| Parkinson's disease(PD) is the second most prevalent neurodegenerative disease just after Alzheimer's disease.There are about 1.7-2.0 billion people affected by PD in our country.The incidence is close to 1.7%in the people over 65.Along with the aging of our society,PD is becoming an overwhelming problem.Since the exact etiology of the PD is unknown.Neuropathological hallmarks of the disease include the degeneration and loss of dopaminergic neurons in the substantia nigra(SN) and the subsequent dopamine(DA) depletion in the striatum.However,lots of researches suggest that multiple factors might be involved,such as heredity,environment, oxidative stress,inflammation and decline in growth factors levels.So we cannot prevent and treat effectively,all of the efferts used in the clinic today aims to alleviate the symptoms.It is of great important solving the problems of what cause PD.Flavonoids are plant polyphenolic compounds with a variety of biological functions.Several studies had carried out to investigate the protective effects of flavonoids on neurodegenerative disease.Curcumin is a well-defined flavonoid.It has a neuroprotective effect due to its free radical scavenging,iron-chelating and anti-inflammatory actions in different tissues.The aim of present study is to explore the prevention of curcumin against Parkinson's disease,as well as the protectctive effect on dopaminergic neurons.In the present study,we selected rat and MES23.5 dopaminergic cells as our model,by a combination of high-performance liquid chromatograph electrochemical detection(HPLC-ECD) and immunohistochemistry, the toxic effects of 6-hydroxydopamine(6-OHDA) on the SN-Str projection system were investigated.Effects of curcumin on 6-OHDA-mediated neurotoxic effects were further investigated;MTT assay,FCM and western blots were used to detect the effect of curcumin on the viability of 6-OHDA-induced MES 23.5 cells,the△Ψm changes, the ROS levels and the expressions of SOD and NF-κB protein level.The results were as follows:1.After 6-OHDA lesions,the DA and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) contents in the Str decreased compared with that of control,while the homovanillic acid(HVA) level was not changed.In the curcumin treatment group, the DA and its metabolites contents increased compared with that of 6-OHDA treatment group.2.The numbers of tyrosine hydroxylase(TH) positive neurons in the SN decreased after 6-OHDA lesions compared with that of control group.Curcumin significantly inhibited 6-OHDA-induced decrease of the numbers of TH positive neurons.3.After 6-OHDA lesions,the numbers of iron-staining cells increased in the SN compared with that of control group.Curcumin significantly inhibited 6-OHDA-induced increase of the numbers of iron-staining cells.4.A decrease viability of MES 23.5 cell was oberserved after 6-OHDA treatment for 24h(P<0.01),it was dose-dependently.The viability of cells treated with 25μmol/L and 50μmol/L 6-OHDA for 24 h reduced by 13%and 12%compared with that of the control.When treated with 100μmol/L and 150μmol/L 6-OHDA, cell viability reduced significantly by 44%and 65%.The viability of cells was unchanged when they were treated with curcumin up to 15μmol/L compared to that of the control.5.The protective effect of curcumin against 6-OHDA-induced cell death was assessed in cultured MES23.5 cells by MTT test.The viability of MES23.5 cells was dramatically reduced in 6-OHDA group,while pretreatment with 10μmol/L curcumin coulds ignificantly increase the cell viability compared to 6-OHDA treatment.Similar results were observed in measuring the leakage of the cytosolic LDH using LDH assay6.Mitochondrial membrane potential changes are markers of mitochondria function.and are ofen associated with ROS generation.When treated with 6-OHDA,MES23.5 cells showed a significant decrease of△Ψm.Pretreatment with curcumin could abolish this reduction.This suggested that curcumin could protect cells from 6-OHDA-induced oxidative stress by restoring the mitochondria function. 7.Since ROS played an important role in cell death and changes of mitochondrial membrane potential were considered to be involved in ROS production,we next investigated the intracellular ROS formation using a fluorescent sensitive probe (H2DCF-DA).After curcumin pretreatment,the levels of ROS dramatically decreased compared to the 100μmol/L 6-OHDA treated group.8.Since SOD is a highly potent protective agent against cell injury during oxidative stress,we examined SOD expression in the MES23.5 cells.In 100μmol/L 6-OHDA-treated cells,Cu/Zn-SOD protein level was down-regulated.However, after 10μmol/L curcumin pretreatment,the protein level could be up-regulated to 75%of control.9.The mechanism of 6-OHDA-induced cell damage was also investigated by examining NF-κB translocation in MES23.5,using polyclonal antibody of NF-κB. In control,NF-κB was preferentially located in the cytoplasm.However,upon 6-OHDA administration(100μmol/L),NF-κB was considerably increased in the nucleus.Curcumin pretreatment inhibited the translocation of NF-κB from cytoplasm to the nucleus.The results suggest that curcumin showed protective effect against 6-OHDA-induced neurodegeneration in rat,increased the content of DA and its metabolites in the Str, decreased the turnover rate of DA.The neuroprotective action of curcumin is mediated via a reduction of oxidative stress due to its iron-chelating property. Curcumin mitigated the damage of MES 23.5 cell from 6-OHDA.Curcumin exerts neuroprotective effects against 6-OHDA-induced cell death,by a mechanism, believed to improve the activity of SOD and increasing the△Ψm,which suppressed an increase in ROS and inhibited the translocation of NF-κB.The present study provides experimental evidence to explore the possible use of curcumin,a very low toxic natural compound as a therapeutic approach in PD.
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