Paraoxonase-1介导硫化氢的抗甲醛神经毒性作用

我们以往的研究工作证实了硫化氢(hydrogen sulfide,H2S)对甲醛神经毒性和氧化应激具有拮抗作用.Paraoxonase-1(PON-1)是机体重要的内源性抗氧化剂.本研究的目的是探讨PON-1是否可介导H2S的抗甲醛神经毒性作用.采用甲醛损伤PC12细胞为甲醛神经毒性的细胞模型.硫氢化钠(NaHS,一种H2S的供体)不仅可以上调PC12细胞PON-1的活力,还可恢复甲醛对PC12细胞PON-1表达与活力的抑制作用.2-hydroxyquinoline(2-HQ)是一种选择性PON-1抑制剂,它可显著降低H2S对甲醛细胞毒性、凋亡和活性氧(reactive oxygen species,ROS)累积的抑制作用.而且,2-HQ可阻止H2S逆转甲醛激活PC12细胞caspase-3和下调PC12细胞bcl-2表达.结果提示H2S依赖PON-1去保护PC12细胞对抗甲醛的神经毒性.我们的这一发现表明PON-1有希望成为防治甲醛神经损伤的新靶点.     

Metallothionein Inducers Protect Against 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Neurotoxicity in Mice

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a drug that induces parkinsonism in human and non-human primates. Free radicals are thought to be involved in its mechanism of action. Recently, the participation of metallothionein as scavenger of free radicals has been proposed. In this work, we studied the effect of metallothionein inducers in MPTP neurotoxic action. Male swiss albino mice were pretreated either with cadmium (1 mg/kg) or dexamethasone (5 mg/kg), two well-known inducers of metallothionein synthesis, and 5 hours later with an MPTP administration (30 mg/kg). Treatment schedule was repeated daily for either 3 or 5 consecutive days. All animals were killed 7 days after the last administration, and striatal dopamine and homovanillic acid contents were analyzed as an end-point of MPTP neurotoxicity. Striatal dopamine content of cadmium plus MPTP-treated animals (3-days) increased by 32%, and 48% (5-days) vs MPTP-alone animals. Dexamethasone plus MPTP-treated group also showed increased dopamine levels 28% (3-days) and 43% (5-days). MPTP treatment reduced striatal metallothionein concentration (49% vs control animals). Dexamethasone and cadmium increased metallothionein concentrations in MPTP-treated groups, by 77% and 82% respectively. Results suggest that metallothionein induction provide a significant resistance factor against the deleterious effect of MPTP.     

Evidence for Hydroxyl Radical Scavenging Action of Nitric Oxide Donors in the Protection Against 1-Methyl-4-phenylpyridinium-induced Neurotoxicity in Rats

In the present study we provide evidence for hydroxyl radical (^•OH) scavenging action of nitric oxide (NO^•), and subsequent dopaminergic neuroprotection in a hemiparkinsonian rat model. Reactive oxygen species are strongly implicated in the nigrostriatal dopaminergic neurotoxicity caused by the parkinsonian neurotoxin, 1-methyl-4-phenylpyridinium (MPP⁺). Since the role of this free radical as a neurotoxicant or neuroprotectant is debatable, we investigated the effects of some of the NO^• donors such as S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine hydrochloride (SIN-1), sodium nitroprusside (SNP) and nitroglycerin (NG) on in vitro ^•OH generation in a Fenton-like reaction involving ferrous citrate, as well as in MPP⁺-induced ^•OH production in the mitochondria. We also tested whether co-administration of NO^• donor and MPP⁺ could protect against MPP⁺-induced dopaminergic neurotoxicity in rats. While NG, SNAP and SIN-1 attenuated MPP⁺-induced ^•OH generation in the mitochondria, and in a Fenton-like reaction, SNP caused up to 18-fold increase in ^•OH production in the latter reaction. Striatal dopaminergic depletion following intranigral infusion of MPP⁺ in rats was significantly attenuated by NG, SNAP and SIN-1, but not by SNP. Solutions of NG, SNAP and SIN-1, exposed to air for 48 h to remove NO^•, when administered similarly failed to attenuate MPP⁺-induced neurotoxicity in vivo. Conversely, long-time air-exposed SNP solution when administered in rats intranigrally, caused a dose-dependent depletion of the striatal dopamine. These results confirm the involvement of ^•OH in the nigrostriatal degeneration caused by MPP⁺, indicate the ^•OH scavenging ability of NO^•, and demonstrate protection by NO^• donors against MPP⁺-induced dopaminergic neurotoxicity in rats.     

