S-adenosylmethionine (SAMe) modulates interleukin-10 and interleukin-6, but not TNF, production via the adenosine (A2) receptor

S-adenosylmethionine (SAMe) is the first product in methionine metabolism and serves as a precursor for glutathione (GSH) as well as a methyl donor in most transmethylation reactions. The administration of exogenous SAMe has beneficial effects in many types of liver diseases. One mechanism for the hepatoprotective action is its ability to regulate the immune system by modulating cytokine production from LPS stimulated monocytes. In the present study, we investigated possible mechanism(s) by which exogenous SAMe supplementation modulated production of TNF, IL-10 and IL-6 in LPS stimulated RAW 264.7 cells, a murine monocyte cell line. Our results demonstrated that exogenous SAMe supplementation inhibited TNF production but enhanced both IL-10 and IL-6 production. SAMe increased intracellular GSH level, however, N-acetylcysteine (NAC), the GSH pro-drug, decreased the production of all three cytokines. Importantly, SAMe increased intracellular adenosine levels, and exogenous adenosine supplementation had effects similar to SAMe on TNF, IL-10 and IL-6 production. 3-Deaza-adenosine (DZA), a specific inhibitor of S-adenosylhomocysteine (SAH) hydrolase, blocked the elevation of IL-10 and IL-6 production induced by SAMe, which was rescued by the addition of exogenous adenosine. Furthermore, the enhancement of LPS-stimulated IL-10 and IL-6 production by both SAMe and adenosine was inhibited by ZM241385, a specific antagonist of the adenosine (A(2)) receptor. Our results suggest that increased adenosine levels with subsequent binding to the A(2) receptor account, at least in part, for SAMe modulation of IL-10 and IL-6, but not TNF production, from LPS stimulated monocytes.     

Protective effect of S-adenosylmethionine on cellular and mitochondrial membranes of rat hepatocytes against tert-butylhydroperoxide-induced injury in primary culture

Accumulating evidence that administration of S-adenosylmethionine (SAMe) protects hepatocytes against oxidative stress-mediated injury led us to evaluate the effect of SAMe on hepatocyte injury induced in culture by oxidant substance tert-butylhydroperoxide (1.5 mM tBHP) with regard to prevent mitochondrial injury. The pretreatment of hepatocyte culture with SAMe in doses of 0.25, 0.5, 1, 2.5, 5, 10, 25 and 50 mg/l for 30 min prevented the release of LDH from cells incubated for 30 min with tBHP in a dose dependent manner. The inhibitory effect of SAMe on lipid peroxidation paralleled the effect on cell viability. SAMe also moderated the decrease of the mitochondrial membrane potential induced by tBHP. Our results indicate that the inhibition of lipid peroxidation by SAMe can contribute to the prevention of disruption of both cellular and mitochondrial membranes. While the protective effect of SAMe against tBHP-induced GSH depletion was not confirmed, probably the most potent effect of SAMe on membranes by phospholipid methylation should be verified.     

The effect of S-adenosyl-L-methionine (SAM) on membrane permeability in the perfused rat liver

S-adenosilmethionine is present in most human tissues and is an important factor for transmethylation, transulphuration and aminopropylation reactions. The compound improves the biological, morphological and histochemical aspects of rat liver following CCl4 intossication. At the same time has been successfully used during chronic liver disease in man. With the aim to better clarify the action mechanism of SAMe some aspects concerning its effects on cell permeability in rat liver, by using the perfusion technique, have been investigated. In particular the capacity of this compound to prevent the enzymatic loss (GPT and GOT) during liver perfusion has been studied. 30 perfusions without SAMe, as control, and 6 by infusing 2 mg of compound during the perfusion time have been accomplished. Varing the perfusion time from 0 to 120 min it has been observed that at any time the presence of the SAMe reduced by about 50% the loss of GOT. Similarly the activity of GPT ranging from 2 to 6 mU/ml indicate that no appreciable enzyme output occurs in presence of SAMe.     

