Protective Effects And Mechanism Of Icariin On 6-OHDA-induced Parkinson's Mice Model

Objective: To study the protective effect of Icariin(ICA)on 6-OHDA-induced dopamine(DA)neuronal injury and discuss whether the mechanism is through Nrf2 signaling pathway.Methods: 1.In vivo: Male wild-type and Nrf2 knockout mice were randomly divided into control,ICA(60 mg/kg),6-OHDA(4 ?g)and 6-OHDA+ICA groups.The mice received intragastric administration with ICA once daily for 10 consecutive days.On the third day,mice were injected stereotactically with 6-OHDA into the substantia nigra(SN)to prepare DA neuronal damage model.The mice behavior changes were detected by rotarod test and open field test;tyrosine hydroxylase(TH),ionized calcium-binding adapter molecule-1(Iba-1)and glia fibrillary acidic protein(GFAP)expression were measured by immunofluorescence staining and Western blotting;DA,DOPAC and HVA levels in striatal tissues were detected by UHPLC;Nuclear factor erythroid-2 related factor 2(Nrf2),Kelchlike ECH-associated protein 1(Keap1),heme oxygenase-1(HO-1)and NADPH quinone oxidoreductase 1(NQO1)expression were analyzed by real-time RT-PCR and Western ting;inflammatory cytokines release,such as tumor necrosis factor-?(TNF-?)and inducible nitric oxide synthase(iNOS)were tested by Western blotting;neurotrophic factors expression,such as glia cells line-derived neurotrophic factor(GDNF)and brain-derived neurotrophic factor(BDNF)were measured by Western blotting.2.In vitro: Primary neuron-glia co-cultures and primary neuron-enriched cultures were randomly divided into control,ICA(0.1 ?M),6-OHDA(40 ?M)and 6-OHDA+ICA groups.Cells were pretreated with ICA for 30 min and added 6-OHDA.Seven days later,TH protein expression was measured by Western blotting.To further investigate whether glial cells Nrf2 is involved in ICA-mediated DA neurons protection,primary glial cells were seeded in Transwells and treated with Nrf2-siRNA for 24 hours.Then,Transwells were transferred into plates filled with primary neurons,and the reconstructed neuron-glia co-cultures divided into control,control-siRNA(40 nmol/L),Nrf2-siRNA(40 nmol/L),ICA(0.1 ?M),6-OHDA(40 ?M),6-OHDA+ICA,6-OHDA+ICA+Nrf2-siRNA groups.Cells were pretreated with ICA for 30 min and added 6-OHDA.Seven days later,DA neuronal damage was analyzed by immunofluorescence staining and Western blotting.Results: 1.In vivo: In the wild type mice,compared with the control group,6-OHDA significantly decreased behavioral ability,DA neurons number,DA,DOPAC and HVA levels.6-OHDA activated microglia and astroglia,increased TNF-? and iNOS protein expressions.Moreover,6-OHDA up-regulated Nrf2 signaling pathway gene and protein expressions.After ICA treatment,DA neurons number was increased,DA and its metabolites were increased,glial cells activation and inflammatory cytokines release were decreased,and Nrf2 signaling pathway gene and protein expressions were further up-regulated.However,in Nrf2 knockout mice,ICA-elicited neuroprotection disappeared,glial cells activation and inflammatory cytokines release could not be inhibited.In addition,there was no significant change in Nrf2 signaling pathway gene and protein expressions.2.In vitro: In primary neuron-enriched cultures and primary neuron-glia co-cultures,6-OHDA reduced TH protein expression.After ICA treatment,ICA exhibited neuroprotection in primary neuron-glia co-cultures but not in neuron-enriched cultures(without glial cells presence).After Nrf2-siRNA treatment to glial cells,Transwells re-constructed neuron-glia co-cultures found that ICA has no protective effect on DA neurons.Conclusion: ICA attenuated glial cells-mediated neuroinflammation and evoked DA neuroprotection via an Nrf2-dependent manner.