21-Aminosteroids Interact with the Dopamine Transporter to Protect Against 1-Methyl-4-Phenylpyridinium-Induced Neurotoxicity

U-78518F, a 21-aminosteroid from the novel family of lipid peroxidation inhibitors (lazaroids), increased survival of dopamine (DA) neurons in mesencephalic cell cultures incubated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Protection against DA neuron death occurred with increasing concentrations of U-78518F up to 30 microM. Non-specific toxicity produced with higher concentrations of MPP+ was not affected by the lazaroid. U-78518F inhibited cellular uptake of [3H]MPP+ and [3H]DA, but not that of gamma-[3H]aminobutyric acid. In human striatal membrane preparations, U-78518F competed with [3H]mazindol for binding to the DA transporter, with a calculated Ki value of 10 microM. Two of four lazaroids tested inhibited [3H]DA uptake in the cell culture system. The protective effects of 21-aminosteroids in MPP(+)-induced neurotoxicity are, in part, a function of the interaction of these agents with the DA transporter.     

Nrf3通路参与星形胶质细胞对抗鱼藤酮毒性的保护作用

目的：寻找星形胶质细胞在对抗由鱼藤酮导致的氧化应激中发挥保护作用的相关分子并探讨其作用机制。 方法：小鼠多巴胺能MN9D细胞分别在星形胶质细胞条件培养液（ACM）与星形胶质细胞用新鲜培养基中培养24小时后加入鱼藤酮作用48小时。细胞计数,评价星形胶质细胞条件培养液对MN9D细胞的保护作用。利用基因芯片技术寻找MN9D细胞在ACM处理后发生表达上调或下调基因,并对这些基因进行分析,找出有意义基因。结果： 在不同作用时间和鱼藤酮浓度梯度下,经过ACM处理的MN9D细胞活性显著高于在普通培养基中培养的细胞。初步得到104个差异表达基因,其中62个表达上调基因,42个表达下调基因。这些基因主要与凋亡、肿瘤、细胞周期、代谢、信号转导、转录调节、翻译调节和传递蛋白等相关。对其中的Atp5a1,Nrf3基因进行分析,发现Nrf3通路参与了ACM的保护作用。结论： ACM能保护MN9D细胞抵抗鱼藤酮所致的细胞毒性, Atp5a1,Nrf3,GCL,NQO1等基因经ACM处理后发生差异表达,可能是星形胶质细胞保护作用的部分下游信号通路。     

内源性气体硫化氢在中枢神经系统中的作用

内源性硫化氢(H2S)可以刺激神经细胞cAMP水平增加,提高NMDA受体介导的突触后兴奋性电位,提高诱导海马长时程增强。H2S不仅具有神经调节剂的功能,还有神经保护剂的功能。H2S自身并不能将细胞从氧化应激中解救出来,但是它能通过提高胞内有效的抗氧化剂——还原型谷胱苷肽的含量而起到保护神经元的作用。对H2S的研究刚刚起步,对其在神经系统中的作用机制开展研究将有助于了解其在神经元保护方面所起的作用。     

Neuroprotective Effects of Farnesene Against Hydrogen Peroxide-Induced Neurotoxicity In vitro

Oxidative stress is highly damaging to cellular macromolecules and is also considered a main cause of the loss and impairment of neurons in several neurodegenerative disorders. Recent reports indicate that farnesene (FNS), an acyclic sesquiterpene, has antioxidant properties. However, little is known about the effects of FNS on oxidative stress-induced neurotoxicity. We used hydrogen peroxide (H₂O₂) exposure for 6 h to model oxidative stress. Therefore, this experimental design allowed us to explore the neuroprotective potential of different FNS isomers (α-FNS and β-FNS) and their mixture (Mix-FNS) in H₂O₂-induced toxicity in newborn rat cerebral cortex cell cultures for the first time. For this aim, both MTT and lactate dehydrogenase assays were carried out to evaluate cell viability. Total antioxidant capacity (TAC) and total oxidative stress (TOS) parameters were used to assess oxidative alterations. In addition to determining of 8-hydroxy-2-deoxyguanosine (8-OH-dG) levels in vitro, the comet assay was also performed for measuring the resistance of neuronal DNA to H₂O₂-induced challenge. Our results showed that survival and TAC levels of the cells decreased, while TOS, 8-OH-dG levels and the mean values of the total scores of cells showing DNA damage (comet assay) increased in the group treated with H₂O₂ alone. But pretreatment of FNS suppressed the cytotoxicity, genotoxicity and oxidative stress, which were increased by H₂O₂ in clear type of isomers and applied concentration-dependent manners. The order of antioxidant effectiveness for modulating H₂O₂-induced oxidative stress-based neurotoxicity and genotoxicity is as β-FNS > Mix-FNS > α-FNS.     