扩散性抑制对脑缺血后海马迟发性神经元死亡的影响

目的为了研究阻断大鼠局灶性脑缺血诱导的扩散性抑制对同侧海马迟发性神经元死亡的影响。方法颈内动脉插线法制备大鼠大脑中动脉缺血再灌注模型,采用电生理学方法记录扩散性抑制波,尼氏染色和TUNEL染色检测海马迟发性神经元死亡;观察阻断局灶性脑缺血再灌注诱导的扩散性抑制对海马迟发性神经元死亡的影响。结果不给予SD阻断剂,大脑中动脉缺血模型有39%的动物出现海马迟发性神经元死亡;用MK-801阻断扩散性抑制后仅10%的动物出现海马迟发性神经元死亡,机率明显减小。结论局灶性脑缺血引起的海马迟发性神经元死亡可能与扩散性抑制由缺血区不断向远隔部位播散有关。     

The effect of S-adenosyl-l-methionine on ischemia-induced disturbances of brain phospholipid in the gerbil

Brain ischemia was produced in gerbils (Meriones unguiculatus) by the bilateral ligation of the carotid arteries with reported procedures. Changes in the energy status of brain demonstrated that carotid ligation was effective. At different time intervals from ligation, groups of gerbils were given either saline of S-Adenosyl-L-methionine (SAMe) by the intraventricular (i.v.) route (1.6 mg/Kg body wt. twice, at each 10 min interval), or by the intraperitoneal (i.p.) administration (200 mg/Kg body wt.) or subcutaneously (s.c.) with 40 mg/Kg body wt, daily, for two weeks. Control animals, with and without SAMe, together with the ischemic groups, were decapitated directly into liquid nitrogen, 10 min after ligation. Brain neutral and polar lipid, together with free fatty acids, which were all labeled in vivo by the intraventricular injection of [1-14C]arachidonic acid 2 hr prior to ligation, were extracted, purified and separated by conventional procedures. SAMe when injected i.v. or i.p. noticeably corrected the changes in polar lipid by reversing the decrease of brain phosphatidylcholine and choline plasmalogen, as well as of their labeling, which was due to ischemia. Concurrently with this action, SAMe treatment (i.v. and i.p.) also provided to some extent to re-establish the normal level of labeling of ethanolamine lipids. When SAMe was given s.c., no effect was present. SAMe had no effect on the increase of free fatty acid and diglyceride due to ischemia. The prevention by SAMe of the changes of choline lipids suggests that a stimulation of the methyltransferase reaction may occur in the ischemic brain, due to increased substrate (SAMe) availability. This effect may be important for cell survival, since membrane phospholipid derangements alter the properties of the membrane.     

Delayed Neuronal Death After Brief Histotoxic Hypoxia In Vitro

Abstract: The effect of three metabolic inhibitors—iodoacetate, potassium cyanide, and potassium arsenate—on neuronal viability was studied in primary rat cortical and hippocampal CA1 neuronal cultures. Iodoacetate (0.1 m M ) applied for 5 min to 8-day-old cultures resulted in delayed neuronal death within 3–24 h in cortical and hippocampal CA1 neurons. Neuronal degeneration was preceded by transient inhibition of energy metabolism to ∼40% and a permanent inhibition of protein synthesis to ∼50%. The inhibition of protein synthesis and the neuronal death were prevented by the free radical scavenger vitamin E but not by the glutamate antagonist MK-801. Removal of calcium during iodoacetate exposure could not protect against toxicity, and there was no increase of intracellular calcium concentration during and shortly after iodoacetate treatment. Cyanide and arsenate produced only partial neuronal degeneration, even at a dose of 10 m M . These observations demonstrate that brief exposure of neurons to low concentrations of iodoacetate produces a delayed type of neuronal death that is not mediated by either calcium or glutamate. The therapeutic effect of vitamin E points to a free-radical mediated injury and suggests that this type of pathology may also be involved in delayed neuronal death after transient energy depletion in vivo.     

