Mechanisms Of Cholesterol And 24S-hydroxycholesterol On Iron Metabolism And Cell Damage In Neurons

Objective:Selective loss of dopaminergic neurons in the Substantia nigra(SN)is the main pathological feature of Parkinson’s disease(PD),and nigral iron deposition is a key factor in the pathogenesis of PD.Excessive iron can lead to increased production of reactive oxygen species(ROS)in cells and cause cell damage.Cholesterol is abundant in the brain.Excessive cholesterol can cause imbanlance of iron metabolism and cell death in the brain,which is closely related to the pathogenesis of PD.Cholesterol derivative 24SHydroxycholesterol(24S-OHC)is significantly increased in plasma and cerebrospinal fluid of PD patients.It has been considered as a potential therapeutic target in PD.However,the mechanisms of cholesterol and 24S-OHC on neuronal iron metabolism and cell damage are still unclear.The aim of this study is to investigate the effects of cholesterol and 24S-OHC on iron metabolism and cell damage in neurons,as well as the related mechanisms,in order to provide new experimental support for the prevention and treatment of PD.Methods:The viability of SH-SY5 Y neurons and primary cultured ventral mesencephalon(VM)neurons was detected by cell counting kit-8(CCK-8)kit.The intracellular ROS levels were detected by flow cytometry.The expressions of proteins related with ferroptosis,apoptosis and necroptosis were detected by western blot.The mRNA expression of pro-inflammatory cytokines was detected by real-time PCR.Results:1.Effect of cholesterol on SH-SY5 Y neurons.1.1 The cell viability increased in SH-SY5 Y neurons treated with cholesterol of 100 μM compared with the control group(P < 0.05).Then cholesterol concentrations of 10 μM,100μM were selected for subsequent experiments.The intracellular ROS levels were increased in SH-SY5 Y neurons treated with 100 μM cholesterol compared with the control group(P< 0.001).Co-treatment with the iron chelator deferoxamine(DFO)partially inhibited the elevated ROS levels(P < 0.05).1.2 The expression of iron regulatory protein 1(IRP1)was increased in SH-SY5 Y neurons treated with 100 μM cholesterol(P < 0.001),compared with the control group.Pretreatment with ROS inhibitor N-acetyl-L-cysteine(NAC)inhibited the increase of IRP1 protein expression(P < 0.01).The expressions of transferrin receptor 1(Tf R1),divalent metal transporter 1(DMT1)and ferritin increased(P < 0.05,P < 0.001)and ferroportin 1(FPN1)expression decreased(P < 0.001).The contents of divalent iron and total iron increased(P< 0.01,P < 0.001),while the protein expression of glutathione peroxidase 4(GPX4)had no significant change in SH-SY5 Y neurons(P > 0.05),which indicating there was no ferroptosis occurred.1.3 The phosphorylation levels of the extracellular signal-regulated kinase(Erk)and nuclear factor kappa-B(NF-κB)increased in SH-SY5 Y neurons treated with 100 μM cholesterol,compared with the control group(P < 0.001).Co-treatment with DFO inhibited the increased phosphorylation of Erk and NF-κB(P < 0.01,P < 0.001).1.4 The expression of interleukin-1 beta(IL-1β)and interleukin-6(IL-6)mRNA in SHSY5 Y neurons treated with 100 μM cholesterol was up-regulated,compared with the control group(P < 0.001),which were inhibited by the addition of Erk inhibitors PD98095,DFO and NAC(P < 0.001).2.Effect of 24S-OHC on VM neurons.2.1 Cholesterol increased the expression of cholesterol 24-hydroxylase(CYP46A1)protein in SH-SY5 Y cells treated with 50 μM and 100 μM cholesterol(P < 0.05,P < 0.001).2.2 The cell viability decreased in primary cultured VM neurons treated with 24S-OHC of0.05 μM compared with the control group(P < 0.001).Then 24S-OHC concentrations of0.01 μM,0.1 μM were selected for subsequent experiments.The ROS levels in the primary cultured VM neurons treated with 0.01 μM and 0.1 μM 24S-OHC were significantly increased compared with the control group(P < 0.05,P < 0.01).2.3 The expressions of IRP1,Tf R1,DMT1 