Naringenin Protects Neuron Against6-OHDA-induced Parkinsonism-like Neurotoxicity Via Activation Of The Nrf2/ARE Signaling Pathway

BackgroundParkinson’s disease (PD) is characterized by progressive and selective loss of dopaminergic neurons in substantia nigra pars compacta (SNc), but the pathogenesis remain elusive. There is increasing evidence showing that oxidative stress is critically involved in the pathogenesis of Parkinson’s disease (PD), pharmacological targeting of the antioxidant machinery is currently of fundamental interest.An extremely promising pathway that confers protection to a variety of oxidative stress-related neurodegenerative insults is the nuclear factor erythroid-2related factor2(Nrf2)/antioxidant response element (ARE) pathway. The transcription factor Nrf2is a key regulator in the coordinated induction of a battery of cytoprotective genes, including those encoding for endogenous antioxidant such as hemeoxygenase (HO-1), glutathione cysteine ligase regulatory subunit (GCLC) and glutathione cysteine ligase modulatory subunit (GCLM).The redox sensitive transcription factor nuclear factor erythroid-2related factor2(Nrf2) plays a key role in the cellular defense against oxidative stress. There is increasing evidence showing that induction of Nrf2-ARE pathway can confer protection against Parkinson’s Disease (PD) injury. Our preliminary data showed that naringenin(NG) isolated from Dracocephalum herb is robust Nrf2activator and have potential therapeutic effects against6-OHDA injury through activation of the ARE. The objective of the study is to investigate whether Naringenin can ameliorate6-OHDA injury in SH-SY5Y cellular and animal models of PD and how Nrf2-ARE pathway is involved. We used SH-SY5Y cells to examine the protective potential of Naringenin, and test whether it can attenuate6-OHDA injury in PD model of C57BL/6mice. This study will be valuable for future development of new therapeutic strategies for the treatment of PD. ObjectivesThe objective of this study is to investigate whether naringenin (NG) can ameliorate6-OHDA-induced neurotoxicity injury in SH-SY5Y cellular and animal models of PD and how Nrf2-ARE pathway is involved.Materials and MethodsIn vitro studies1. Western blot is used to detect the expression of Nrf2protein levels,8h later and the downstream (GCLC, GCLM, HO-1) protein levels,24h later respectively, after the different doses of naringenin are added to the SH-SY5Y cells. To detect whether naringenin can activate Nrf2-ARE pathway. To test GSH protein levels24h later after given different doses of naringenin (40μM,80μM) to the cells.2. Different doses of naringenin are added to the SH-SY5Y cell,2h later,6-OHDA is given to injure cells for24h. Then, cell viability is determined by MTT assay. Hoechst33258staining is to test cells apoptosis. LDH levels is examined by the chemical chromogenic assay. The fluorescent staining is to detect ROS generation. To detect whether naringenin has a protective effect on SH-SY5Y cell damage caused by6-OHDA.3. Different doses of naringenin are added to the SH-SY5Y cell,2h later,6-OHDA is given to injure cells for24h. Then, Western blot is performed to detect proteins levels involved in apoptosis pathway (p-JNK, p-p38, caspase-3). To test whether naringenin can block JNK/p38apoptotic signaling pathway activated by6-OHDA.4. si-RNA is used to knock down the Nrf2expression. Naringenin is added to the cell.2h later, Western blot is performed to detect Nrf2protein levels.24h later, downstream (GCLC, GCLM, HO-1) protein levels is tested. To test whether naringenin can activate the Nrf2-ARE pathway after Nrf2is knocked down.5. si-RNA is used to knock down the Nrf2expression, naringenin is added to the SH-SY5Y cell,2h later,6-OHDA is given to injure cells for24h. Then, Western blot is performed to detect proteins levels involved in apoptosis pathway (p-JNK, p-p38, caspase-3).To test whether naringenin can block JNK/p38apoptotic signaling pathway activated by6-OHDA after Nrf2is knocked down. 6. si-RNA is used to knock down the Nrf2expression, naringenin is added to the SH-SY5Y cell,2h later,6-OHDA is given to injure cells for24h. Then, cell viability is determined by MTT assay. To detect whether naringenin still has a protective effect after Nrf2is knocked down. To determine whether naringenin’s protective effect is involved in Nrf2-ARE pathway.In vivo studies1. Naringenin is given to C57BL/6mice by gavage.5days later, different brain regions and the striatum are taken at different time points.Western blot is performed to detect the Nrf2and downstream (GCLM HO-1GCLC,) protein expression levels, which is extracted from the tissues mentioned above.2. Naringenin is given to C57BL/6mice by gavage.5days later,6-OHDA is injected into striatum by stereotactic injection method to construct a PD model. Apomorphine-induced changes in animal behavior is detected at given time points,7d,14d, and21d later, respectively.3. Brain tissue is taken from the PD model mice1d and7d later respectively, then test striatum GSH and ROS protein levels.4. Mice were sacrificed7d after the PD model is succeed. Western blot is performed to detect proteins levels involved in apoptosis pathway (p-JNK, p-p38, caspase-3). To test whether naringenin can block JNK/p38apoptotic signaling pathway activated by6-OHDA in striatum.5.21d after the PD model is succeed, HPLC is performed to test the levels of DA and its metabolites DOPAC and HVA in the striatum. Immunohistochemical method and Western blot are used to test TH protein levels in striatum and substantia nigra. To observe whether naringenin can reduce the lack of dopamine neurons in PD mouse model.ResultsThe main conclusions were as follows:1. Naringenin induces nuclear translocation of Nrf2and the expression of ARE-regulated genes in SH-SY5Y cells2. Naringenin induces glutathione synthesis and inhibits6-OHDA-induced ROS production in SH-SY5Y cells3. Naringenin induces nuclear translocation of Nrf2and the expression of ARE-regulated genes in C57BL/6mice 4. Naringenin induces nuclear translocation of Nrf2and the expression of ARE-regulated genes in different brain regions of C57BL/6mice5. Naringenin induces glutathione synthesis and inhibits6-OHDA-induced ROS production in C57BL/6mice6. Naringenin protects cells against6-OHDA-induced cell death and reduces the lost of dopamine neurons in PD mice7. The protective effect of naringenin involves the Nrf2-ARE pathwayConclusionsThe results of our study provide compelling evidence for the protective effect of naringenin to activate Nrf2/ARE pathway for the treatment of neurodegenerative diseases PD.