阻断缝隙连接对脑缺血后海马迟发性神经元凋亡的影响

目的探讨阻断缝隙连接(gap junction)通讯对大鼠局灶性脑缺血后海马迟发性神经元死亡(delayed neuronal death,DND)及Bcl-2蛋白表达的影响。方法术前2h左侧脑室注射缝隙连接阻断剂甘珀酸(carbenoxolone,CBX),对照组左侧脑室注射生理盐水,颈内动脉插线法制备大鼠大脑中动脉缺血再灌注模型,采用DNA原位末端标记TUNEL技术及免疫荧光技术,观察阻断缝隙连接对大鼠局灶性脑缺血3d后海马迟发性神经元死亡及BCL-2蛋白表达的影响。结果不给予缝隙连接阻断剂,大脑中动脉缺血模型有45%的大鼠在术后3d出现海马迟发性神经元死亡;用甘珀酸阻断缝隙连接后,30%的大鼠出现海马迟发性神经元死亡,其发生率明显减小(P<0.01);与对照组相比,干预组Bcl-2蛋白的表达较高(P<0.01),两组Bcl-2蛋白的表达均高于假手术组(P<0.01)。结论阻断缝隙连接通讯可以减少局灶性脑缺血后海马迟发性神经元死亡的发生率,Bcl-2参与了局灶性脑缺血后海马神经元凋亡的调节。     

S-adenosylmethionine exerts a protective effect against thioacetamide-induced injury in primary cultures of rat hepatocytes

S-adenosylmethionine (SAMe) has been shown to protect hepatocytes from toxic injury, both experimentally-induced in animals and in isolated hepatocytes. The mechanisms by which SAMe protects hepatocytes from injury can result from the pathways of SAMe metabolism. Unfortunately, data documenting the protective effect of SAMe against mitochondrial damage from toxic injury are not widely available. Thioacetamide is frequently used as a model hepatotoxin, which causes in vivo centrilobular necrosis. Even though thioacetamide-induced liver necrosis in rats was alleviated by SAMe, the mechanisms of this protective effect remain to be verified. The aim of our study was to determine the protective mechanisms of SAMe on thioacetamide-induced hepatocyte injury by using primary hepatocyte cultures. The release of lactate dehydrogenase (LDH) from cells incubated with thioacetamide for 24 hours, was lowered by simultaneous treatment with SAMe, in a dose-dependent manner. The inhibitory effect of SAMe on thioacetamide-induced lipid peroxidation paralleled the effect on cytotoxicity. A decrease in the mitochondrial membrane potential, as determined by Rhodamine 123 accumulation, was also prevented. The attenuation by SAMe of thioacetamide-induced glutathione depletion was determined after subsequent incubation periods of 48 and 72 hours. SAMe protects both cytoplasmic and mitochondrial membranes. This effect was more pronounced during the development of thioacetamide-induced hepatocyte injury that was mediated by lipid peroxidation. Continuation of the SAMe treatment then led to a reduction in glutathione depletion, as a potential consequence of an increase in glutathione production, for which SAMe is a precursor.     

细胞周期调控对大鼠全脑缺血后海马迟发性神经元死亡的影响

目的观察细胞周期调控对大鼠全脑缺血再灌流后海马区迟发性神经元死亡(delayed neuronal death,DND)以及星形胶质细胞的活化、增殖的影响.方法建立大鼠短暂性全脑缺血再灌流模型,利用尼氏染色、TUNEL、免疫组织化学方法观察再灌流后细胞周期素依赖的蛋白激酶(cyclin depedent kinase, CDK)抑制剂Olomoucine对海马DND以及星形胶质细胞活化增殖的影响.结果全脑缺血再灌流后3d、7d、30d海马神经元明显脱失,部分CA1、CA2区神经元凋亡;星形胶质细胞数目增多,GFAP表达上调,应用Olomoucine后TUNEL阳性神经元数目明显减少,幸存神经元数目增加;星形胶质细胞数目无明显增多,GFAP表达明显下调.结论 CDK抑制剂Olomoucine可有效抑制大鼠全脑缺血后海马神经元DND以及星形胶质细胞活化增殖.     

