N-acetyl Cysteine Protects Motor Neurons Against Glutamate Exitotoxicity And The Mechnisms Involved In

Objective:Amyotrophic lateral sclerosis (ALS) is an adult-onset, progressive and lethal neurodegenerative disease, characterized by the degeneration of motor neurons from cerebral cortex, brainstem, and spinal cord. ALS is one of the most common neurodegenerative disorders, occurring both sporadically (sALS) and as a familial disorder (fALS) with inherited cases accounting for about 10% of patients. Using the current standard therapy, the typical survival time for patients after diagnosis is 3 to 5 years, although large deviation has been observed. Present evidence indicates that loss of neurons in ALS could not be due to a single factor,but results from a complex interplay among oxidative stress, glutamate excitotoxicity, dysfunction of autoimmunity, genetic factors,aggregation and/or dysfunction of critical proteins. Recent investigations support that among these mechanisms,oxidative stress is a common final pathway.So far,an effective disease-modifying treatment for this disabling and fatal disease is lacking.That is,proven therapeutic options are limited to riluzole,a glutamate release inhibiting drug that only extends the survival in humans by approximately 3 months without showing any therapeutic effect on motor function,muscle strength or motor symptoms.Hence,the development of innovative and more effective neuroprotective strategies for this devastating disease is of utmost importance.Green tea is a world wide beverage.It has been used in the oriental countries for a long time and traditionally thought to have diverse effects.Recently,it has been demonstrated that green tea contains many compounds that have many pharmacologic activities..Flavonoids,major polyphenolic compounds found in green tea, including epigallocatechin gallate (EGCG), epicatechin (EC), epigallocatechin (EGC) and epicatechin gallate (ECG) ,protect neuronal cells from oxidative stress.EGCG is one of the most well-known green tea polyphenolics (GTPs) with diverse effects,such as anti-apoptotic, anti-cancer, anti- mutagenic,and anti-inflamentory effects.The exact mechanisms of neuroprotective effect of EGCG have not been clearly understood.Previous studies suggest that EGCG protect neuronal cells via free radical scavenging,metal chelation,the enhancing expression of cell survival gene and inhibition of apoptotic gene,the regulation of cell signal transduction pathway,mitochondrial function,and ubiquitin-protesome system.Previously,in the threohydroxyaspartate (THA) induced glutamate exitotoxicity organotypic spinal cord culture model,we pretreated explants with EGCG, and then discovered that EGCG could not only protect motor neurons,alleviate the extent of cell injury and the level of lipoperoxidation,but also regulate the glutamate concentration in the culture medium.This is an exiciting result,because in the in vivo exitotoxicity model,EGCG have both anti-oxidative and glutamate regulating effects.However,owing to the simultaneous antioxidative effect of EGCG,we do not know whether the regulation of glutamate of EGCG is related to its anti-oxidative effect.N-acetyl cysteine (NAC) is a well-known antioxidative agent.It plays antioxidative role directly or provides substrate to synthesis of glutathione (GSH),which is a potent scanvenging agent of reactive oxygen species (ROS) and lipoperoxidative products.So,in the current study,the protection to motor neurons of NAC and the mechanisms involved in are tested.Methods:Organotypic spinal cord cultures were prepared using lumber spinal cord slices from 7-day-old rat pups.Lumber spinal cords were collected under sterile condition and sectioned transversly at 350μm intervals with a McIIwain tissure chopper.Slices were carefully placed on the surface of Millipore Millicell-CM insert (five slices per membrane).Cultures were incubated at 37℃in the 5%CO2 and 95% humidified environment.To establish the glutamate exitotoxicity spinal cord organotypic culture model,the culture medium were continuously added with 100μmol/L THA,an inhibitor of glutamate transporter.After one week of recovery,explants were treated with 100μmol/L NAC for 48 hours,and then the combination of 100μmol/L THA and 100μmol/L NAC for 3 weeks.Then collect the explants and culture medium to be tested.Motor neurons in the ventral horn were identified and evaluated by SMI-32 immunohistochemical staining.The lactate- dehydrogenase (LDH) activity in the culture medium was assayed to evaluate the extent of cell injury.The malondialdehyde (MDA) content in the spinal cord explants were assayed to evaluate the level of lipoperoxidation.The glutamate concentration in the culture medium was assayed to determine whether NAC could regulate glutamate level.Results:The spinal cord slices of normal control group had excellent organtypic cellular organization and their size became larger.There are about 24.13±3.44 motor neurons in the ventral horn in control group.The number of motor neurons in the ventral horn in THA group was markedly reduced compared with the control,only 6.27±1.62(P<0.05). Moreover,the glutamate concentration and LDH activity in the culture medium ,the MDA content in the spinal cord explants were significantly increased(P<0.05). Conversely,pretreatment with NAC significantly increased the number of motor neurons in the ventral horn ,19.29±2.13(P<0.05), and decreased the activity of LDH in the culture medium and the MDA content in the explants (P<0.05)compared with those in THA group. However, the glutamate concentration in the culture medium had no significant change(P>0.05).Conclusion:In the glutamate exitotoxicity spinal cord organotypic culture model,antioxidative agent NAC can protect motor neurons,and simultaneously alleviate the extent of cell injury and lipoperoxidation.However,NAC had no effect on the glutamate level in the culture medium.That is,the regulation of glutamate concentration in the culture medium of EGCG is not an additional result of antioxidation.In contrast,it is EGCG itself that regulate the glutamate level in the culture medium.