The Mechanisms Of6-OHDA-induced Neurotoxicity In PC12Cells And Establishment Of The Model Of Parkinson’s Disease With Depression

Part ⅠThe effects of6-Hydroxydopamine on apoptosis in PC12cellsObjective:To study the effect of6-Hydroxydopamine (6-OHDA) on apoptosis in PC12cells, and the potential effects of N-acetyl cysteine (NAC) and Vesicular Monoamine Transporter (VMAT) inhibitors on apoptosis.Methods:Flow cytometry was used to measure apoptosis in PC12cells treated with NAC (20mmol/L) or reserpine (VMAT inhibitor,50,100,400,1600nmol/L)) in the presence of6-OHDA (25,50,100,200μmol/L) for0,12,24,36, and48h, respectively. Methyl thiazolyl tetrazolium (MTT) and Western-blot analysis were used to measure the cell viability and the expression of apoptosis-related proteins Bcl2and Bax.Results:The cell viability was decreased in a time-dependent manner, while the apoptotic rates were increased in PC12cells with the concentration of6-OHDA increasing. Western-blot analysis showed that the expression of Bcl2was down-regulated, whereas the expression of Bax was up-regulated in a time-dependent manner with the concentration of6-OHDA increasing. The treatment of NAC attenuated the decrease in the cell viability and the increase in the apoptotic rates in PC12cells treated with6-OHDA. NAC treatment also modulated the dysregulation of Bcl2and Bax induced by6-OHDA in PC12cells. However, the treatment of reserpine further decreased the cell viability and increased the apoptotic rates in PC12cells. Reserpine treatment also boost the dysregulated expression of Bcl2and Bax in PC12cells treated with6-OHDA.Conclusion:6-OHDA could induce decline in the cell viability and increased apoptosis in PC12cells in a time-and concentration-dependent manner.6-OHDA reduced the expression of Bcl2protein but increased the expression of Bax protein in PC12cells. NAC could protect against6-OHDA-induced neurotoxicity, while the inhibitor of VMAT aggravated the neurotoxicity. The protective effects of NAC and the toxic effects of reserpine could be related to the change of the expression of Bcl2and Bax proteins in PC12cells. Part ⅡThe mechanisms of6-Hydroxydopamine-induced apoptosis in PC12cellsObjective:To explore the possible involvement of the changes of mitochondrial memberane potential (△Ψm) and intercellular free calcium concentration ([Ca2+]i) in6-Hydroxydopamine (6-OHDA)-induced apoptosis in PC12cells. To explore the potential effects of N-acetyl cysteine (NAC) and Vesicular Monoamine Transporter (VMAT) inhibitors on△Ψm and [Ca2+]i.Methods:The△Ψm was measured by JC-1fluorescence staining using flow cytometry and the [Ca2+]i was measured by analyzing the fluorescent intensity (FI) using laser scanning confocal microscopy in PC12cells treated with NAC(20mmol/L) or reserpine (VMAT inhibitor,50,100,400,1600nmol/L)) in the presence of6-OHDA (25,50,100,200μmol/L) for0,12,24,36, and48h, respectively.Results:The percentage of the cells with△Ψm decline was increased in PC12cells treated with6-OHDA in a concentration-and time-dependent manner. However, the [Ca2+]i were siginiciantly increased in PC12cells treated with6-OHDA in a concentration-and time-dependent manner. The treatment of NAC significantly reduced the percentage of the cells with△Ψm decline in PC12cells and prevented the elevation of [Ca2+]i induced by6-OHDA in PC12cells. However, the treatment of teserpine further increased the percentage of the cells with△Ψm decline and aggraved the elevation of [Ca2+]i induced by6-OHDA in PC12cells.Conclusion:The neurotoxicity of6-OHDA, the protective effects of NAC, and the toxic effects of reserpine in PC12cells may be closed related to the changes of△Ψm and[Ca2+]jin PC12cells. Part ⅢThe effect of6-hydroxydopamine on monoamine neurotransmittters and the genes of synthesis rate-limiting enzymes in PC12cellsObjective:To study the effects of6-hydroxydopamine (6-OHDA), N-acetyl cysteine (NAC) and inhibitors of vesicular monoamine transporter (VMAT) on5-HT. NE, DA and the expression of TpH mRNA、DβHmRNA、TH mRNA in PC12cells.Methods:Enzyme-linked immunosorbent assay (ELISA) was used to measure the content of5-HT, NE, and DA, and RT-PCR was used to detect the levels of TpH mRNA、DβH mRNA、TH mRNA in PC12treated with NAC (20mmol/L) or reserpine (VMAT inhibitor,50,100,400,1600nmol/L)) in the presence of6-OHDA (25,50,100,200μmol/L) for0,12,24,36, and48h, respectively.Results:1) The content of5-HT in PC12cells did not change with the