Delayed Postconditionig Initiates Additive Mechanism Necessary for Survival of Selectively Vulnerable Neurons After Transient Ischemia in Rat Brain

1. The aim of this study was to validate the role of postconditioning, used 2 days after lethal ischemia, for protection of selectively vulnerable brain neurons against delayed neuronal death.2. Eight, 10, or 15 min of transient forebrain ischemia in rat (four-vessel occlusion model) was used as initial lethal ischemia. Fluoro Jade B, the marker of neurodegeneration, and NeuN, a specific neuronal marker were used for visualization of changes 7 or 28 days after ischemia without and with delayed postconditioning.3. Our results confirm that postconditioning if used at right time and with optimal intensity can prevent process of delayed neuronal death. At least three techniques, known as preconditioners, can be used as postconditioning: short ischemia, 3-nitropropionic acid and norepinephrine. A cardinal role for the prevention of death in selectively vulnerable neurons comprises synthesis of proteins during the first 5 h after postconditioning. Ten minutes of ischemia alone is lethal for 70% of pyramidal CA1 neurons in hippocampus. Injection of inhibitor of protein synthesis (Cycloheximide), if administered simultaneously with postconditioning, suppressed beneficial effect of postconditioning and resulted in 50% of CA1 neurons succumbing to neurodegeneration. Although, when Cycloheximide was injected 5 h after postconditioning, this treatment resulted in survival of 90% of CA1 neurons.4. Though postconditioning significantly protects hippocampal CA1 neurons up to 10 min of ischemia, its efficacy at 15 min ischemia is exhausted. However, protective impact of postconditioning in less-sensitive neuronal populations (cortex and striatum) is very good after such a damaging insult like 15 min ischemia. This statement also means that up to 15 min of ischemia, postconditioning does not induce cumulation of injuries produced by the first and the second stress.     

Bradykinin Postconditioning Protects Pyramidal CA1 Neurons Against Delayed Neuronal Death in Rat Hippocampus

Aims The present study was undertaken to evaluate possible neuroprotective effect of bradykinin against delayed neuronal death in hippocampal CA1 neurons if applied two days after transient forebrain ischemia in the rat. Methods Transient forebrain ischemia was induced in male Wistar rats by four-vessel occlusion for 8 min. To assess efficacy of bradykinin as a new stressor for delayed postconditioning we used two experimental groups of animals: ischemia 8 min and 3 days of survival, and ischemia 8 min and 3 days of survival with i.p. injection of bradykinin (150 μg/kg) applied 48 h after ischemia. Results We found extensive neuronal degeneration in the CA1 region at day 3 after ischemia/reperfusion. The postischemic neurodegeneration was preceded by increased activity of mitochondrial enzyme MnSOD in cytoplasm, indicating release of MnSOD from mitochondria in the process of delayed neuronal death. Increased cytosolic cytochrome c and subsequently caspase-3 activation are additional signs of neuronal death via the mitochondrial pathway. Bradykinin administration significantly attenuated ischemia-induced neuronal death, and also suppressed the release of MnSOD, and cytochrome c, and prevented caspase-3 activation. Conclusions Bradykinin can be used as an effective stressor able to prevent mitochondrial failure leading to apoptosis-like delayed neuronal death in postischemic rat hippocampus.     

Sendai virus vector-mediated gene transfer of glial cell line-derived neurotrophic factor prevents delayed neuronal death after transient global ischemia in gerbils.

We have developed a cytoplasmic replicating virus vector of Sendai virus (SeV) that infects and replicates in most mammalian cells, including neurons, and directs high-level gene expression. To investigate the protective effect of SeV vector-mediated gene transfer of glial cell line-derived neurotrophic factor (GDNF) on the delayed neuronal death caused by transient global ischemia in gerbils, SeV vectors carrying either GDNF (SeV/GDNF) or enhanced green fluorescent protein gene (SeV/GFP) were stereotaxically microinjected into the lateral ventricle. Four days after injection, occlusion of the bilateral common carotid arteries for 5 min produced transient global forebrain ischemia. Treatment with SeV/GDNF significantly decreased the delayed neuronal death of the hippocampal CA1 pyramidal neurons observed 6 days after the operation. TUNEL staining demonstrated that SeV/GDNF treatment markedly reduced the number of apoptotic cells in the hippocampal CA1 neurons, indicating that SeV/GDNF treatment prevented apoptosis. Furthermore, delayed neuronal death on the contralateral side of the hippocampal CA1 was also prevented to a similar extent as that on the ipsilateral side. These results suggest that SeV/GDNF prevents the delayed neuronal death induced by ischemia and is potentially useful for gene therapy for stroke.     

