The Protective Effect Of Uric Acid On The Damage Of PC12 Cells Induced By 6-OHDA

Parkinson`s disease (PD) is a common neruodegenerative disease characterized slow progress, along with prevalenc and incidence rate that increases significantly with age. There are about 2 million patients in China.An estimated 1.07% of population older than 55 year, 2.5% of population older than 75 year has PD.There will be about 5million patients with PD in 2030 in China.PD is characterized by selective degeneration of dopaminergic cells in the substania nigra and the nigrostriatal pathway.This progress is contribute to a considerable amount of oxidative stress and mitochondrial dysfunction.When patiant show the motion disorder, the dopaminergic neurons in substantia nigra nigrostriatal lost more than 50% and the same time the level of dopamine in nigrostriatal decreased more than 80%.Because there is not a kind of way for cure ,as early as using neuroprotective treatment is very important for patients with PD.Uric acid (UA) is an end-rpoduct ofadenosine and metabolismpurine which is an important natural antioxidant.UA can scavenge 60% free radical in vivo include O2-,OH, O2- et al. It also can prevent degradation of SOD.SOD could prevent the reaction of NO and O2- from nitrite and can prevent protein from nitroing.UA is an iron chelator which inhibits the damage by oxidation stress depending on iron and ascorbate. Furthermore UA can protect DA cells through glial cells.Recently research showed that the level of plasm urate were associated with the progress of PD.PC12 cells, a cell line derived from rat adrenal pheochromocytomam cells, possess intracellular substrates for dopamine(DA) synthesis, metabolism and transport. 6-Hydroxydopamine(6-OHDA), a hydroxylated analogue of dopamine, can induce apoptosis and necrosis of dopaminergic neurons. 6-OHDA could be detected in brain and urine of patients with PD. Our research is to investigate whether UA can protect PC12 cells from the damage of 6-OHDA.Part one: The Effect of Uric Acid and 6-Hydroxydopamine(6-OHDA) On PC12 cellsAim: To investigate the effect of uric acid and 6-Hydroxydopamine(6-OHDA) On PC12 cellsMethods: PC12 cells were cultured with DMEM content 10% calf serum. The culture medium was changed every 2~3 days.Effect of 6-OHDA and UA on the PC12 cells were determined by MTT assay. In MTT assay, PC12 cells were divided into 6-OHDA group: control group(DMEM), 25, 50, 75, 100, 125μmol/L6-OHDA group and 100,200,300, 400,500μmol/L UA group. Each group was treated with 6-OHDA or UA for 24 hours. Results:By MTT assay, PC12 cells were treated with 25,50,75,100, 125μmol/L 6-OHDA for 24 hours, the survial rate decreased:93.3±6.0%, 79.86±5.20%, 55.92±3.72%, 47.05±2.06% and 46.56±3.43% (P<0.01);The PC12 cells were treated with 100,200, 300, 400, 500μmol/L UA for 24 hours, the survival rate:are 93.3±6.0%,98.9±6.8%, 105.0±4.0%, 106.6±5.0% and 131.5±16.4%.100~400μmol/L of UA had no significant effect on the PC12 cells,500μmol/L UA significantly increased the survial compared with the contorl group.(P<0.01)Conclusions: 6-hydroxy-dopamine has toxicity on PC12 cells.UA of certain concentration have no significant effect on the survial rate of PC12 cells. Part two:The protective effect of uric acid on the cell damage of PC12 cells induced by 6-OHDAAim: To investigate the toxicity of 6-hydroxy-dopamine on PC12 cells and protective effect of UA against it.Methods: PC12 cells were cultured with DMEM content 10% calf serum. The culture medium was changed every 2~3 days.Toxicity of 6-hydroxy-dopamine on PC12 cells was detected by MTT assay, and protective action of UA on them was detected under the same conditions. PC12 cells were treated with 6-OHDA, UA for 6,12 and 24 hours. In MTT assay, PC12 cells were divided groups:control group(DMEM),6-OHDA(50μmol/L)group, UA(100, 200, 300,400μmol/L)group and UA(100μmol/L)+6-OHDA(50μmol/L)group, UA(200μmol/L)+6-OHDA(50μmol/L)group,UA(300μmol/L)+6-OHDA(50μmol/L) group, UA(400μmol/L)+6-OHDA(50μmol/L)group.Choose one of UA concentration which has no significant effect on PC12 cells survial rate.After PC12 cells treated for 24h with 6-OHDA or/and UA, to detect the caspase-3 activity in PC12 cells with immunofluorescence technology and calculate apoptosis rate by flow cytometry(FCM) technique with Annexin V/PI double staining. PC12 cells were divided groups:control group(DMED), 6-OHDA(50μmol/L)group,UA(200μmol/L)group and UA(200μmol/L)+6-OHDA(50μmol/L)group PC12 cells were treated with 6-OHDA, UA for 24 hours.Results:50μmol/L of 6-OHDA decreased the survival rates of of PC12 cells 95.5±2.4%,88.7±5.1% and 73.8±10.0% at 6, 12, 24h.Compared with control group, the survial rates of PC12 cells treated with 6-OHDA for 12 and 24 hours decreased significantly. 100~400μmol/L of UA has no significant effect on the survival rates of PC12 cells.100~400μmol/L of UA reversed the decrase of PC12 cells induced by 6-OHDA.The check of caspase-3 activity showed dim red in the control group and UA group, intensive red in 50μmol/L 6-OHDA group and weaken red in 200μmol/L UA+50μmol/L 6-OHDA group.This indicated the caspase-3 protein level increased after 6-OHDA (50μmol/L) treatment for 24h, which were decreased by UA(200μmol/L) treatment. Annexin V/PI calculating apoptosis rate using FCM discovered 3.5±1.3% and 3.2±0.8% in the control group and 200μmol/LUA group, which were not statistics different.Compared with 9.8±2.3% in 50μmol/L6-OHDA group, the apoptosis rate 6.0±1.5% in 200μmol/LUA+50μmol/L 6-OHDA group decreased significantly. Conclusions 6-OHDA can decrease the survival rate of PC12 cells and increase the apoptosis of PC12 cells.Uric acid can protect PC12 cells from injury induced by 6-OHDA.Overall Conclusions:1.6-hydroxy-dopamine of certain concentration has toxicity on PC12 cells.2.Uric acid of certain concentration has no significant affect on the survial rate of PC12 cells.3.The appropriate doeses of UA can reversed the decrease of the survival rate of PC12 cells induced by 6-OHDA.