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Edaravone Protects Against 1-methyl-4-phenylpyridinium Induced Cytotoxicity in Rat Primary Cultured Astrocytes

Author: ChenZuo
Tutor: HuGang
School: Nanjing Medical University
Course: Pharmacology
Keywords: edaravone astrocyte apoptosis reactive oxygen species NADPH Oxidase
CLC: R96
Type: Master's thesis
Year: 2008
Downloads: 104
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Abstract

Edaravone (Eda) is a potent radical scavenger, exerting multiple benefical effects on patients with acute ischemic stroke. Eda can efficiently scavenge free radicals via transfering an electron to oxygen free radicals or fat free radicals. Previouse researches revealed that Eda have potent antioxidant effects against ischemia-reperfusion-induced injury, delayed neuronal death and concomitant neurological deficits. These evidences also suggested that Eda provides neuroprotective effects via other mechanisms which have not been found.Astrocytes, the most abundant glial cell type in the brain, provide metabolic and trophic support to neurons and modulate synaptic activity as well as survival of neurons. Increasing evidence indicates that glial cells form neural networks. The brain pathology is, in some sense, a pathology of glia. Glia cells are important for the survival of neurons and the defense of central nervous system. Therefore, modulation of astrocytic function may provide a novel therapeutic strategy for neurodegenerative disorders.Oxidative stress has been implicated in a range of degenerative diseases. Astrocytes play an important role in clearing production of reactive oxygen species (ROS) in the brain. Astrocytes may have a close relation with the selective vulnerability of neurons by scavenging ROS and releasing the precursor ofγ-L-glutamyl-L-cysteinyglycine (GSH) synthesis in neurons. Furthermore, alteration in astrocyte GSH level may be an important contributor to the pathogenesis of neurodegenerative disease.Mitochondria are not only major source of ROS generation in aerobic cells, but they are also sensitive target for the damaging effects of oxygen radicals. Mitochondria participate in the regulation of both energy metabolism and cell death. Excessive ROS production impairs mitochondria membrane system and activates mitochondrial permeability transition pore (PTP), resulting in the release of pro-apoptotic proteins which subquently initiated mitochondrial apoptotic pathway. Mitochondria dysfunction is a prominent feature in apoptosis, and release of pro-apoptotic proteins from the mitochondrial intermembrane space has been considered to be a critical event that occurs during apoptosis. Restoring mitochondrial function helps to provide neuroprotective effects on neurodegenerative disordersPrevious studies demonstrated that Eda exerted neuroprotective effects by inhibiting endothelial injury and ameliorating neuronal damage after brain ischemia insult. As astrocytes play critical roles in decreasing the oxidative damage in the brain and restoring neuronal function, we hypothesized that Eda promotes the survival of neurons via regulating astrocytic function. The neurotoxin 1-methyl-4-phenylpyridinium (MPP~+) is a high affinity inhibitor of mitochondrial complex I and is commonly used as a neurotoxin to induce cellular injury. In the present study, we investigated the effect of Eda on the MPP~+-induced cytotoxicity and ROS production in rat primary cultured astrocytes, focusing on mitochondrial function.AIM: To investigate the effects and possible mechanisms of Eda on MPP~+-induced cytotoxicity in rat primary cultured astrocytes.METHODS: Astrocytic apoptosis was determined by staining with Hoechst 33324 staining and LDH assay. The production of ROS was measured by DCFH-DA probe. GSH assay was performed by test kits purchased from Jian Cheng Bioengineering Institute. Mitochondria respiratory function was measured by using a Clark Electrode provided by Hansatech. Molecular probe JC-1 was used to detect mitochondrial membrane potential (△Ψ_m). We observed changes in mRNA levels of gp91 and p47 by RT-PCR semi-quantitative analysis. We introduced Western-blotting for the analyses of AIF, cyto c.RESULTS: 1) Treatment with MPP~+ (200μM) significantly induced astrocytic apoptosis and the release of LDH, Eda inhibited astrocytic apoptosis and reduced the LDH release induced by MPP~+; 2) Eda inhibited production of ROS through preventing GSH depletion and down-regulating mRNA expressions of NADPH Oxidase membrane subunit gp91 and membrane-translocated subunit p47 induced by MPP~+ (200μM); 3) Treatment with MPP~+ (50μM) significantly induced mitochondria oxidative respiratory dysfunction, Eda (1 and 10μM) prevented the decreases of ST3 and RCR induced by MPP~+ which inhibited ROS production of mitochondria source in astrocytes; 4) Eda protected astrocytes against MPP~+-induced apoptosis by inhibiting△Ψ_m loss and subsequently release of pro-apoptotic factors (cyto c and AIF) induced by MPP~+.In the present study, we found that Eda inhibited astrocytic apoptosis and reduced the LDH release induced by MPP~+. Eda could inhibit production of ROS through preventing GSH depletion and down-regulating mRNA expressions of NADPH Oxidase membrane subunit gp91 and membrane-translocated subunit p47. Furthermore, Eda not only ameliorated mitochondrial respiratory function but also prevented△Ψ_m loss, thereby inhibiting subsequent release of cyto c and AIF. These findings reveal that Eda protects astrocytes against MPP~+-induced apoptosis via modulating ROS level and mitochondrial apoptotic pathway.CONCLUSION: These results indicated that Eda could inhibit MPP~+-induced astrocytic apoptosis, the decrease of antioxidation and mitochondria dysfunction, suggesting that Eda may be a regulator of the astrocytic function.In summary, the present study firstly reveals that Eda protects against MPP~+-induced cytotoxicity in astrocytes via decreasing ROS production and inhibiting mitochondria apoptotic pathways. These findings provide a new perspective for Eda used as a neuroprotective agent.

Edaravone Protects Against 1-methyl-4-phenylpyridinium Induced Cytotoxicity in Rat Primary Cultured Astrocytes

Abstract

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