Carnosine protects neurons against oxidative stress and modulates the time profile of MAPK cascade signaling

Carnosine is a known protector of neuronal cells against oxidative injury which prevents both apoptotic and necrotic cellular death. It was shown earlier that carnosine serves as an intracellular buffer of free radicals. Using the model of ligand-dependent oxidative stress in neurons, we have shown that homocysteine (HC) initiates long-term activation of extracellular signal regulated kinase, isoforms 1 and 2 (ERK 1/2) and Jun N-terminal kinase (JNK) which corresponds to exitotoxic effect resulting in cellular death. L-carnosine (β-alanyl-L-histidine) protects neurons from both excitotoxic effect of homocysteine and cellular death. Its analogs, β-alanyl-D-histidine (D-carnosine) and L-histidyl-β-alanine, restricted accumulation of free radicals and delayed activation of ERK1/2 and JNK in neuronal cells, but did not promote neuronal viability.     

Endogenously released DOPA is a causal factor for glutamate release and resultant delayed neuronal cell death by transient ischemia in rat striata

Glutamate is implicated in neuronal cell death. Exogenously applied DOPA by itself releases neuronal glutamate and causes neuronal cell death in in vitro striatal systems. Herein, we attempt to clarify whether endogenous DOPA is released by 10 min transient ischemia due to four-vessel occlusion during rat striatal microdialysis and, further, whether DOPA, when released, functions to cause glutamate release and resultant delayed neuronal cell death. Ischemia increased extracellular DOPA, dopamine, and glutamate, and elicited neuronal cell death 96 h after ischemic insult. Inhibition of striatal L-aromatic amino acid decarboxylase 10 min before ischemia increased markedly basal DOPA, tripled glutamate release with a tendency of decrease in dopamine release by ischemia, and exaggerated neuronal cell death. Intrastriatal perfusion of 10-30 nM DOPA cyclohexyl ester, a competitive DOPA antagonist, 10 min before ischemia, concentration-dependently decreased glutamate release without modification of dopamine release by ischemia. At 100 nM, the antagonist elicited a slight ceiling effect on decreases in glutamate release by ischemia and protected neurons from cell death. Glutamate was released concentration-dependently by intrastriatal perfusion of 0.3-1 mM DOPA and stereoselectively by 0.6 mM DOPA. The antagonist elicited no hypothermia during and after ischemia. Endogenously released DOPA is an upstream causal factor for glutamate release and resultant delayed neuronal cell death by brain ischemia in rat striata. DOPA antagonist has a neuroprotective action.     

Effect of opioids on delayed neuronal death in the gerbil hippocampus

The effect of opioids on delayed neuronal death was evaluated in the gerbil hippocampus. Male Mongolian gerbils were subjected to transient forebrain ischemia and neuronal density was evaluated in the hippocampus 7 days following ischemia. When hypothermia during and after ischemia was prevented treatment with morphine, U-50488H, or naloxone provided no significant protection. In contrast, a spontaneous drop in rectal temperature to 32°C at the end of ischemia produced near-complete protection of CA₁ pyramidal neurons. No opioids modulate the protective effect of hypothermia.     

DLPC and SAMe prevent alpha1(I) collagen mRNA up-regulation in human hepatic stellate cells, whether caused by leptin or menadione

We previously reported that the combination of dilinoleoylphosphatidylcholine (DLPC) and S-adenosylmethionine (SAMe), which have antioxidant properties and antifibrogenic actions, prevented leptin-stimulated tissue inhibitor of metalloproteinase (TIMP)-1 production in hepatic stellate cells (HSCs) by inhibiting H2O2-mediated signal transduction. We now show that DLPC and SAMe inhibit alpha1(I) collagen mRNA expression induced by leptin or menadione in LX-2 human HSCs. We found that DLPC and SAMe prevent H2O2 generation and restore reduced glutathione (GSH) depletion whether caused by leptin or menadione. Blocking H2O2 signaling through ERK1/2 and p38 pathways resulted in a complete inhibition of leptin or menadione-induced alpha1(I) collagen mRNA. The inhibition of collagen mRNA by DLPC and SAMe combined is at least two times more effective than that by DLPC or SAMe alone. In conjunction with the prevention of TIMP-1 production, the ability of DLPC and SAMe to inhibit alpha1(I) collagen mRNA expression provides a mechanistic basis for these innocuous compounds in the prevention of hepatic fibrosis, because enhanced TIMP-1 and collagen productions are associated with hepatic fibrogenesis and their attenuation may diminish fibrosis.     

