褪黑素对过氧化氢损伤心肌细胞一氧化氮的影响

目的　探讨氧化损伤时心肌细胞一氧化氮 (NO)生成的变化和褪黑素 (MT)的保护作用。方法　分离培养原代心肌细胞 ,用 NO试剂盒检测细胞培养液中 NO含量 ,硫代巴比妥酸法测定细胞中丙二醛 (MDA)含量 ,荧光探针 DCFH- DA标记细胞 ,用激光共聚焦显微镜观察心肌细胞内总的自由基水平。结果　与对照组相比 ,1 0 0 μmol/ L的 H2 O2 作用 60 min可使培养液中 NO浓度、心肌细胞中 MDA含量和心肌细胞内总的自由基水平明显增加 (分别为 2 2 .4± 2 .6vs97.3± 2 .4nmol/ L、1 .2 3± 0 .0 5vs3.65± 0 .2 0 nmol/ 1 0 6 cells和 86± 6.9vs742± 1 3.4,P<0 .0 1 ) ;加入 MT干预后 ,H2 O2 引起的 NO、MDA和细胞内总的自由基水平的升高被明显抑制 ,两组相比显著差异 (P<0 .0 1 )。结论　 H2 O2 造成心肌细胞氧化损伤时 ,心肌细胞合成的 NO增多并参与了损伤机制 ,MT不仅能使心肌细胞内总的自由基水平下降 ,也能抑制 NO的过度增加 ,从而起到保护心肌细胞的作用。     

褪黑素对过氧化氮损伤心肌细胞一氧化氮的影响

目的探讨氧化损伤时心肌细胞一氧化氮(NO)生成的变化和褪黑素(MT)的保护作用.方法分离培养原代心肌细胞,用NO试剂盒检测细胞培养液中NO含量,硫代巴比妥酸法测定细胞中丙二醛(MDA)含量,荧光探针DCFH-DA标记细胞,用激光共聚焦显微镜观察心肌细胞内总的自由基水平.结果与对照组相比,100 μmo1/L的H2O2作用60min可使培养液中NO浓度、心肌细胞中MDA含量和心肌细胞内总的自由基水平明显增加(分别为22.4±2.6 vs 97.3±2.4 nmol/L、1 23±0.05 vs 3.65±0.20 nmol/106cells和86±6.9 vs 742±13.4,P＜0.01);加入MT干预后,H2O2引起的NO、MDA和细胞内总的自由基水平的升高被明显抑制,两组相比显著差异(P＜0.01).结论H2O2造成心肌细胞氧化损伤时,心肌细胞合成的NO增多并参与了损伤机制,MT不仅能使心肌细胞内总的自由基水平下降,也能抑制NO的过度增加,从而起到保护心肌细胞的作用.     

褪黑素对心肌细胞氧化损伤保护作用的实验研究

目的　探讨氧化损伤时褪黑素 (MT)对心肌细胞形态学变化、细胞存活率、自身一氧化氮 (NO)生成的影响 ,以及NO水平与其它氧化指标的关系。方法　分离培养原代心肌细胞 ,分为对照组、H2 O2 处理组、MT组、MT干预组和N 硝基 L 精氨酸 (L NAME)干预组五组进行对照。用外源性H2 O2 造成氧化损伤 ,用生化法检测培养液中乳酸脱氢酶 (LDH)浓度 ,台盼蓝排斥试验检测细胞存活率 ,硫代巴比妥酸法检测细胞中丙二醛 (MDA)含量 ,用试剂盒检测细胞培养液中NO含量。结果　H2 O2 处理组和L NAME干预组中的细胞存活率、NO含量、LDH含量和MDA含量与对照组比较差异有非常显著性 (P <0 .0 1) ,H2 O2 处理组四项均比对照组明显增高 ,而L NAME干预组中NO含量比对照组明显低。MT干预组与L NAME干预组比较LDH含量和MDA含量明显低 (P <0 .0 1) ,与H2 O2 组加入MT干预后 ,培养液中NO和LDH及MDA含量明显降低 (P <0 .0 1)。结论　心肌细胞在受到H2 O2 损伤时 ,心肌细胞自身产生的NO增加 ,并且在一定程度上参与了对心肌细胞的氧化损伤 ;MT不仅能抑制LDH和MDA的升高 ,还能抑制NO的产生。     

