The Role Of SIRT1-Nrf2 Axis In Regulating Ferroptosis Of Neurons In Rotenone-induced Neurotoxicity

Background:Rotenone（Rot）is a naturally occurring botanical insecticide with the formula C₂₃H₂₂O₆.Studies have shown that long-term exposure to rotenone can damage dopaminergic neurons and increase the risk of Parkinson’s disease（PD）.However,the specific mechanism by which rotenone causes dopaminergic neuronal damage and even death is still unclear.Ferroptosis is a form of non-apoptotic regulated cell death（RCD）discovered in recent years and characterized by iron-dependent accumulation of lipid peroxides,leading to oxidative stress and cell death.Recent studies have shown that Ferroptosis plays an important role in the pathology of neurological diseases.Previous studies have also confirmed that rotenone can induce mitochondrial damage in dopaminergic neurons,which in turn induce Ferroptosis.Sirtuin 1（SIRT1）is a nicotinamide adenine dinucleotide-dependent histone deacetylase,which deacetylates Nrf2 and increases the activity of Keap/Nrf2.Recently,SIRT1 was found to be considered a protective agent against oxidative stress damage and lipid peroxidation in cells by mediating the expression of Nrf2 and its downstream target heme oxygenase-1（HO-1）.However,whether Rot can regulate dopaminergic Ferroptosis through the SIRT1-Nrf2 signaling pathway to exert neurotoxicity has not been reported yet.Aims:The rotenone-infected SH-5Y5Y cell model and rat primary midbrain nerve cell model were established to investigate the role of SIRT1-Nrf2 axis in Rot-induced dopaminergic neuronal ferroptosis,and to elucidate the molecular mechanism of Rot-induced dopaminergic neurotoxicity,so as to provide experimental basis and new targets for the prevention and treatment of neurotoxicity caused by rotenone.Methods:1.Establishment of Rot-infected SH-SY5Y cell model:SH-SY5Y cells were randomly divided into control group（Con）,Rot low-dose group（Rot-0.5μM）,Rot high-dose group（Rot-1μM）,Rot infection for 24h.2.Activator intervention in SH-SY5Y cell model establishment:SH-SY5Y cells were trained by Nrf2 activator（Oltipraz）,SIRT1 activator（CAY10602）,iron vegetation group（DFO）;Ferrostatin-1,Z-VAD-FMK,N-ace,Nec-1 were pretreated for 30 minutes,and then were exposed to 1μM Rot for 24 hours.3.Activator intervention in the establishment of primary cell culture model:SH-SY5Y cells were randomly divided into control group（Con）,Rot group（Rot-10n M）,model and Nrf2 activator group（Rot-10n M+Oltipraz）,model and SIRT1 activator group（Rot-10n M+CAY10602）,model which exposed to Nrf2 activator and ammonium ferric citrate group(ROT-10n M+Oltipraz+Fe²⁺),the model which exposed to SIRT1activator and ammonium ferric citrate group(Rot-10n M+CAY10602+Fe²⁺)for 72hours.4.MTT cell viability detection:detect the toxic effect of different concentrations of Rot（0.0625μM,0.125μM,0.5μM,1μM,2μM,4μM）in SH-SY5Y cells.5.qRT-PCR assay:detect the m RNA levels of iron regulators（FTH-1,TFR,HO-1,IRP1,IRP2,etc.）and antioxidant regulators（SLC7A11,GPX4,COX2,ACSL4）.6.Western blot analysis:detect the proteins expression level of SIRT1-Nrf2signaling pathways（Keap1,Nrf2,SIRT1,Fpn-1,GPX4）in each group.7.C11 BODIPY 581/591 ROS fluorescence assay:detect the lipid peroxidation levels.8.Fluorescent staining assay of Ferro Orange-Fe²⁺content:Fe²⁺probe was used to detect Fe²⁺content of SH-SY5Y cell models with different exposure.9.Detection of MDA and GSH levels:The concentrations of MDA and GSH in cells of different groups were detected according to the instructions.10.Immunohistochemistry measures the number of dopaminergic neurons:Rat primary cells were extracted and the changes in the number and morphology of cells after reinfection with Nrf2 activator（Oltipraz）and SIRT1 activator（CAY10602）were