| BackgroundParkinson's disease(PD)is one of the most common motor deficit diseases and the second most common chronic progressive neurodegenerative disease in the world.Neurodegeneration and loss of dopaminergic neuron in substantia nigra pars compacta(SNpc)and the emergence of misfolded a-synuclein(a-syn)inclusion(also known as Lewy body)are the hallmarks of PD pathology.The pathological mechanisms of PD remain unclear.Decades of studies show that many mechanisms are contributing to the damage of dopaminergic neurons,including oxidative stress,neuro-inflammation,a-syn proteostasis and mitochondrial dysfunction.Nrf2(Nuclear transcription factor NF-E2-related factor 2,Nrf2)is a transcription factor for an inducible oxidative stress regulatory system.Nrf2 remains in cytoplasm under normal physiological condition.When the redox hemeostasis changes,Nrf2 translocates into the neucleus and binds to the antioxidant responsive element(antioxidant responsive element,ARE).the specific DNA sites,and initiates the transcription of protective proteins,such as Heme Oxygenase-1(HO-1),and glutathione(GSH)related regulatory enzymes to protect against the oxidative stress.Studies reveal that oxidative stress contributes greatly to PD and anti-oxidative stress is a promising way for the treatment of PD.Nrf2-ARE signaling pathway as one of the most robust anti-oxidative pathways plays a vital role in mitigating the oxidative damage of PD.Finding a new Nrf2 activator with no side effect will improve the quality of life for PD patients.It has been shown that n-3 polyunsaturated fatty acids(n-3 PUFAs)intake correlates with a lower incidence of PD.Docosahexaenoic acid(DHA)is the main component of n-3 PUFAs.We investigate whether n-3 PUFAs act as an antioxidant by activating the Nrf2-ARE pathway and explore the protective effect of n-3 PUFAs in 6-hydroxydopamine(6-OHDA)and a-syn-induced PD models.ObjectivesTo investigate the protective effect of n-3 PUFAs on 6-OHDA and a-syn-induced PD mice models.MethodsIn vitro:1.SH-SY5Y cells were pretreated with different concentrations of DHA for 24 h,and the proliferation of cells were detected by MTT assay.2.SH-SY5Y cells were pretreated with different concentrations of DHA for 8 h or treated with 80?M of DHA for different time points.The expression of Nrf2 and its downstream protein HO-1 were detected by western blotting.3.SH-SY5Y cells were pretreated with different concentrations of DHA for 8 h.then the cells were exposed to 6-OHDA for another 24 h,the cell viability was detected by MTT assay.Cell apoptosis and intracellular ROS level were detected by flow cytometry.TUNEL assay was used to detect the apoptosis of SH-SY5Y cells.4.BV2 microglial cells were pretreated with different concentrations of DHA for 8 h,and then exposed to LPS for another 24 h,real time RT-PCR analysis were used to detect the mRNA levels of inflammatory cytokines to assess whether DHA can reduce the inflammatory reactions.5.The small interfering RNA of Nrf2(Nrf2 siRNA)was used to knockdown the expression of Nrf2.The efficiency of Nrf2 siRNA was verified by western blot analysis.SH-SY5Y cells were pretreated with different concentrations of DHA for 8 h,and 6-OHDA was added for another 24 h.Apoptosis was detected by flow cytometry to evaluate whether the protective effect of DHA on SH-SY5Y cells was mediated by Nrf2-ARE pathway.In vivo:1.Fish oil was administered to C57BL/6 mouse by oral gavage for 35 days,and the animal model of PD was established by stereological injection of 6-OHDA into the mice striatum.One day after 6-OHDA injection,mice were sacrificed and the striatum was taken.The expression of Nrf2 was detected by western blotting.2.Fish oil was administered to C57BL/6 mouse by oral gavage for 35 days,and the animal model of PD was established by stereological injection of 6-OHDA into the mice striatum.21 days after 6-OHDA injection,mice were sacrificed,brains were taken for frozen sections,tyrosine hydroxylase(TH)immunostaining was performed to observe the changes of dopaminergic neurons in mice SN and striatum.3.Fish oil was administered to C57BL/6 mouse by oral gavage for 35 days,and the animal model of PD was established by stereological injection of 6-OHDA into the striatum.7 days and 14 days after the injection of 6-OHDA,mice were sacrificed,brains were taken for frozen sections.Anti-GFAP and IBA1 primary antibodies were used for immunofluorescence staining to observe the neuro-inflammation.4.Fish oil was administered to C57BL/6 mouse by oral gavage for 35 days,and the animal model of PD was established by stereological injection of 6-OHDA into the mice striatum.21 days after the injection of 6-OHDA,mice were sacrificed and the striatum were collected for the measurement of dopamine(DA),dihydroxyphnylacetic acid(DOPAC)and homovanillic acid(HVA)by HPLC-MS.5.Fish oil was administered to C57BL/6 mouse by oral gavage for 35 days,and the animal model of PD was established by stereological injection of 6-OHDA into the mice striatum The mice were subcutaneously injected with apomorphine at 7 days,14 days and 21 days after 6-OHDA injection.The apomorphine-induced circling was documented for behavioral evaluation.6.Fish oil was administered to C57BL/6 mouse by oral gavage for 14 days,the animal model of PD were established by stereological injection of adeno-associated virus-a-synuclein(AAV-a-syn)into the mice SN,then fish oil were administered for another 21 days.Mice were painlessly sacrificed 10 weeks after virus injection,brains were removed and frozen sections were stained with TH to observe the dopaminergic neuron loss in the SN.7.Fish oil was administered to C57BL/6 mouse by oral gavage for 14 days,the animal model of PD were established by stereological injection of AAV-a-syn into the mice SN,then fish oil were administered for another 21 days.Mice were subjected to open field test at 14 days and 21 days after the virus injection,and the behavioral changes were recorded.Results1.Different concentration of DHA does not affect the cell viability.2.DHA can upregulate the expression of Nrf2 and HO-1 in SH-SY5Y and BV2 cells in a concentration and time-dependent manner.3.DHA can reduce 6-OHDA-induced cell death,attenuate cell apoptosis and decrease intracellular ROS levels.DHA can also reduce LPS-induced inflammation in BV2 cells.4.When Nrf2 is knocked down by Nrf2 siRNA,the protective effect of DHA disappears.5.n-3 PUFAs can induce the expression of Nrf2 in C57BL/6 mice,n-3 PUFAs can remarkably attenuate 6-OHDA-induced neurodegeneration in the striatum and SN.6.n-3 PUFAs can improve the dopaminergic neuron loss,alleviate the inflammatory response and improve motor behavior in a-syn-induced PD mice.Conclusionn-3 PUFAs can ameliorate dopaminergic neurotoxicity in 6-OHDA and a-syn-induced PD animal models.This protective effect may be related to the activation of Nrf2-ARE pathway. |
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