褪黑素对心肌细胞氧化损伤的保护作用

目的 :探讨褪黑素 （MT）对过氧化氢 （H2 O2 ）损伤乳鼠心肌细胞的保护作用。方法 :胰酶消化法分离培养原代心肌细胞 ,用生化法检测培养液中乳酸脱氢酶 （L DH）浓度 ,台盼蓝排斥试验检测细胞存活率 ,硫代巴比妥酸法测定细胞中丙二醛 （MDA）含量。结果 :10 0 μmol/ L 的 H2 O2 使培养液 L DH漏出量和心肌细胞中 MDA含量明显增加 （分别为 6 .5± 1.6 vs 2 4.8± 1.7U / m l和 1.2 3± 0 .0 5 vs 3.6 5± 0 .2 0 nmol/ 10 6 cells,P<0 .0 1） ,心肌细胞存活率明显下降 （96 .0 %± 1.8% vs 6 0 .0 %± 3.2 % ,P<0 .0 1）。加入 MT后 ,培养液 L DH漏出量和细胞 MDA含量明显降低（分别为 10 .2± 1.3U / ml和 1.5 9± 0 .0 9nm ol/ 10 6 cells） ,细胞存活率明显增高 （90 .0 %± 2 .2 % ） ,与 H2 O2 处理组相比均有显著差异 （均 P<0 .0 1）。结论 :MT能对抗过氧化氢所致的心肌细胞损伤 ,其保护作用与其抗氧化作用有关。     

葡萄糖对心肌细胞游离钙的影响

大量临床及流行病学资料表明，随着糖尿病的发生发展，冠心病发生率日趋增高，但糖尿病患者没有明显微血管病变时也可出现心力衰竭，提示糖尿病性心肌病存在［1，2］。迄今为止，糖尿病性心肌病发生的确切机制尚不清楚，我们观察葡萄糖对培育心肌细胞内游离钙的影响，旨在探讨糖尿病性心肌病的可能发病机制。材料和方法1．主要试剂与材料：DMEM培养基（Gibco产品）；CollagenaseIV（胶原酶IV，美国Sigma产品）；Fura－2／AM（钙荧光探针指示剂，美国Sigma产品）；其余试剂为国产分析纯。2．心肌细胞培养：取10只2～3天龄的乳鼠心脏，…     

褪黑素干预急性胰腺炎的实验研究进展

褪黑素是一种具有抗氧化作用、免疫调节作用的吲哚类激素,可以下调急性炎症反应过程中炎症介质的表达,减轻急性炎症反应.此文就近年来的褪黑素干预急性胰腺炎的实验研究作一综述.     

二甲基硫脲对心肌细胞抗过氧化氢损伤的实验研究

为探讨二甲基硫脲（ＤＭＴＵ）保护心肌细胞抗过氧化氢（Ｈ２Ｏ２）损伤。３２瓶培养乳鼠心肌细胞随机分４组，每组８瓶：（１）对照组；（２）Ｈ２Ｏ２（５ｍｍｏｌ）组；（３）ＤＭＴＵ（２０ｍｍｏｌ）组；（４）Ｈ２Ｏ２（５ｍｍｏｌ）＋ＤＭＴＵ（２０ｍｍｏｌ）组。在３７℃、５％ＣＯ２的ＭＥＭ培养４小时。结果发现：（１）与对照组比，Ｈ２Ｏ２组乳酸脱氢酶（ＬＤＨ，Ｕ／１００ｍｌ）释放多（２８２．３８±４７．２８比７７．２５±１８．２５，Ｐ＜０．０１）、ＴＢＡ反应物（ＴＢＡＲＳ，ｎｍｏｌ／ｍｇＰｒ．）产生多（２．２５±０．５３比０．７９±０．３６，Ｐ＜０．０１）；（２）与Ｈ２Ｏ２组比，Ｈ２Ｏ２＋ＤＭ－ＴＵ组ＬＤＨ释放少（９９．２５±４１．８８比２８２．３８±４７．２８，Ｐ＜０．０１）；ＴＢＡＲＳ产生少（０．５９±０．１８比２．２５±０．５３，Ｐ＜０．０１）；此外，我们还发现ＤＭＴＵ组超氧化物歧化酶（ＳＯＤ，μｇ／ｍｇＰｒ．）比对照组高（８．４９±３．６５比１．９２±１．４０，Ｐ＜０．０１）。ＤＭＴＵ能保护心肌细胞抗Ｈ２Ｏ２损伤，机制与灭活羟自由基（·ＯＨ）、保护ＳＯＤ活性有关     