observed.Results:1.Compared with the control group,MTT experimental results demonstrated that the activity of SH-SY5Y cells in the Rot-0.5μM,Rot-1μM,Rot-2μM and Rot-4μM groups was significantly reduced（P<0.05）,so 0.5μM and 1μM concentrations of Rot were selected for the next experiment.2.Iron homeostasis disorder caused by Rot dose-dependence:compared with the control group,in the Rot-1μM group,the content of Fe²⁺was significantly increased（P<0.001）,and the levels of TFR,DMT1,IRP1,IRP2 m RNA were significantly increased（P<0.001）,,while the level of FTH-1 m RNA and FPN-1 protein were significantly reduced（P<0.0001）.3.Iron dependence induced lipid peroxidation in SH-SY5Y nerve cells:Compared with the Con group,lipid peroxidation and MDA levels in SH-SY5Y nerve cells in the Rot-1μM group were significantly increased（P<0.001）,and iron-phylocates deferoxamine DFO significantly inhibited the increase of lipid peroxidation induced by Rot（P<0.01）;Compared with the Con group,the GSH level decreased significantly in SH-SY5Y nerve cells at the Rot-1μM group（P<0.001）,and DFO inhibited the decrease of GSH level at the Rot-1μM group（P<0.01）.4.Rot induces SH-SY5Y nerve cell Ferroptosis:The results of RT-q PCR showed that compared with the Con group,the expression levels of ferrozotic antioxidant regulators COX2 and ACSL4 m RNA in the Rot-1μM group increased significantly（P<0.01）.The expression of ferrozotic regulators SLC7A11 and GPX4 in the Rot-1μM group was significantly reduced（P<0.01）.MTT results show that antioxidants such as caspase inhibitors（Z-VAD-FMK）,iron-phylocates deferoxamine（DFO）and Ferrostatin-1 both can effectively inhibit SH-SY5Y cell death caused by 1μM Rot infection（P<0.01）.5.Rotenone inhibits the activation of SIRT1-Nrf2 signaling pathway in SH-SY5Y cells:Compared with the control group,Western blot experimental results demonstrated that the expression levels of Nrf2,SIRT1 protein and SIRT1-Nrf2 signaling pathway downstream factors NQO-1 and HO-1 m RNA in the Rot-1μM group cells were significantly reduced（P<0.01）,SIRT1 activator CAY10602 could significantly reduce the decrease of Nrf2 proteins,NQO-1 and HO-1 m RNA expression induced by Rot（P<0.01）,Compared with the Con group,the expression level of Keap1 protein in the Rot-1μM group cells was significantly increased（P<0.05）,and CAY10602 could significantly reduce the increase of Keap1（P<0.05）expression induced by Rot.6.Activation of SIRT-1-Nrf2 pathway alleviates Rot-induced lipid peroxidation of SH-SY5Y nerve cells:Compared with the Rot group,in the Nrf2 activator and SIRT1activator co-treated with Rot,the ROS level and MDA content of cells decreased significantly（P<0.001）,while GSH content increased significantly（P<0.001）.7.Activation of SIRT1-Nrf2 pathway inhibits Rot-induced iron metabolism disorders in SH-SY5Y cells:Compared with Rot-exposed group,the cell activity of Nrf2 activator and SIRT1 activator co-treated with Rot increased significantly（P<0.001）,the content of Fe²⁺decreased significantly（P<0.001）,and the abnormal m RNA level of iron transport related factors was significantly alleviated（P<0.01）.8.Activation of SIRT1-Nrf2 reduces the Ferroptosis and injury of primary dopaminergic neurons induced by Rot:Compared with Rot-exposed group,the number of dopaminergic neurons and synapses increased significantly（P<0.01）and the abnormal expression of iron death regulator m RNA was significantly relieved（P<0.05）when Nrf2 activator（Oltipraz）and SIRT1 activator（CAY10602）were co-treated with Rot.Conclusions:1.Rot exposure can induce Ferroptosis in dopaminergic neurons.2.Rot exposure can obviously inhibit the activation of SIRT1-Nrf2 signaling pathway.3.Activation of SIRT1-Nrf2 signaling pathway can obviously alleviate the Ferroptosis of dopaminergic neurons induced by Rot.