Studies on the Neurotoxicity of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine: Inhibition of NAD-Linked Substrate Oxidation by Its Metabolite, 1-Methyl-4-Phenylpyridinium

Abstract: The effects of the parkinsonism-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its 4-electron oxidation product 1-methyl-4-phenylpyridinium (MPP⁺) were studied in isolated mitochondria and in mouse brain striatal slices. ADP-stimulated oxidation of NAD-linked substrates was inhibited in a time-dependent manner by MPP⁺ (0.1–0.5 m M ), but not MPTP, in mitochondria prepared from rat brain, mouse brain, or rat liver. Under identical conditions, succinate oxidation was relatively unaffected. In neostriatal slices prepared from the mouse, a species susceptible to the dopaminergic neurotoxicity of MPTP, incubation with either MPP⁺ or MPTP caused metabolic changes consistent with inhibition of mitochondnial oxidation, i.e., an increase in the formation of lactate and accumulation of the amino acids glutamate and alanine with concomitant decreases in glutamine and aspartate levels. The changes resulting from incubation with MPTP were prevented by the monoamine oxidase inhibitor pargyline, which blocks formation of MPP⁺ from MPTP. The results suggest that compromise of mitochondrial function and its metabolic sequelae within dopaminergic neurons could be an important factor in the neurotoxicity observed after MPTP administration.     

Effects of 1-Methyl-4-Phenyl- 1,2,3,6-Tetrahydropyridine and 1 -Methyl-4-Phenylpyridinium Ion on ATP Levels of Mouse Brain Synaptosomes

Mouse brain synaptosomes, essentially devoid of mitochondrial contamination, were used as a model to study the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) on the levels of ATP of neuronal terminals. Similar to known inhibitors of ATP synthesis, both MPTP and MPP+ caused a dramatic depletion of synaptosomal ATP. This depletion was dose dependent and occurred as a relatively early biochemical event in the absence of any apparent damage to synaptosomal membranes. MPP+ was more effective than its parent compound in decreasing ATP; it induced a significant loss at concentrations (10-100 microM) similar to those it reaches in the brain in vivo. MPTP-induced ATP depletion was completely prevented by the monoamine oxidase B inhibitor deprenyl, which, on the contrary, was ineffective against MPP+. As expected in view of the heterogeneous population of nerve terminals present in our synaptosomal preparations, the catecholamine uptake blocker mazindol did not significantly affect the ATP loss caused by both compounds. Data indicate that (1) administration of MPTP may cause a depletion of ATP within neuronal terminals resulting from the generation of MPP+, and (2) exposure to the levels of MPP+ reached in vivo may cause biochemical changes that are nonselective for dopaminergic terminals.     

Role of Lipoamide Dehydrogenase and Metallothionein on 1-Methyl-4-phenyl-1,2,3,6- tetrahydropyridine-induced Neurotoxicity

In the present study, we investigated the effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on lipoamide dehydrogenase activity and metallothionein content. Lipoamide dehydrogenase is a flavoprotein enzyme, which reduces lipoamide and low molecular weight thiols. This enzyme has also been involved in the conversion of ubiquinone (coenzyme Q-10, oxidized form) to ubiquinol (reduced form). Lipoamide dehydrogenase activity was measured spectrophotometrically following its incubation with different doses of MPTP, MPP⁺, and divalent metals. MPTP at higher concentrations inhibited the lipoamide dehydrogenase activity, whereas it’s potent toxic metabolite 1-methyl-4-phenylpyridinium (MPP⁺) had a similar effect at lower concentration. Calcium and copper did not affect the enzyme activity at any of the doses tested, whereas, zinc dose dependently enhanced the lipoamide dehydrogenase activity. Additionally, levels of metallothionein in the mouse nigrostriatal system were measured by cadmium affinity method following administration of MPTP. Metallothionein content was significantly reduced in the substantia nigra (SN), and not in the nucleus caudatus putamen (NCP) following a single administration of MPTP (30 mg/kg, i.p.). Our results suggests that both lipoamide dehydrogenase activity and metallothionein levels may be critical for dopaminergic neuronal survival in Parkinson’s disease and provides further insights into the neurotoxic mechanisms involved in MPTP-induced neurotoxicity.     