褪黑素通过激活Akt信号通路缓解高糖诱发的原代心肌细胞损伤

目的 明确高糖对心肌细胞损伤的影响;揭示褪黑素（Mel）在高糖诱发乳鼠原代心室肌细胞损伤中的作用及机制。 方法 体外培养乳鼠原代心室肌细胞,分为4组:正常葡萄糖浓度组(NG)、高糖组(HG,HG=25 mmo/L)、高糖+褪黑素组（HG+Mel,HG=25 mmo/L;Mel=30 μmo/L）、高糖+褪黑素+ PI3K/Akt抑制剂组（HG+Mel,HG=25 mmo/L;Mel=30 μmo/L;LY294002=50 μmo/L）组,采用Western Blotting、RT-PCR和免疫荧光技术检测心肌细胞凋亡相关蛋白以及PI3K/p-Akt等指标,评价褪黑素对高糖诱发心肌细胞凋亡的作用及机制。 结果 与NG组比较,HG组心肌细胞Cl -Caspase3、Caspase9的mRNA及蛋白表达明显升高,伴有p-Akt的mRNA及蛋白表达降低和PI3K蛋白表达降低;与HG组相比,HG+Mel组心肌细胞Cl-Caspase3、Caspase9的mRNA及蛋白表达下调伴有p-Akt的mRNA及蛋白表达回升和PI3K蛋白表达升高;与HG+Mel组相比,HG+Mel+LY294002组心肌细胞的Cl-Caspase3、Caspase9的mRNA及蛋白表达上升伴有p-Akt蛋白表达水平显著降低和PI3K蛋白表达降低。免疫荧光的结果与Western Blotting、RT-PCR的结果趋势一致。 结论 褪黑素通过激活PI3K/Akt信号通路缓解高糖诱导的原代心肌细胞损伤。     

缬沙坦对过氧化氢损伤乳鼠心肌细胞的保护作用

目的探讨缬沙坦对过氧化氢（H2O2）致心肌细胞氧化损伤的保护作用及其机制。方法用H2O2作用于新生SD大鼠心肌细胞,建立心肌细胞H2O2损伤模型。用缬沙坦预处理后,观察心肌细胞结构,测定心肌细胞存活率、乳酸脱氢酶（LDH）、丙二醛（MDA）及一氧化氮（NO）水平。结果缬沙坦提高心肌细胞过氧化损伤的细胞存活率（P〈0.05）,减少损伤时LDH、MDA和NO的生成（P〈0.05）。结论缬沙坦通过抗氧化、抗脂质过氧化反应,对心肌细胞过氧化损伤起保护作用。     

维拉帕米对心肌细胞肥大的影响

目的研究维拉帕米对心肌细胞肥大的影响及其机制。方法测定培养的心肌细胞［Ｃａ２＋］ｉ和３Ｈ－亮氨酸（３Ｈ－Ｌｅｕ）参入。结果２×１０－７ｍｏｌ／Ｌ维拉帕米对培养的心肌细胞［Ｃａ２＋］ｉ和３Ｈ－Ｌｅｕ参入无明显影响（Ｐ＞０．０５），２×１０－６～１０－５ｍｏｌ／Ｌ维拉帕米则可显著抑制培养的心肌细胞［Ｃａ２＋］ｉ和３Ｈ－Ｌｅｕ参入。与对照组相比，心肌细胞［Ｃａ２＋］ｉ和３Ｈ－Ｌｅｕ参入分别减低１０％～２７％（Ｐ＜０．０１）和１２％～２５％（Ｐ＜０．０５）。结论维拉帕米可以抑制培养的心肌细胞肥大，作用机制可能与其降低细胞内游离钙和细胞收缩性有关。     

Influence of melatonin on free radical-induced changes in rat pancreatic beta-cells in vitro

Free radicals may produce cytotoxicity to pancreatic islets under pathophysiological conditions. The aim of our in vitro investigations was to compare functional and morphological changes in pancreatic beta-cells induced by reactive oxygen species (ROS) generated by alloxan or xanthine oxidase/hypoxanthine (XO/HX), respectively. We demonstrate that short-term exposure to alloxan or to XO/HX leads to a temporarily elevated insulin release from isolated pancreatic islets. On application of alloxan, this effect is caused by beta-cell necrosis and can be prevented by administration of melatonin, while in contrast, XO/HX did not lead to long-term morphological changes in the majority of the cells. Among the cells destroyed by alloxan, only necrosis could be detected, while in contrast, some apoptotic cells were identified by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) reaction and electron microscopic examinations of cells treated with XO/HX. Melatonin was able to prevent the changes caused by alloxan, but failed to influence the alterations caused by XO/HX. Using electron spin resonance and lipid peroxidation assay, respectively, it was confirmed that melatonin effectively detoxifies hydroxyl radicals. Therefore, we believe that hydroxyl radicals are the toxic principle of alloxan, but not of XO/HX toxicity.     

