| BackgroundParkinson’s disease(PD)is becoming more and more common.The main characteristics of PD are damage of dopaminergic(DA)neurons in substantia nigra(SN)and loss of DA in striatum.However,the damage of locus coeruleus noradrenergic(LC/NE)neurons in PD patients was even greater,and the level of norepinephrine(NE)released by LC/NE neurons in the brain were also significantly reduced.NE is a major regulatory neurotransmitter in the central nervous system,including anti-inflammation,providing neurotrophic support and antioxidation.However,whether the damage to LC/NE neurons and the decrease of NE content in the brain further promote the development of PD and the molecular mechanism still remain unclear.The factors leading to occurrence of PD are complex,including genetics,environment,aging,inflammation,oxidative stress and abnormal iron content.Ferroptosis is a cell death caused by iron-dependent oxidative stress injury.However,whether loss of NE in early stage of PD promotes iron accumulation in dopaminergic neurons and ferroptosis in cells is not known.Therefore,our study will give new ideas about damage of dopaminergic neurons.ObjectivesBefore using rotenone(ROT)to establish a mouse of PD model,we use N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine(DSP4),a toxic substance to LC-NE neurons,to induce LC/NE neurons in mice damage and NE loss.Observing the changes of mouse motor function through gait behavior testing and detecting the pathological effect of DSP-4 in SN of PD mice by molecular biology experiments.To clarify the protective effect of NE on dopaminergic neurons and the molecular mechanism of NE regulating the dopaminergic neurons,the ferroptosis inhibitor lipoxstatin-1(LIP-1)was used to inhibit the occurrence of cell ferroptosis for explore the role of ferroptosis in dopaminergic neurons.MethodsAfter 7 days,mice were split into three groups randomly: control group(Con),ROT group and DSP-4+ ROT group.Rotenone(ROT)was gave to mice everyday for21 days,the dose is 1.5 mg/kg.The dose of DSP-4 is 50 mg/kg,and treat mice one week before ROT administration.Another batch of mice were split into Con,ROT,DSP-4+ ROT and ferroptosis inhibitor(LIP-1)treatment groups(LIP+DSP-4+ ROT).The dose of LIP-1 is 10mg/kg,treating 30 minutes before ROT administration.Then,the mice were anesthetized.Perfusing mice with saline for 5 minutes,we used 4% paraformaldehyde to fix brains for 10 minutes,following dehydrating with sucrose,made frozen sections,and then detected by immunohistochemical staining.Two mice of each group were perfused with 2.5% glutaraldehyde,and brain tissue was extracted.Put SN into 2.5% glutaraldehyde fixed solution and preserved at 4 degrees.The other mice’s striatum and midbrain regions were isolated and put in-80℃.The expression of ferroptosis homeostasis regulatory proteins(FPN-1,TFR,Ferritin,IRP1/IRP2)and oxidative stress related proteins(GPX-4,XCT,COX2,ACSL4,g P91 phox,p47phox)in mouse midbrain were detected by WB.Iron level and lipid peroxidation level in mouse midbrain were detected by test kits.The changes of motor function of mice were detected by gait test.Prism8.0 software was used to analyze the experimental results.Results1.Effects of NE removal on iron content in midbrain tissues of ROT-exposed mice: Compared with Con group,iron content in midbrain of ROT group was significantly increased(P <0.01).And DSP-4 further increased the iron content in midbrain(P <0.01).Previous studies have verified DSP-4 treatment alone has no significant effect on iron content in mouse midbrain tissues.2.Effects of NE removal on ferroptosis related proteins in midbrain of ROT-exposed mice : WB showed the expression of Fpn-1 in midbrain was decreased,while DSP-4 further decreased the protein expression of Fpn-1(P < 0.05).Compared with the Con group,the protein levels of TFR,Ferritin,IRP1 and IRP2 were increased,and DSP-4 further increased the protein expression levels,the difference was significant(P < 0.05).3.Effects of NE removal on mitochondrial morphology of dopaminergic neurons:compared with Con,ROT made mitochondria of DA neurons atrophied,the density of mitochondria membrane was increased,the nuclear size was normal,and the nucleoli was enlarged.Compared with ROT,the nuclei in the ROT +DSP-4 group were deformed,chromatin pyknosis and marginalization appeared and the mitochondria shrinked further.4.Effects of NE removal on lipid peroxidation in ROT-exposed mice: Compared with Con,ROT increased the level of MDA in midbrain(P <0.001),and DSP-4 further increased the level of MDA in the midbrain(P <0.01).Compared with Con,ROT reduced the level of GSH(P <0.001),while DSP-4 further reduced the content of GSH.5.Effects of NE removal on lipid peroxidation: ROT decreased GPX4’s expression,and DSP-4 further decreased GPX4’s expression(P < 0.001).Expression of XCT,ACSL4,COX2,gp91-phox and p47-phox in the midbrain of mice were increased,and DSP-4 further increased these protein expression levels,with significant difference(P < 0.05).6.Effects of NE removal on dopaminergic system: Immunohistochemical experiment showed TH cells in LC and SN regions in ROT group were decreased(P <0.05),and DSP-4 further decreased TH cells in LC and SN regions(P < 0.05).The results of striatum region were consistent with those of SN and LC region.Western Blot analysis of TH protein content in the midbrain and striatum of mice showed that Rot decreased expression of TH(P < 0.01),and DSP-4 further significantly decreased the TH protein content in the midbrain and striatum of mice(P < 0.001).7.Effect of LIP-1 on iron content in midbrain of ROT mice with NE deficiency:Iron content kit test showed LIP-1 reduced iron content of mice’s midbrain(P <0.001).8.Effects of LIP-1 on lipid peroxidation in the midbrain of NE deficient ROT mice: LIP-1 increased GSH level of mice’s midbrain(P < 0.001).And LIP-1 decreased MDA level of mice’s midbrain(P < 0.001).9.Effects of LIP-1 on the dopaminergic nervous system of NE deficient ROT mice: Immunohistochemical staining results showed that LIP-1 significantly inhibited the degeneration and death of TH cells in LC and SN region(P < 0.001).And the expression of TH protein in the midbrain and striatum of mice was increased compared with that in the DSP-4+ ROT group(P < 0.01).10.Effect of LIP-1 on gait test: ROT decreased the limb stride mice,and increased the forelimb and hindlimb distance(P < 0.001).However,DSP-4+ ROT group showed no significant changes in limb stride and fore-hind limb distance when controlled with ROT mice,which may be due to the maximum changes of ROT on motor function of mice.LIP-1 treatment alleviated ROT-induced gait abnormity.Compared with ROT group,LIP-1 increased the limb stride of mice,while decreased the fore-hind limb distance(P < 0.05).Conclusions1.NE deficiency aggravated the ferroptosis of dopaminergic neurons in Rot-exposed mice.2.Ferroptosis inhibitors alleviated the damage of DA neurons induced by NE deficiency.3.Ferroptosis inhibitors alleviated the abnormal motor function induced by NE deficiency. |
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