities that were induced in the colon tissues by DNBS administration. Reduced MMP-9 and MMP-2 activities were associated with reduced expression of TNF- α . We conclude that melatonin's ability to reduce DNBS-induced colon injury in rats is related to a reduction in proMMP-9 and MMP-2 activities and expression.     

Modulating Action of Melatonin on Serotonin-Induced Aggravation of Ethanol Ulceration and Changes of Mucosal Blood Flow in Rat Stomachs

Effects of melatonin and serotonin on ethanol ulceration and mucosal blood flow in the rat stomach were investigated. Melatonin and serotonin (5-HT) administration did not produce observable gastric injury in the ex vivo stomach, but the 5-HT dose dependently reduced glandular mucosal blood flow (GMBF) in this organ. Ethanol depressed GMBF and induced visible glandular mucosal injury. The latter effect was prevented by melatonin preincubation. Serotonin pretreatment aggravated the gastric mucosal injury and GMBF changes induced by ethanol; these actions were partially reversed by melatonin. The findings indicate that the GMBF and gastric injury are related; the reduction in FMBF, however, may not be the sole factor responsible for ulceration. The antagonistic effects of melatonin on 5-HT action on the stomach suggest that melatonin may act as a modulator for 5-HT action on the gastrointestinal tract.     

Protective effect of melatonin on indomethacin-induced gastric injury in rats

The gastric injury associated with nonsteroidal anti-inflammatory drug (NSAID) therapy has been linked to the detrimental effects of the agents on the processes of prostaglandin synthesis, neutrophil (PMN) activation. and oxygen free radical generation. In the present study, we investigated the in vivo protective effects of melatonin on indomethacin-induced gastric lesions in the rat. Peroxidation of lipids and changes in the activities of related enzymes such as glutathione peroxidase (GSH-px) and myeloperoxidase (MPO), as a marker of PMNs infiltration, were also studied. Intraperitoneal (i.p.) injection of melatonin (0.25. 0.5, 1 mg kg(-1)) 30 min before indomethacin administration prevented gastric injury. The mean ulcer indices significantly (P < 0.05) decreased. Thiobarbituric acid (TBA) reactive substances in the gastric mucosa as an index of peroxidation, was increased after indomethacin administration and this increase was inhibited by melatonin. In addition, pretreatment with melatonin resulted in a significant increase of the enzymatic GSH-px activity up to the control levels; however, inhibition of ulceration by melatonin was not associated with a significant reduction in PMN infiltration. These results suggest that the protection afforded by the pineal hormone against indomethacin-induced gastric injury may be, in addition to other possible mechanisms, to its radical scavenging activity.     

Decreased MT1 and MT2 melatonin receptor expression in extrapineal tissues of the rat during physiological aging

Abstract:  Aging is a complex process associated with a diminished ability to respond to stress, a progressive increase in free radical generation and a decline in immune function. Melatonin, a molecule with a great functional versatility exerts anti-oxidant, oncostatic, immunomodulatory, and anti-aging properties. Melatonin levels drop during aging and it has been speculated that the loss of melatonin may accelerate aging. This study was designed to elucidate whether aging involves responsiveness to reduced melatonin. Melatonin membrane receptor (MT1 and MT2) expression and MT1 protein expression were analyzed in extrapineal tissues (thymus, spleen, liver, kidney, and heart) of 3- and 12-month-old rats using real time polymerase chain reaction and western blotting analysis. Moreover, melatonin in tissues was measured by high performance liquid chromatography. We report for the first time, an age-related reduction in mRNA MT1 and MT2 expression levels as well as MT1 protein expression in all tissues tested except the thymus, where surprisingly, both melatonin receptor levels were significantly higher in 12-month-old rats and MT1 protein expression maintained unchanged with age. Diminished melatonin concentrations were measured in spleen, liver, and heart during aging. As a conclusion, physiological aging seems to exert responsiveness to melatonin and consequently, the loss of this potent anti-oxidant may contribute to onset of aging.     

褪黑激素对胃黏膜的保护作用及其机制

目的：探讨褪黑激素和血清素对鼠胃酒精性溃疡及黏膜血流的影响。方法：制备胃在体动物模型。分别皮下注入血清素及同容积生理盐水。用褪黑激素及同容积蒸馏水作为浸育液分别放入各组鼠的胃腔中。30min未毕毕胃黏膜血流后,采用40％的酒精作为浸育液分别放入各组鼠胃腔中,继续检测胃黏膜血流并测量胃黏膜损伤指数。结果：褪黑激素和血清素两者均不能损伤鼠胃黏膜,但血清素可降低胃黏膜血流,且与其剂理相关。褪黑激素可减轻由酒精引起的胃黏膜损伤和胃黏膜血流减少,而血清素可加重酒精引起的胃黏膜损伤和胃黏膜血流减少,但可被褪黑激素部分逆转。结论：胃黏膜的损伤与胃黏膜血流有关,但不是溃疡形成的唯一因素。因此,能拮抗血清的褪黑激素可作为在胃肠道作用的调节剂。     

Melatonin regulates the calcium‐buffering proteins,parvalbumin and hippocalcin,in ischemic brain injury

