| Objective:To explore the neuroprotective of Ellagic acid（EA）on rotenone（ROT）-induced DA neuronal damage and its potential mechanisms.Methods:1.Wild type c57 male mice were randomly divided into the following five groups（n=6）:Control,EA alone（100 mg/kg）,ROT（1 mg/kg）,ROT+EA（20mg/kg）and ROT+EA（100 mg/kg）.Mice were given subcutaneous injection of ROT（1 mg/kg）six times a week for consecutive five weeks followed by daily intragastric administration of EA for consecutive another five weeks.Mice behavior changes were assessed by open field and rotarod tests.Then,the protein expression of tyrosine hydroxylase（TH）in mice substantia nigra（SN）was analyzed by western blot and Immunohistochemistry assays.The mRNA and protein expressions of nuclear factor erythroid-2 related factor 2（Nrf2）,heme oxygenase-1（HO-1）and NADPH quinone oxidoreductase 1（NQO1）in mice midbrain were detected via real-time RT-PCR and western blot assays.2.MN9D-enriched cultures,MN9D-BV2 and MN9D-C6 co-cultures via transwells were randomly divided into Control,EA alone（1μM）,ROT（0.1μM）,ROT+EA（0.1μM）and ROT+EA（1μM）groups.EA was pretreated for 30 min and then incubated with ROT for 24 h.MTT assay was used to analyze the viability of MN9D cells.The expression of TH was detected by western blot and immunofluorescence staining.The mRNA and protein expressions of Nrf2,HO-1 and NQO1 were determined by real-time RT-PCR and western blot assay in C6 cells.Then,C6 cells were treated with Nrf2-siRNA（50 nmol/L）,the silence efficiency was validated by real-time RT-PCR and western blotting,DA neuronal damage was determined by TH-positive neuronal number counting and TH protein expression detection in MN9D-C6 co-cultures.3.Nrf2-/-male mice were randomly divided into the following five groups（n=6）:Control,EA alone（100 mg/kg）,ROT（1 mg/kg）,ROT+EA（20 mg/kg）and ROT+EA（100 mg/kg）,and the same way of administration as wild-type c57 mice.Nrf2-/-mice behavior changes were assessed by open field and rotarod tests.Western blotting was used to test knockout efficiency in Nrf2-/-mice.The mRNA and protein expressions of HO-1 and NQO1 in Nrf2-/-mice midbrain were detected via real-time RT-PCR and western blot assays.Then,the protein expression of TH in Nrf2-/-mice substantia nigra（SN）was analyzed by western blot and Immunohistochemistry assays.Results:1.In the wild type male mice,compared with the control group,ROT could significantly decreased motor activity,TH-positive neuronal number and TH protein expression.After EA（100 mg/kg）treatment,behavioral ability,TH-positive neuronal number and TH protein expression were improve.Furthermore,we found that EA elicited Nrf2 activation,compared with ROT group,the higher protein and mRNA expressions of Nrf2,HO-1 and NQO1 in ROT+EA group.2.In MN9D cell enriched cultures and MN9D-BV2 and MN9D-C6 co-cultures,ROT reduced MN9D cell viability and TH protein expression.After EA（1μM）treatment,EA-mediated neuroprotection was indicated in MN9D-C6 co-cultures but not in either MN9D-enriched or MN9D-BV2 co-cultures.In addition,Nrf2-siRNA in C6 cells was performed,Transwells re-constructed MN9D-C6 co-culture found that EA-generated DA neuroprotection from ROT-induced neurotoxicity was neutralized in TH-positive neuronal counting and TH protein expression assays.3.In Nrf2-/-male mice,the knockout efficiency was verified by Nrf2 protein level detection.Compared with ROT group,there was no significant difference of the genes and proteins expressions of HO-1 and NQO1 in the midbrain after EA treatment.In addition,EA failed to improve the motor activity.TH-positive neuronal counting and TH protein expression detection showed EA-mediated DA neuroprotection was abolished in Nrf2-/-mice.Conclusion:Ellagic acid confers DA neuroprotection via the activation of Nrf2 in astroglia. |