concentration of6-OHDA increasing, but decreased in a time-dependent manner, and the5-HT concentration was lowest in36h. The NE concentration decreased with the concentration elevating of6-OHDA, the change have the time dependence. The content of DA in PC12cells was significantly decreased with the concentration of6-OHDA increasing. The treatment of NAC increased the content of5-HT, NE and DA in PC12cells in a dose-and time-dependent manner. The treatment of reserpine significantly decreased the content of5-HT, NE and DA in PC12cells.2) The levels of TpH mRNA, DβH mRNA and TH mRNA in PC12cells were significantly decreased in a time-dependent manner with the concentration of6-OHDA increasing. The treatment of NAC significantly increased the levels of TpH mRNA, DβH mRNA and TH mRNA in a dose-and time-dependent manner in PC12cells. However, the treatment of reserpine further decreased the levels of TpH mRNA, DβH mRNA and TH mRNA in a dose-and time-dependent manner in PC12cells.Conclusion:6-OHDA could damage the synthesis of neurotrasmitters such as5-HT, NE, and DA, and decrease the expression of TpH mRNA, DβH mRNA, and TH mRNA in a dose-and time-dependent manner. Reserpine could aggravate but NAC could prevent these changes. PartIVThe establishment of the model of depression in PD and the changes of monoamine neurotransmittters in the different brain regionsObjective:To explore the changes of depressive behavior and pathologies in6-hydroxydopamine (6-OHDA)-induced PD model or in the acute and chronic depression model of PD induced by6-OHDA.Methods:70female adult rats were randomly divided into normal group (8rats), PD group (10rats), PD with chronic depression group (15rats), chronic depression group (10rats), PD with acute depression group (15rats), and acute depression group (12rats). The normal rats or6-OHDA-induced PD rats received3-weeks unpredictable chronic mild stress (CUMS) according to the literatures to establish the chronic depression model, or received3-days forced-swim tests to establish acute depression model. On the1st,7th,14th, and21st day, the body weight of the rats was dynamically weighed. Open-field test (OFT), sugar water and food consumption test, and passive avoidance test were used to evaluate depression-like behavior and the ability of learning. The morphological change in prefrons cortex, substantia nigra, hippocampus and locus ceruleus of the rats were examined by hematoxylin&eosin (HE) staining. Enzyme-linked immunosorbent assay was used to measured the content of5-HT, NE, and DA in the brains of the rats.Results:1) Compared with the normal group, the growing percentage of the body weight of the rats in PD group and chronic depression group was significantly decreased on7th,14th, and21st day. The growing percentage of the body weight of the rats in PD with chronic depression group was lowest at the above time points. The growing percentage of the body weight of the rats in PD with acute depression group and acute depression group was not examined because of the fact that the time to make the model was too short.2) In OFT, the horizontal total distance and vertical movement activities of the rats in the PD with chronic depression group were significantly lower than that of the rats in the normal group, PD group and chronic depression group on1st,7th,14th, and21th day, especially on the1st,7th days. The horizontal total distance and vertical movement activities of rats in the acute depression group were significantly lower than those of normal group, PD group and chronic depression group on1st day. The scores had significant declining tendency on3rd day. The scores of PD with acute depression group were significantly lower than those of others groups on1st and3rd day.3) Starting from7th day, the rats in PD group and chronic depression group showed obviously a decline in the sugar water consumption compared with the normal group. But there was not a significant difference in food consumption at the above time points among all the groups. From1st day, the sugar water and food consumption of rats in PD with chronic depression group was significantly decreased compared with other groups. The sugar water and food consumption of rats in the PD with acute depression group and acute depression group were not evaluated as the time of establishing the model is too short.4) In the passive avoidance test, compared with the normal group, the T1value was significantly increased and the