Neurochemical Evidence for Agmatine Modulation of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP) Neurotoxicity

Agmatine treatment is known to exert neuroprotective effects in several models of neurotoxic and ischemic brain and spinal cord injuries. Here we sought to find out whether agmatine treatment would also prove to be neuroprotective in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson’s disease. Concomitant daily treatment (intraperitoneal injections) with agmatine (100 mg/kg for 5 days) and MPTP (40 mg/kg for 2 days) exacerbated MPTP-related toxicity as evidenced by a larger reduction in dopamine uptake into striatal synaptosomes (42.4% as compared to 58.3% of control, respectively). In contrast, agmatine treatment commencing after MPTP, produced partial protection (31%) against MPTP dopaminergic toxicity. The findings implicate agmatine in mechanisms regulating MPTP neurotoxicity, but underscore the characteristic neuroprotective efficacy of agmatine when applied after the insult.     

Modulatory Effects of Testosterone on 1-Methyl-4-Phenyl- 1, 2, 3, 6-Tetrahydropyridine-Induced Neurotoxicity

Abstract: In this experiment, we examined the modulatory effects of testosterone on the parkinsonism-inducing drug 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) in two strains of mice. Orchidectomized male CD-1 and C57/BI mice were implanted with either empty Silastic capsules or capsules containing testosterone and subsequently treated with MPTP. A small area of the corpus striatum was removed for determination of dopamine (DA) content, whereas the remainder was superfused and used to measure L-DOPA (5 μ M )-evoked DA release. In animals treated with MPTP, L-DOPA-evoked DA release was reduced significantly in CD-1 mice, but not in C57/BI mice, treated with testosterone. No differences in L-DOPA-stimulated DA release between MPTP- versus vehicle-treated mice was observed in either the CD-1 or C57/BI mice receiving empty Silastic capsules. Corpus striatum DA contents were more severely depleted in the MPTP-sensitive C57/BI versus the CD-1 mouse strain irrespective of hormone treatment. These results confirm previous results demonstrating differences in these two mouse strains in response to the neurotoxic effects of MPTP upon corpus striatum DA content. More interestingly, they show an important differential modulatory effect of testosterone upon L-DOPA-evoked DA release as a function of MPTP treatment and indicate that testosterone significantly alters the neurotoxic effects of MPTP in the CD-1 mouse.     

1-Methyl-4-phenyl-pyridinium ion-induced oxidative stress, c-Jun phosphorylation and DNA fragmentation factor-45 cleavage in SK-N-SH cells are averted by selegiline

Parkinson's disease is a progressive neurodegenerative disorder, associated with the selective loss of dopaminergic neurons in the substantia nigra pars compacta. Recent studies have shown that c-Jun-N terminal kinase pathways might be involved in the oxidative stress-induced neuronal demise. In addition, there are several studies demonstrating that selegiline protects neural cell degeneration. In view of the above, the toxic effects of MPP(+) and the protective roles of selegiline were studied in cultures of human neuroblastoma (SK-N-SH) cell lines in the present study. MPP(+) significantly decreased cell viability but increased reactive oxygen species formation and lipid peroxidation, and the said effects were attenuated by selegiline. MPP(+) did not change the total levels of c-Jun but enhanced phosphorylation of c-Jun at Ser73 and cleavage of DNA fragmentation factor 45, which were diminished by selegiline. MPP(+)-treated SK-N-SH cells exhibited an irregularly shaped nuclear chromatin or DNA fragmentation, which was abolished by selegiline. These data suggest that c-Jun-N terminal kinase pathways are involved in oxidative stress-induced dopaminergic neuronal degeneration and pretreatment with selegiline affords neuroprotection by inhibiting these cell death-signaling pathways.     

EGb761 Pretreatment Reduces Monoamine Oxidase Activity in Mouse Corpus Striatum During 1-Methyl-4-Phenylpyridinium Neurotoxicity

EGb761 produces reversible inhibition of both monoamine oxidase (MAO) isoforms in the central nervous system. 1-Methyl-4-phenylpyridinium (MPP+) neurotoxicity is prevented by treatment with the MAO inhibitor pargyline. We investigated EGb761's effect on striatal MAO activity during MPP+ neurotoxicity. C-57 black mice were pretreated with EGb761 (10 mg/kg) daily for 17 days followed by administration of MPP+ (0.72 mg/kg). MPP+ enhanced striatal MAO (30%) activity at 6 h, and EGb761 prevented this effect. MAO-B activity in striatum was enhanced (70%) 6 h after MPP+ administration and was reduced to almost normal levels in EGb761 + MPP+ group compared to MPP+ group. Pretreatment with EGb761 partially prevented (32%) the striatal dopamine-depleting effect of MPP+ and prevented the reduction in striatal tyrosine hydroxylase activity (100%). Results suggest that EGb761 supplements may be effective in reducing MAO activity as well as enhancement in dopamine metabolism, thereby preventing MPP+-neurotoxicity.     

