Study On Ferroptosis Of Primary Cortical Neurons In Rats Induced By BPDE

Objective:Benzo[a]pyrene（B[a]P）,as an environmental pollutant,can induce neuron loss in humans and animals,leading to neurobehavioral loss.Ferroptosis is a newly discovered way of cell death.The aim of this study was to investigate the mechanism of ferroptosis in neurons induced by 7,8-dihydroxy9,10-epoxybenzo[a]pyrene（BPDE）,a product of B[a]P metabolism,and to provide a new idea for the study of the neurotoxic mechanism of B[a]P.Methods:The primary cerebral cortex cells were extracted from SD rats within one day of birth,and the Neurons were isolated and cultured for 7 days.The cells were divided into solvent control group（DMSO）and BPDE infected group（0.125,0.25,0.5μmol/L BPDE）for 24 h.Another four groups were taken from the same group as above,and each dose group was added with 20μmol/L apoptosis inhibitor（Z-VAD-FMK）,10μmol/L ferroptosis inhibitor（Fer-1）,100μmol/L deferriamine（DFO）,and 20μmol/L necrotizing apoptosis inhibitor（Nec-1）for intervention.The survival rate of primary cortical neurons in rats was detected by CCK-8 method.MDA content,SOD activity,GSH content,GSH-Px activity and Fe²⁺content in cells were detected by the assay kit.The protein expression levels of iron death markers GPX4,ACSL4,SLC7A11,COX2,ferroptosis channels and autophagy-related proteins TFR,TF,FTH1,FTL,DMT1,ZIP8,NCOA4,P62 and LC3B were detected by Western blot.Results:With the increase of BPDE exposure dose,the survival rate of neuronal cells in rats showed a decreasing trend(P_trend<0.05).Morphological observation showed that the neuronal cell body atrophy and synaptic rupture after BPDE exposure.The ultrastructure of primary cortical neurons of rats exposed to BPDE was observed by transmission electron microscopy.The mitochondria of cells exposed to 0.25μmol/L and 0.5μmol/L doses of BPDE showed obvious changes.Severe crest reduction or disappearance was found in mitochondria of the 0.25μmol/L dose group.In the 0.5μmol/L dose group,the mitochondria of neurons became smaller and pyknotic,and the membrane density increased and mitochondrial ridges disappeared.The levels of lipid ROS,MDA,total Fe²⁺and mitochondrial Fe²⁺all increased gradually with the increase of BPDE exposure dose(P_trend<0.01).Especially in the highest dose of0.5μmol/L exposure oxidation level was significantly higher than both the control group and the other two groups of lower dose.The content of GSH,the activity of GSH-PXs and the activity of SOD all showed a decreasing trend with the increase of exposure dose(P_trend<0.01).The antioxidant levels at 0.25μmol/L and 0.5μmol/L doses were significantly lower than those in the control group.The results showed that the protein expression level of GPX4 decreased gradually with the increase of the exposure dose（P<0.01）;The expression level of COX2 and ACSL4 at0.25μmol/L and 0.5μmol/L was significantly higher than that in the control group.Compared with the control group,SLC7A11 protein expression was significantly decreased（P<0.01）;The results showed that BPDE exposure significantly increased transferrin（TF）expression compared with the control group（P<0.01）,zinc ferric ion transporter 8（ZIP8）was significantly increased only in the 0.25μmol/L dose group（P<0.05）,the levels of ferritin heavy chain（FTH1）protein were significantly decreased at 0.25μmol/L and 0.5μmol/L doses（P<0.05）.Compared with the control group,the expression level of nuclear receptor coactivator 4（NCOA4）in all dose groups was significantly increased（P<0.01）;The expression of transferrin receptor（TFR）was not significantly changed in 0.125μmol/L and 0.25μmol/Lgroups,but decreased in 0.5μmol/L group（P<0.05）.Divalent metal ion transporter 1（DMT1）and ferritin light chain（FTL）protein levels were not different at different doses compared with those in the control group.After the intervention of four cell death inhibitors,the cell survival rate showed that the apoptosis inhibitor（Z-VAD-FMK）could significantly increase the cell survival rate of 0.25μmol/L and 0.5μmol/L groups（P<0.01）.Deferriamine（DFO）significantly increased cell viability and decreased ROS level in 0.25μmol/L and 0.5μmolmol/L groups（P<0.01）,increased GSH level and SOD activity in 0.5μmol/L group（P<0.01）,decreased Fe²⁺level and MDA content in 0.125μmol/L,0.25μmol/L and 0.5μmol/L groups（P<0.01）;Ferroptosis inhibitor（Fer-1）significantly increased cell survival rate and decreased ROS level in 0.25μmol/L and 0.5μmol/L groups（P<0.01）,increased GSH level and SOD activity in 0.5μmol/L group（P<0.01）,decreased Fe²⁺level and MDA content in 0.125μmolmol/L,0.25μmol/L and 0.5μmol/L groups（P<0.01）;The survival rate of cells exposed to BPDE was not significantly changed by necrotizing apoptosis inhibitor（NEC-1）（P>0.05）.Conclusion:Exposure to BPDE can induce ferroptosis of primary cortical neurons in rats.The mechanism may be related to the blocked production and depletion of GSH,decreased GSH-PXS activity,and the disorder of iron balance.