Apoptosis-inducing factor is involved in the regulation of caspase-independent neuronal cell death

Caspase-independent death mechanisms have been shown to execute apoptosis in many types of neuronal injury. P53 has been identified as a key regulator of neuronal cell death after acute injury such as DNA damage, ischemia, and excitotoxicity. Here, we demonstrate that p53 can induce neuronal cell death via a caspase-mediated process activated by apoptotic activating factor-1 (Apaf1) and via a delayed onset caspase-independent mechanism. In contrast to wild-type cells, Apaf1-deficient neurons exhibit delayed DNA fragmentation and only peripheral chromatin condensation. More importantly, we demonstrate that apoptosis-inducing factor (AIF) is an important factor involved in the regulation of this caspase-independent neuronal cell death. Immunofluorescence studies demonstrate that AIF is released from the mitochondria by a mechanism distinct from that of cytochrome-c in neurons undergoing p53-mediated cell death. The Bcl-2 family regulates this release of AIF and subsequent caspase-independent cell death. In addition, we show that enforced expression of AIF can induce neuronal cell death in a Bax- and caspase-independent manner. Microinjection of neutralizing antibodies against AIF significantly decreased injury-induced neuronal cell death in Apaf1-deficient neurons, indicating its importance in caspase-independent apoptosis. Taken together, our results suggest that AIF may be an important therapeutic target for the treatment of neuronal injury.     

Melatonin is protective in necrotic but not in caspase-dependent, free radical-independent apoptotic neuronal cell death in primary neuronal cultures.

To assess the neuroprotective potential of melatonin in apoptotic neuronal cell death, we investigated the efficacy of melatonin in serum-free primary neuronal cultures of rat cortex by using three different models of caspase-dependent apoptotic, excitotoxin-independent neurodegeneration and compared it to that in necrotic neuronal damage. Neuronal apoptosis was induced by either staurosporine or the neurotoxin ethylcholine aziridinium (AF64A) with a delayed occurrence of apoptotic cell death (within 72 h). The apoptotic component of oxygen-glucose deprivation (OGD) unmasked by glutamate antagonists served as a third model. As a model for necrotic cell death, OGD was applied. Neuronal injury was quantified by LDH release and loss of metabolic activity. Although melatonin (0.5 mM) partly protected cortical neurons from OGD-induced necrosis, as measured by a significant reduction in LDH release, it was not effective in all three models of apoptotic cell death. In contrast, exaggeration of neuronal damage by melatonin was observed in native cultures as well as after induction of apoptosis. The present data suggest that the neuroprotectiveness of melatonin strongly depends on the model of neuronal cell death applied. As demonstrated in three different models of neuronal apoptosis, the progression of the apoptotic type of neuronal cell death cannot be withhold or is even exaggerated by melatonin, in contrast to its beneficial effect in the necrotic type of cell death.     

Calpain activates caspase-3 during UV-induced neuronal death but only calpain is necessary for death

While caspases have been strongly implicated in delayed neuronal death in a variety of experimental paradigms, other proteases such as calpain can also contribute to neuronal death. To evaluate the relative roles of caspase and calpain, we used a model system wherein UV treatment induced moderate or severe delayed cortical neuronal death, as quantified by propidium iodide and calcein AM. UV treatment led to increases in both caspase and calpain activation. Calpain inhibitor III (MDL-28170) reduced caspase activation, suggesting that caspase activation was mediated by calpain. Calpain contributed to neuronal death, as indicated by strong neuroprotection provided by calpain inhibitor III, calpeptin, or Ca2+-free medium. In contrast, caspase inhibitors were not neuroprotective. These results suggest that UV neurotoxicity is mediated by a loss of Ca2+ homeostasis which leads to a calpain-dependent, caspase-independent cell death. That calpain, but not caspase, may mediate death in instances involving the activation of both proteases may have relevance to other neuronal death models.