and Ferritin in primary cultured VM neurons treated with 0.1 μM 24S-OHC increased(P < 0.05,P < 0.01)while FPN1 expression decreased,compared with the control group(P < 0.01).Hepcidin mRNA expression was up-regulated when cells were treated with 0.01 μM 24S-OHC(P < 0.001).The intracellular divalent iron and total iron concentrations increased(P < 0.01),while the expressions of GPX4 and ferroptosis suppressor protein 1(FSP1)had no significant changes in the primary cultured VM neurons treated with 0.01 μM and 0.1 μM 24S-OHC(P > 0.05),indicating there is no ferroptosis occurred.2.4 Cleaved caspase-3 protein expression increased(P < 0.01),and the ratio of B-cell lymphoma-2(Bcl-2)/BCL2-associated X(BAX)protein expression decreased in primary cultured VM neurons treated with 0.1 μM 24S-OHC,compared with the control group(P< 0.05),indicating cell apoptosis occurred.The expression of receptor-interacting protein kinase 1(RIPK1),RIPK3 and phosphorylated mixed lineage kinase domain-like(p-MLKL)increased in primary cultured VM neurons treated with 0.1 μM 24S-OHC,compared with the control group(P < 0.01,P < 0.001).When pretreated with necrostatin-1(Nec-1),the expressions of RIPK1,RIPK3 and p-MLKL were inhibited(P < 0.001),indicating necroptosis occurred;After co-treatment with DFO,the increase of Cleaved caspase-3protein expression was inhibited(P < 0.05),while the expression of p-MLKL protein was not affected(P < 0.001).The result of electron microscope showed that different degrees of nuclear membrane rupture,chromatin condensation,cytoplasmic vacuolization,organelle swelling,membrane rupture and content outflow were observed in primary cultured VM neurons treated with 0.1 μM 24S-OHC,which further proved that the cells underwent apoptosis and necroptosis.3.Effects of 24S-OHC on SH-SY5 Y neurons.3.1 The cell viability increased in SH-SY5 Y neurons treated with 24S-OHC of 5 μM compared with the control group(P < 0.001).Then cholesterol concentrations of 2 μM,5μM were selected for subsequent experiments.The expressions of IRP1 and DMT1 increased in the 5 μM 24S-OHC treated SH-SY5 Y neurons,compared with the control group(P < 0.01).The expressions of Tf R1 and FPN1 decreased(P < 0.05,P < 0.01),while GPX4 protein expression remained unchanged in the 5 μM 24S-OHC treated SH-SY5 Y neurons(P > 0.05),indicating no ferroptosis occurred.3.2 The expression of Cleaved caspase-3 increased(P < 0.001)and the ratio of Bcl-2/ Bax protein levels decreased(P < 0.001)in SH-SY5 Y neurons treated with 5 μM 24S-OHC than that in the control group,indicating the cells underwent apoptosis;The decrease of cell viability was inhibited when pretreated with Nec-1(P < 0.001),indicating the cells underwent necroptosis.3.3 The expression of IL-1β and IL-6 mRNA in SH-SY5 Y neurons treated with 5 μM 24 SOHC was up-regulated,compared with the control group(P < 0.01,P < 0.001).Conclusion:1.Cholesterol causes an increase in ROS levels in SHSY-5Y cells.Activation of IRP1 by elevated ROS increases iron influx and reduces iron outflow,leading to an increase in intracellular iron content,but no ferroptosis occurs in the cells;Cholesterol increases proinflammatory factors IL-1βand IL-6,which may be related to Erk and NF-κB pathway,and this process can be partially inhibited by DFO.2.24S-OHC increases ROS levels in primary cultured VM neurons and SHSY-5Y cells,and increases iron influx and decreases iron outflow through IRP1 activation,resulting in an increase in intracellular iron content;24S-OHC does not cause ferroptosis but can induce cell apoptosis and necroptosis.DFO could partially inhibit cell apoptosis but not necroptosis.This experiment observed the effects of different concentrations of cholesterol and 24 SOHC on SH-SY5 Y neurons and primary cultured VM neurons,and the related mechanisms of cholesterol on cell iron metabolism and cell damage,providing new experimental support for the prevention and treatment of neurodegenerative diseases.