Existence of a Hydrogen Sulfide:Ferric Ion Oxidoreductase in Iron-Oxidizing Bacteria

The existence of a hydrogen sulfide:ferric ion oxidoreductase, which catalyzes the oxidation of elemental sulfur with ferric ions as an electron acceptor to produce ferrous and sulfite ions, was assayed with washed intact cells and cell extracts of various kinds of iron-oxidizing bacteria, such as Thiobacillus ferrooxidans 13598, 13661, 14119, 19859, 21834, 23270, and 33020 from the American Type Culture Collection, Leptospirillum ferrooxidans 2705 and 2391 from the Deutsche Sammlung von Mikroorganismen, L. ferrooxidans BKM-6-1339 and P3A, and moderately thermophilic iron-oxidizing bacterial strains BC1, TH3, and Alv. It was found that hydrogen sulfide:ferric ion oxidoreductase activity comparable to that of T. ferrooxidans AP19-3 was present in all iron-oxidizing bacteria tested, suggesting a wide distribution of this enzyme in iron-oxidizing bacteria.     

Effects of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine and 1-Methyl-4-Phenylpyridinium Ion on Activities of the Enzymes in the Electron Transport System in Mouse Brain

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium ion (MPP+) on activities of enzyme complexes in the electron transport system were studied using isolated mitochondrial preparations from C57BL/6J mouse brains. Both MPTP and MPP+ dose-dependently inhibited activity of NADH-ubiquinone oxidoreductase (EC 1.6.5.3). The inhibition was reversible. Preincubation of freeze-thawed mitochondria with MPTP or MPP+ had no effect on the inhibition; however, when nonfrozen mitochondria were used, NADH-ubiquinone oxidoreductase activity was reduced to 46% of that in the nonincubated sample after a 5-min preincubation with MPTP and to 77% of that in the nonincubated sample after a 5-min preincubation with MPP+. Kinetic analyses revealed that inhibition of MPTP was noncompetitive and that of MPP+ uncompetitive with respect to NADH. On the other hand, inhibition of MPTP was uncompetitive and that of MPP+ noncompetitive with respect to ubiquinone. Succinate-ubiquinone oxidoreductase (complex II), dihydroubiquinone-cytochrome c oxidoreductase (complex III), and ferrocytochrome c-oxygen oxidoreductase (EC 1.9.3.1) activities were either slightly inhibited or not inhibited by MPTP or MPP+. The significance of these findings is discussed in relation to the mechanism of MPTP-induced neuronal degeneration.     

RIPK1/RIPK3-Mediated Necroptosis is Involved in Sevoflurane-Induced Neonatal Neurotoxicity in the Rat Hippocampus

Cellular and Molecular Neurobiology - Recent studies have shown that exposure to sevoflurane in developing brains causes neuronal apoptosis and cognitive dysfunction. “Necroptosis” is a...     

1-Methyl-4-phenyl-pyridinium ion-induced oxidative stress,c-Jun phosphorylation and DNA fragmentation factor-45 cleavage in SK-N-SH cells are averted by selegiline

Parkinson’s disease is a progressive neurodegenerative disorder, associated with the selective loss of dopaminergic neurons in the substantia nigra pars compacta. Recent studies have shown that c-Jun-N terminal kinase pathways might be involved in the oxidative stress-induced neuronal demise. In addition, there are several studies demonstrating that selegiline protects neural cell degeneration. In view of the above, the toxic effects of MPP⁺ and the protective roles of selegiline were studied in cultures of human neuroblastoma (SK-N-SH) cell lines in the present study. MPP⁺ significantly decreased cell viability but increased reactive oxygen species formation and lipid peroxidation, and the said effects were attenuated by selegiline. MPP⁺ did not change the total levels of c-Jun but enhanced phosphorylation of c-Jun at Ser73 and cleavage of DNA fragmentation factor 45, which were diminished by selegiline. MPP⁺-treated SK-N-SH cells exhibited an irregularly shaped nuclear chromatin or DNA fragmentation, which was abolished by selegiline. These data suggest that c-Jun-N terminal kinase pathways are involved in oxidative stress-induced dopaminergic neuronal degeneration and pretreatment with selegiline affords neuroprotection by inhibiting these cell death-signaling pathways.