T2value was significantly decreased in a time-dependent manner in PD group and chronic depression group from the7th day. Compared with the other three groups, the T1value was significantly increased and the T2value was significantly decreased in a time-dependent manner in PD with chronic depression group from the1st day. The passive avoidance test was not performed in the PD with acute depression group and acute depression group as the time of establishing the model is too short.5) In prefront cortex, the cell number of the brain regions of the rats in PD group, chronic depression group, acute depression group and PD with acute depression group was significantly decreased compared with the normal group, but there was not a significant difference among these groups. The cell number of the brain regions of the rats in PD with chronic depression group was significantly decreased as compared with the above four groups. In substantia nigra, hippocampus, and locus ceruleus, the cell number was not significant different among PD group, chronic depression group and acute depression group, but the cell number of the brain regions in these groups was decreased compared with that of the normal group. The cell number of the brain regions of the rats in PD with acute depression group and PD with chronic depression group was significantly decreased compared with that of the above groups.6) The content of5-HT and NE was the highest in locus ceruleus and the content of DA was the highest in substantia nigra among all the groups. In prefront cortex, substantia nigra, hippocampus and locus ceruleus, the content of5-HT, NE and DA was not significantly different among PD group, chronic depression group and acute depression group, but was significantly decreased compared with the normal group. The content of5-HT, NE and DA in PD with acute depression group and PD with chronic depression group was significantly decreased compared with the above groups.Conclusion:The symptoms of depression and pathology alteration can be observed in PD rats. The core symptoms of depression such as anhedonia and underactivity, and impaired learning and memory ability were expressly observed both in PD with chronic depression rat model and PD with acute depression rat model. The rats of both groups can also be found pathology alteration in cells number and monoamine neurotransmittter concentration. This study demonstrated that the mechanism of PD with depression is due to endogenous factors, and ectogenic factors promote and aggravate the neurotoxicity of endogenous factors. Summary1. This study demonstrated that6-OHDA could induce a decline in cell viability, induce cellular apoptosis, down-regulated the expression of Bcl2and up-regulated the expression of Bax in PC12cells in a dose-and time-dependent manner. The mechanisms of6-OHDA could involve increased oxidative stress, impaired△Ψm and overloaded calcium ion. The inhibition of vesicular monoamine transporter activity aggravated the neurotoxicity, but NAC protected against the damage.2. This study demonstrated that6-OHDA could decrease the content of5-HT, NE, and DA in adose-and time-depentedent manner. NAC could prevent the change but reserpine could aggravate the change.3. This study demonstrated that6-OHDA could decrease the expression of TpH mRNA, DβH mRNA and TH mRNA levels in adose-and time-depentedent manner. NAC could prevent the change but reserpine could aggravate the change.4. This study demonstrated that6-OHDA not only could induce cellular apoptosis, but also could decrease the content of monoamine neurotransmitters and the synthetic rate-limiting enzyme gene of them, which may be the biochemistry base of PD with depression. The mechanism may be oxidative stress.5. The symptoms of depression and pathology alteration can be observed in PD rats. The core symptoms of depression were expressly observed both in PD with chronic depression rat model and PD with acute depression rat model. The rats of both groups can be found pathology alteration in cells number and monoamine neurotransmittter concentration. This study demonstrated that the mechanism of PD with depression is due to endogenous factors, and ectogenic factors promote and aggravate the neurotoxicity of endogenous factors.6. With good operability, more judgment criterion and closer to the course of human, the rats PD following chronic depression group model is a more ideal model for the PD depression research.