Protective Effects Of Resveratrol On Primary Spinal Cord Astrocytes Induced By Hydrogen Peroxide

Neurodegenerative diseases include a number of different pathological conditions, like Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's diseases.By far, the aetiology and pathology of these disease is unclear, Some of the therapeutic approaches that have used to the clinical arena cannot effectively control the progression of disease. It is extremely important to find a key therapeaic strategy and effective drug .Accumulating evidence suggests that astrocytes are associated with pathology and development of neuro- degenerative diseases referred to as taupathies. Astrocytes activation is the common pathology characteristic. Astrocytes contribute to a variety of functions of neurons, including release nutrition factor and remove glutamate of synaptic cleft, support and protect motor neuron. But when astrocytes are activatived, the shape and function is changed, the crosstalk between astrocytes and motor neuron is disordered, which can acc- elerate the deth of motor neuron.Imbalances in the generation of ROS and cellular antioxidant defenses lead to oxidative stress, which results in oxidative damage. The impairment of glutamate transporters causes excitotoxicity and leads to increase ROS production and consequent cell damage. Oxidative stress and excitotoxicity have long been associated with the development of pathological conditions in degenerative diseases. Both experimental and clinical results have shown that antioxidants are effective in reducing oxidative stress and are promising neuroprotectants in reducing oxidative damage.Recently, there is reported that the activation of Nrf2/ARE signalling pathway could induce a series of antioxidant enzyme, antioxidant protein, antiinflammatory and antitoxic protein. The expression of those antioxidant enzyme and antioxidant proteins in nervous system exhibited broad neuroprotection against injury by glutamate, hydrogen peroxide and dopamine. So Nrf2/ARE signalling pathway is expected a promising drug target to combat oxidative stress in neurodegenerative disorders.Resveratrol is an antioxidant component of grapes and red wine , exhibited a large spectrum of beneficial health effects including antioxidantactivities, cardioprotective, antiproliferat- ive and neuroprotective properties , active sirtuin pathway.It is capable of scavenging lipid hydroperoxyl free radical as well as hydroxyl and superoxide anion radicals.The most important effect is antioxidant activities and neuroprotective properties .H₂O₂ is a representative reactive oxidant species and causes cell death through apoptosis or necrosis. We use the primary spinal cord astrocytes culture to establish the model of oxidant stress, and then to study the protective effects of resveratrol on hydrogen peroxide induced toxicity in primary spinal cord astrocytes culture.We are in the point of antioxidantactivities and antiexcitotoxicity to understand resveratrol neuroprote- ctive properties.Objective: The study was aimed to investigate the antioxidantactivities of resveratrol by observing the protein expression of phase II detoxifying enzyme: NAD(P)H: quinone oxidoreductase 1 (NQO1) , GSH-S-transferase (GST) and NF-E2-related factor 2 (Nrf2) which is a transcription factor involved in the cellular protection against oxidative stress through antioxidant response element (ARE)-directed induction of several phase 2 detoxifying and antioxidant enzymes, the mRNA expression of Nrf2, and the culture medium level of MDA and LDH ,also observing the difference of ROS fluorescence intensity in astrocyte. We investigate the anti- excitotoxicity of resveratrol by observing the protein expression of EAAT1 and EAAT2, and the culture medium level of glutamate.Methods: Primary cultures enriched in astrocytes were established from spinal cord tissue of 1-2-day-old ICR mouse. After 2 weeks, glial cultures contained 95-98% GFAP+ astrocytes, and then purified and passaged the astrocytes .We used the third generation of astrocytes to intervention.The experiment had three group:normal control group;H₂O₂ model group(25uM H₂O₂,1h);RES pretreatment group(15uM RES24h +25uM H₂O₂,1h).We used Western blot to detect the protein expression of phase II detoxifying enzyme:NQO1, GST, NF-E2-related factor 2 (Nrf2) and EAAT1 and EAAT2,and used RT-PCR to detect the mRNA expression of Nrf2,and used confocal to detect the difference of ROS fluorescence intensity in astrocyte,and used assay Kit to detect the culture medium level of LDH,MDA and glutamate.Results:(1) In our study, in the situation of H₂O₂ induced oxidative stress, the protein expression of NQO1, GST and total Nrf2 were decreased (P<0.01) . Pretreatment of astrocytes cells with a non-toxic concentration(15 M) of resveratrol mitigated H₂O₂-induced toxicity upregulating protein of NQO1, GST and total Nrf2(P<0.05). (2) Treatment with resveratrol enhanced the expression of total Nrf2 mRNA compared to the control group(P<0.05). (3) In the situation of H₂O₂ induced oxidative stress, the culture medium level of LDH compared to the control group was increased. (P<0.05). Pretreatment of astrocytes cells with resveratrol decreased level of LDH compared to the H₂O₂ model group(P<0.05).(4) In the situation of H₂O₂, the culture medium level of MDA compared to the control group was increased. (P<0.05). Pretreatment of astrocytes cells with resveratrol decreased the level of MDA compared to the H₂O₂ model group(P<0.01). (5) In the situation of H₂O₂ induced oxidative stress,we observed an increase in ROS production in H₂O₂ model group(P<0.05).Pretreatment of astrocytes cells with resveratrol can decrase the ROS production compared to the H₂O₂ model group(P<0.01 ). (6) In H₂O₂ model group, the protein expression of EAAT1 and EAAT2were decreased compared to the control group (P<0.05). Pretreatment of astrocytes with resveratrol upregulated the protein expression of EAAT1 and EAAT2 compared to the H₂O₂ model group (P<0.05). (7) In the situation of H₂O₂ induced oxidative stress, the culture medium level of glutamate compared to the control group was increased(P<0.01). Pretreatment of astrocytes with resveratrol decreased the level of glutamate compared to the H₂O₂ model group(P<0.05).Conclusions:We use the primary spinal cord astrocytes culture to establish the model of oxidant stress, the production of ROS in astrocyte is increased, the protein expression of phase II detoxifying enzyme: NQO1,GST,totalNrf2,EAAT1 and glutamate transporter 1(GLT1) are decreased , the level of the culture medium level of MDA ,LDH and glutamate are increased. Pretreatment of astrocytes with a non-toxic concentration(15uM) of resveratrol mitigates H₂O₂-induced toxicity by decreaseing the production of ROS in astrocyte, upregulating the protein expression of phase II detoxifying enzyme: NQO1﹑GST , totalNrf2 and EAAT1 and EAAT2,and decreaseing the level of the culture medium level of MDA , LDH and glutamate. Forther more, pretreatment of astrocytes cells with a non-toxic concentration(15uM) of resveratrol can upregulate the the protein expression of EAAT1 and GLT1, the expression of total NRF2 mRNA compared to the control group. These have identified resveratrol as a promising neuroprotective agent by antioxidantactivities and anti- excitotoxicity, and suggest that the activation of Nrf2 signal pathway and depressing excitotoxicity may be new strategy in neurodegeneration disease.It can give a potentinal novel treatment for neurodegenerative disease.