Abstract: Melatonin has anti‐oxidant activity and it exerts a neuroprotective effects during ischemic brain injury. Calcium‐buffering proteins including parvalbumin and hippocalcin are involved in neuronal differentiation and maturation through calcium signaling. This study investigated whether melatonin moderates parvalbumin and hippocalcin expression in cerebral ischemia and glutamate toxicity‐induced neuronal cell death. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). Male Sprague‐Dawley rats were treated with vehicle or melatonin (5 mg/kg) prior to MCAO, and cerebral cortical tissues were collected 24 hr after MCAO. Parvalbumin and hippocalcin levels were decreased in vehicle‐treated animal with MCAO, whereas melatonin prevented the ischemic injury‐induced reduction in these proteins. In cultured hippocampal cells, glutamate toxicity decreased parvalbumin and hippocalcin levels, while melatonin treatment prevented the glutamate exposure‐induced diminished in these proteins levels. Melatonin also attenuated the glutamate toxicity‐induced increase in intracellular Ca²⁺ levels. These results suggest that the maintenance of parvalbumin and hippocalcin levels by melatonin in ischemic injury contributes to the neuroprotective effect of melatonin against neuronal cell damage.     

褪黑激素对应激性溃疡大鼠胃黏膜诱生型一氧化氮合酶表达的影响

背景胃黏膜诱生型一氧化氮合酶(iNOS)的过度表达在应激性溃疡的发生中起重要作用。目的研究褪黑激素(MT)对水浸鄄束缚应激大鼠胃黏膜iNOS表达的影响及其对胃黏膜的保护作用,以进一步阐明MT的作用机制。方法正常对照组不予水浸鄄束缚应激和MT预防,模型组和MT低剂量预防组、MT高剂量预防组于应激前30min分别腹腔注射含1%二甲基亚砜(DMSO)或MT5mg/kg、20mg/kg的等体积生理盐水。应激6h后处死动物,检测各组大鼠胃黏膜一氧化氮(NO)水平、iNOS蛋白和iNOSmRNA的表达,并评估胃黏膜损伤程度。结果水浸鄄束缚应激6h后,大鼠胃黏膜NO水平、iNOS蛋白和iNOSmRNA表达显著增加,胃黏膜病变明显。MT预防组大鼠胃黏膜NO水平、iNOS蛋白和iNOSmRNA表达均显著低于模型组(P<0.01),且溃疡指数(UI)显著下降(P<0.01)。MT高剂量预防组大鼠各检测指标均显著低于MT低剂量预防组(P<0.05),作用呈剂量依赖性。结论MT可预防水浸鄄束缚应激诱导的大鼠胃黏膜损伤,其机制可能与其抑制胃黏膜iNOS过度表达有关。     

Protective effects of chronic melatonin treatment against renal injury in streptozotocin-induced diabetic rats

The aim of this study was to investigate the effects of melatonin as an antioxidant, on prevention and treatment of streptozotocin (STZ)-induced diabetic renal injury in rats. Male Wistar rats were divided into four groups: (1) untreated, (2) melatonin-treated, (3) untreated diabetic (UD), (4) melatonin-treated diabetic (MD). Experimental diabetes was induced by single dose (60 mg/kg, i.p.) STZ injection. For 3 days prior to administration of STZ, melatonin was injected (200 microg/kg/day, i.p.); these injections were continued until the end of the study (4 weeks). Malondialdehyde (MDA) levels as a marker of lipid peroxidation were significantly increased in the renal homogenates of UD animals and decreased after melatonin administration. The activity of the antioxidative enzyme glutathione peroxidase (GSH-Px) was significantly reduced in UD rats. Melatonin treatment reversed STZ-induced reduction of GSH-Px activity without having an effect on blood glucose. Upon histopathological examination, it was observed that the melatonin treatment prevented the renal morphological damage caused by diabetes. Upon immunohistochemical investigation, glomerular anti-laminin beta1 staining decreased in MD rats. Additionally, no tubular anti-IGF-1 staining was observed in melatonin-treated rats. In conclusion, chronically administered melatonin reduced renal injury in STZ-induced diabetic rats and thus it may provide a useful therapeutic option in humans to reduce oxidative stress and the associated renal injury in patients with diabetes mellitus.     

Vascular reactivity in diabetic rats: effect of melatonin

The aim of the present study was to evaluate the in vitro contractile response of rat aorta in mild and severe type I diabetes and the effect of melatonin on it. Aortic rings were obtained from male Wistar rats injected with streptozotocin 8-12 wks earlier. Rats were divided into three groups: non-diabetic rats (NDR), mildly diabetic rats (MDR) and severely diabetic rats (SDR). Dose-response curves for acetylcholine-induced, endothelium-related relaxation of aortic rings (after previous exposure to phenylephrine) and for serotonin-induced vasoconstriction were conducted in the presence or absence of 10-5 mol/L melatonin. This protocol was repeated with rings preincubated in a high glucose solution (44 mmol/L). The contractile response to phenylephrine decreased in SDR, an effect counteracted by preincubation with high glucose. Melatonin decreased phenylephrine-induced vasoconstriction in MDR and counteracted the effect of high glucose in SDR. Acetylcholine-evoked relaxation decreased significantly after exposure to a high glucose in SDR, this effect being counteracted by melatonin. Serotonin-induced vasoconstriction decreased in SDR and augmented in MDR, but only after exposure to high glucose. Melatonin reduced the maximal tension of aortic contraction after serotonin in MDR, both under basal conditions and after preincubation in a high glucose solution. The results support the existence of differences in vasomotor responses as a function of the diabetes state and of an improvement of contractile performance in diabetic rats after exposure to melatonin at a pharmacological concentration (in terms of circulating melatonin levels but not necessarily for some other fluids or tissues).