Scavenging effect of melatonin on hydroxyl radicals generated by alloxan

Alloxan can act as a generator of reactive oxygen species (ROS) as long as sufficient suitable reducing agents (e.g. reduced glutathione) and oxygen are available. Using electron spin resonance-spectroscopy and the oxygen-centered spin trap DEPMPO, we demonstrate that hydroxyl radicals (OH.) are formed in vitro by alloxan in the presence of glutathione (GSH) and chelated divalent iron. Furthermore, peroxidation of polyunsaturated fatty acids from phosphatidylcholine-containing liposomes with concomitant formation of malondialdehyde (MDA) was used as a further indicator for a preceding OH. formation. Melatonin, the main secretory product of the pineal gland, is an effective scavenger of OH.. The 50%-inhibitor concentration (IC50-value) for melatonin to scavenge OH. generated from the alloxan/GSH-reaction in the presence of ferrous ions was 23 micromol/L. In contrast to the ability to effectively scavenge OH., the potential of melatonin to prevent lipid peroxidation is considerably less pronounced.     

Visualization of the antioxidative effects of melatonin at the mitochondrial level during oxidative stress-induced apoptosis of rat brain astrocytes

Oxidative stress-induced mitochondrial dysfunction has been shown to play a crucial role in the pathogenesis of a wide range of diseases. Protecting mitochondrial function, therefore, is vital for cells to survive during these disease processes. In this study, we demonstrate that melatonin, a chief secretory product of the pineal gland, readily rescued mitochondria from oxidative stress-induced dysfunction and effectively prevented subsequent apoptotic events and death in rat brain astrocytes (RBA-1). The early protection provided by melatonin in mitochondria of intact living cells was investigated by the application of time-lapse conventional, confocal, and multiphoton fluorescent imaging microscopy coupled with noninvasive mitochondria-targeted fluorescent probes. In particular, we observed that melatonin effectively prevented exogenously applied H2O2-induced mitochondrial swelling in rat brain astrocytes at an early time point (within 10 min) and subsequently reduced apoptotic cell death (150 min later). Other early apoptotic events such as plasma membrane exposure of phosphatidyl serine and the positive YOPRO-1 staining of the early apoptotic nucleus were also prevented by melatonin. A mechanistic study at the mitochondrial level related to the early protection provided by melatonin revealed that the indole molecule significantly reduced mitochondrial reactive oxygen species (ROS) formation induced by H2O2 stress. Melatonin also prevented mitochondrial ROS generation caused by other organic hydroperoxides including tert-butyl hydroperoxide and cumene hydroperoxide. This antioxidative effect of melatonin is more potent than that of vitamin E. Via its ability to reduce mitochondrial ROS generation, melatonin prevented H2O2-induced mitochondrial calcium overload, mitochondrial membrane potential depolarization, and the opening of the mitochondrial permeability transition (MPT) pore. As a result, melatonin blocked MPT-dependent cytochrome c release, the downstream activation of caspase 3, the condensation and karyorrhexis of the nucleus and apoptotic fragmentation of nuclear DNA. Thus, the powerful mitochondrial protection provided by melatonin reinforces its therapeutic potential to combat a variety of oxidative stress-induced mitochondrial dysfunctions as well as mitochondria-mediated apoptosis in various diseases.     

The utility of melatonin in reducing cerebral damage resulting from ischemia and reperfusion

