Protective Effect Of Combination Of Sulforaphane And Riluzole On Glutamate- Mediated Excitotoxicity

Amyotrophic lateral sclerosis (ALS) is one of the most common neurodegenerative disorders characterized by the progressive and selective death of upper and lower motor neurons. The disease is characterized by progressive musele weakness and the patient's death is usually caused by respiratoy failure,occurring 3-5 years after the first appearance of symptoms. The cause of this process is mostly unknown and there has been no effective treatment. Riluzole, which was the only drug currently registered for ALS, prolonged survival by only approximately three months but its serious makes it impossible to use extensively.Several mechanisms for the pathogenesis of ALS have been proposed: 1)mutations of the copper/zinc superoxide dismutase (Cu/Zn SOD) gene;2)glutamate excitotoxicity;3)mitochondrial dysfunction;4)oxidative stress;5)Immunity and inflammation,et al. It has been demonstrated that several mechanisms have been proposed to work together in contributing to the disease. Excitotoxicity and oxidative stress are the important pathogenic factors. Glutamate is a major excitatory neurotransmitter, which play important role in maintaining the neuron's function. the level of extracellular or synaptic cleft glutamate is tightly controlled by glutamate transporters, . Glutamate excitotoxicity is thought to result mainly from excess Ca2+ entry into neurons triggered by over-stimulation of postsynaptic glutamate receptor, and a large rise in intracellular Ca2+ in these cells causes rapid mitochondrial Ca2+ overload, resulting in mitochondrial damage and mitochondrial generation of reactive oxygen species (ROS). Mitochondrial damage may lead to necrosis and apoptosis, as a result of potential loss of ATP synthesis and release to cytoplasm of apoptogenic factors. Increased ROS levels may cause oxidative damage within motor neurons and also cause oxidative damage and disruption of glutamate transport in surrounding astrocytes, the latter resulting from the release of ROS from injured motor neurons.Riluzole is the only drug proven to slow the disease process in humans and has anti-excitotoxic properties. A growing body of work shows that riluzole has neuroprotective properties in some animal models and in cell cultures. riluzole can reduce glutamate release and enhance the activity of the glutamate transporters.Sulforaphane (SF), which is present in broccoli, can activate the Nrf2-ARE signaling, which functions as one of the most important anti-oxidant defense mechanisms by inducing and up-regulating many cytoprotective and antioxidant phase II enzymes. Therefore, the Nrf2-ARE signaling has been recognized as the novel pharamacological target for ALS treatment. Nrf2 is a nuclear transcription factor in cytoplasm,while antioxidant response element (ARE) , which is present in the promoter regions of many antioxidant genes. Nrf2 is sequestered in the cytoplasm by interacting with two molecules of Keap1. Oxidative stress and other inducers can result in a conformational change that renders Keap1 unable to bind to Nrf2, thereby inducing translocation of Nrf2 to the nucleus and binds to the antioxidant response element (ARE) .In our research, we use the organotypic spinal cord cultures interfered with Threohydroxyaspartate (THA) as our research model. The objective of the current study is to investigate whether the combination of SF and riluzole is superior to either one used alone. Our results suggest that the combination of sulforaphane and riluzone was more effective than each drug used alone in the protective action against glutamate-mediated excitotoxicity. It represents a novel approach in potential combination therapy for ALS.Part I Study the model of glutamate excitotoxicityObjective: To study the model of THA-induced spinal cord organotypic culture.Methods: The SD rat pups spinal cord organotypic cultures were divided into two groups at random: control and 100μmol/L THA group. The number of motor neurons was assessed by immunohistochemistry and the level of LDH,MDA and glutamate in the medium was assayed with ELIASA.Results: At the end of the 3-week THA treatment, we found that the motor neurons number in the group treated with THA was less than the control group and the level of LDH,MDA and glutamate in the medium in THA group is higher than that in the control group.Conclusions: Threohydroxyaspartate (THA) causes glutamate excitotoxicity in motor neurons in organotypic culture of rat spinal cord. So the model is an ideal model for studying chronic glutamate-induced excitotoxicity.PartⅡThe objective of the current study is to investigate whether the combination of SF and riluzole is superior to either one used aloneObjective: To investigate whether SF can act together with riluzole in protecting motor neurons against glutamate-induced excitotoxicity in organotypic spinal cord cultures.Results: In riluzole (5μM) , SF (10μM) and the combination treatment groups, the number of motor neuron is higher than that in THA group, and there was no significant difference among the three groups. In riluzole (5μM) , SF (10μM) and the combination treatment groups, the level of MDA,LDH in the medium is lower than that in THA group, and there was no significant difference among the three groups.There was no difference among the SF (4μM) treatment group,riluzole (2μM) treatment group and the THA group, as measured by the number of motor neuron, medium MDA, and LDH level. In the combination treatment group, the number of motor neuron was significantly higher than that in the THA group, the SF(4μM) group and the riluzole(2μM) group. In the combination treatment group, the level of LDH and MDA was significantly lower than that in the THA group, the SF(4μM) group and the riluzole(2μM) group.Conclusions: The combination treatment is more effective than each drug used alone. PartⅢInvestigate the neuroprotective mechanisms of the combinationObjective: To investigate the neuroprotective mechanisms of the combination of sulforaphane with riluzole.Methods: The SD rat pups spinal cord organotypic cultures were divided into five groups at random: control, the THA group, the SF treatment group,. the riluzole treatment group and the combination treatment group. the explants are harvested for measurement of expression of Nrf2, NADPH: quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1) by immunoblotting analysis. The level of glutamate was assayed with ELIASA.Results: At the end of the treatment, Nrf2 accumulated and the expression of two Phase II enzymes, including HO-1 and NQO1, increased in the SF treatment group and the combination treatment group. In the SF treatment group and the combination treatment group, the level of glutamate in the medium is lower than that in the THA treatment group.Conclusions: combination of SF and riluzole can stimulate the expression of phase II enzymes and reduces glutamate accumulation in the extracellular space.Conclusions: The neuroprotective mechanisms of the combination ar is that combination treatment can stimulate the expression of phase II enzymes and reduces glutamate accumulation in the extracellular space.