The brain is highly susceptible to focal or global ischemia. Unless ischemia is promptly reversed, reperfusion produces further cerebral damage. Acute thrombolysis or defibrinogenation is effective only in selective patients with ischemic stroke and carries a significant risk of bleeding complications. Whereas numerous neuroprotectants were shown to be effective in experimental studies, none of them have been shown to work in clinical trials. The major pathogenetic mechanisms of ischemia/reperfusion injury include excitotoxicity, disturbed calcium ion homeostasis, overproduction of nitric oxide and other free radicals, inflammation, and apoptosis. Nitric oxide and other free radicals, the key mediators of excitotoxicity and disturbed calcium ion homeostasis, cause direct injury and also indirectly damage via inflammation and apoptosis. Melatonin is a potent free radical scavenger and an indirect antioxidant. This mini review summarizes the in vivo and in vitro evidence that melatonin protects against ischemia/reperfusion injury. There is convincing evidence from the literature that melatonin treatment is highly effective in different in vivo and in vitro models of excitotoxicity or ischemia/reperfusion in multiple animal species. Melatonin is safe and non-toxic in humans, and its administration via the oral route or intravenous injection is convenient. While more experimental studies should be conducted to further explore the neuroprotective mechanisms and to document any synergistic or additive protection from combining melatonin with thrombolysis, defibrinogenation or other neuroprotectants, interested clinical scientists should consider planning phase II and III studies to confirm the benefit of melatonin as an acute stroke treatment or a preventive measure for stroke patients.     

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.     

Attenuation of cold-induced apoptosis by exogenous melatonin in carrot suspension cells: the possible involvement of polyamines

Pretreatment with 43 nM (10 ng/mL) to 86 nM melatonin for 5 days significantly attenuated cold-induced apoptosis in carrot suspension cells (Daucus carota L.) as evidenced by the TUNEL procedure, DNA fragmentation and the morphological changes revealed by electronic microscopy observations. The antiapoptotic effect of melatonin was initially thought to be a result of its antioxidant actions. In our study, however, reactive oxygen species (ROS) generation remained unaffected by melatonin treatment, suggesting that melatonin plays its protective role not related to its direct ROS scavenger. At the same time, notable increases in putrescine and spermidine levels were observed in melatonin-treated cells, which may be responsible for the alleviation of the cold-induced apoptosis. The possible involvement of polyamines in the antiapoptotic effect of melatonin was further confirmed by the inhibitory effect of exogenous polyamines on apoptosis as displayed by the DNA laddering assay.     

Extracellular vesicles and melatonin benefit embryonic develop by regulating reactive oxygen species and 5-methylcytosine

Embryo culture conditions are crucial as they can affect embryo quality and even offspring. Oviductal extracellular vesicles (EVs) long been considered a major factor influencing interactions between the oviduct and embryos, and thus its absence is associated with inferior embryonic development in in vitro culture. Herein, we demonstrated that melatonin is present in oviduct fluids and oviduct fluid-derived EVs. Addition of either EVs (1.87 × 10¹¹ particles/mL) or melatonin (340 ng/mL) led to a significant downregulation of reactive oxygen species (ROS) and 5-methylcytosine (5-mC), as well as an increase in the blastocyst rate of embryos, which was inhibited by the addition of luzindole—a melatonin receptor agonist. A combination of EVs (1.87 × 10¹⁰ particles/mL) and melatonin (at 34.3 pg/mL) led to the same results as well as a significant decrease in the apoptosis index and increase in the inner cell mass (ICM)/trophectoderm (TE) index. These results suggest that an EV-melatonin treatment benefits embryonic development. Our findings provide insights into the role of EVs and melatonin during cell communication and provide new evidence of the communication between embryos and maternal oviduct.     

Reduced oxidative damage in ALS by high-dose enteral melatonin treatment

Amyotrophic lateral sclerosis (ALS) is the collective term for a fatal motoneuron disease of different etiologies, with oxidative stress as a common molecular denominator of disease progression. Melatonin is an amphiphilic molecule with a unique spectrum of antioxidative effects not conveyed by classical antioxidants. In preparation of a possible future clinical trial, we explored the potential of melatonin as neuroprotective compound and antioxidant in: (1) cultured motoneuronal cells (NSC-34), (2) a genetic mouse model of ALS (SOD1(G93A)-transgenic mice), and (3) a group of 31 patients with sporadic ALS. We found that melatonin attenuates glutamate-induced cell death of cultured motoneurons. In SOD1(G93A)-transgenic mice, high-dose oral melatonin delayed disease progression and extended survival. In a clinical safety study, chronic high-dose (300 mg/day) rectal melatonin was well tolerated during an observation period of up to 2 yr. Importantly, circulating serum protein carbonyls, which provide a surrogate marker for oxidative stress, were elevated in ALS patients, but were normalized to control values by melatonin treatment. This combination of preclinical effectiveness and proven safety in humans suggests that high-dose melatonin is suitable for clinical trials aimed at neuroprotection through antioxidation in ALS.