Role Of Oxidative Damage And Abnormal Iron Metabolism In Aluminum-induced Ferroptosis In Primary Neurons And Its Molecular Mechanism

Objective:To investigate the role of ferroptosis in aluminum-induced primary neuronal death,to clarify the possible mechanisms of oxidative damage and abnormal iron metabolism in aluminum-induced neuronal ferroptosis,and to provide a theoretical basis for aluminum neurotoxicity studies.Methods:Neonatal rat cerebral cortex was isolated at 24 h of birth in SD rats,and primary neurons were extracted and cultured.After the primary neuron-specific Neurobasal?-A mediumneurons matured,the neurons were stained with 0,25,50,100,200 and 400μmol/L Al（mal）₃ for 12 h,24 h and 48 h.The cell survival rate of each group at different staining times and doses was determined by CCK-8,and the staining doses and staining times at which the primary neurons survival rate was 65%-75%were used for subsequent experiments.The primary neurons were divided into 13 groups as follows:0μmol/L Al（mal）₃ group,0.5%DMSO group,100μmol/L Al（mal）₃ group,50μmol/L Nec-1 group,20μmol/L Z-VAD-FMK group,50μmol/L 3-MA group,and 50μmol/L DFO group,20μmol/L FINO₂group,100μmol/L Al（mal）₃+Nec-1 group,100μmol/L Al（mal）₃+Z-VAD-FMK group,100μmol/L Al（mal）₃+3-MA group,100μmol/L FINO₂ group Al（mal）₃+DFO and 100μmol/L Al（mal）₃+FINO₂ groups.When combined with 0.5%DMSO,50μmol/L Nec-1,20μmol/L Z-VAD-FMK,50μmol/L 3-MA,50μmol/L DFO and 20μmol/L FINO₂,the final concentration was 100μmol/L Al（mal）₃ was added 2 h before pretreatment and cultured in the incubator for 24 h before subsequent experiments.The primary neurons survival rate was determined by CCK-8,and the morphological changes of cells after Microtubule-associated protein 2（MAP2）and DAPI fluorescence staining were observed by inverted microscopy.Solute carrier family 7 member 11（SLC7A11）and monoclonal antibody to glutathione peroxidase 4（GPX4）,which are proteins related to oxidative damage,were detected by Western-blot.Glutathione peroxidase（GSH-PX）activity and Glutathione（GSH）content were measured using glutathione peroxidase test kit and micro-reduced glutathione test kit.The expression levels of iron metabolism-related proteins Transferrin receptor 1（TFR1）,Divalent metal transporter 1（DMT1）,Ferroportin l（FPN1）and Ferritin heavy chain（FTH1）were detected by Western-blot.Quantitative Real-time PCR（q RT-PCR）was used to detect the m RNA expression levels of TFR1,DMT1,FPN1 and FTH1 in neonatal rat cortical primary neurons of SD rats.To explore the possible mechanisms of ferroptosis in aluminum-induced neuronal death by comparing the bivariate effects of Al（mal）₃ alone and its co-staining with DFO and FINO₂on neuronal cortical primary neuronal neurotoxicity indicators,oxidative damage and iron metabolism related indicators in of SD rats.Results:1.Determination of Al（mal）₃ dose and timeThere was no significant change in the survival rate when the primary neurons were contaminated with gradient concentrations of Al（mal）₃ for 12 h（P>0.05）.The primary neurons survival rate decreased significantly after staining with 100μmol/L,200μmol/L and 400μmol/L Al（mal）₃ for 24 h（P<0.05）.The survival rate also decreased significantly after staining primary neurons with 100μmol/L,200μmol/L and 400μmol/L Al（mal）₃ for48 h（P<0.05）,and 24 h of 100μmol/L Al（mal）₃contamination at a survival rate of69.48±0.04 was used as the dose and time of contamination for subsequent experiments.2.Role of Ferroptosis in Aluminum-induced Primary Neuronal Death in SD RatsThe cell survival rate after 24 h Al（mal）₃ alone and combined with DFO and FINO₂ in primary neurons showed that 20μmol/L FINO₂、100μmol/L Al（mal）₃+DMSO and 100μmol/L Al（mal）₃+FINO₂ groups were（18.76±0.002）%,（71.92±0.04）%and（15.42±0.002）%,which were significantly lower than 0μmol/L Al（mal）₃group（100±0.00）%（P<0.05）.Compared with the cell survival rate of 0.5%DMSO group（102.59±0.05）%,the survival rate of 100μmol/L Al（mal）₃+DMSO group（72.81±0.04）%,20μmol/L FINO₂ group（18.76±0.002）%,100μmol/L Al（mal）₃+DMSO group（71.92±0.04）%and 100μmol/L Al（mal）₃+FINO₂ group（15.42±0.002）%were reduced（P<0.05）.Compared with 100μmol/L Al（mal）₃+DMSO group（71.92±0.04）%,the50μmol/L DFO group（113.59±0.05）%and 100μmol/L Al（mal）₃+DFO group（95.42±0.06）%had an increased survival rate（P<0.05）,and 20μmol/L FINO₂ group（18.76±0.002）%,100μmol/L Al（mal）₃+FINO₂ group（15.42±0.002）%were decreased（P<0.05）.After 24 h Al（mal）₃ staining alone and its combined staining with DFO and FINO₂ in primary neurons,the following qualitative results were observed on the morphology of fluorescently stained neurons.The neurons in 0μmol/L Al（mal）₃,0.5%DMSO,and 50μmol/L DFO groups had full and rounded cytosomes with clear and intertwined axons,while the neurons in 100μmol/L Al（mal）₃,100μmol/L Al（mal）₃,100μmol/L Al（mal）₃+DMSO,20μmol/L FINO₂ and 100μmol/L Al（mal）₃+FINO₂ groups,the neuronal cell bodies were smaller,the edges were blurred,and the dendrites were broken,deformed and less interconnected.The neurons in 100μmol/L Al（mal）₃+DFO group recovered better,with some axons shortened and not obviously connected with the surrounding neurons,but most of the cells had rounded cytosomes and long and thick axons,which were interwoven into a dense net visible to the naked eye.Statistical analysis of the immunofluorescence intensity after 24 h of Al（mal）₃ staining alone and its co-staining with DFO and FINO₂ in primary neurons yielded the following quantitative results.The statistical results of immunofluorescence staining on the morphology of primary neurons and neuronal fluorescence intensity after 24 h of Al（mal）₃staining alone or in combination showed that compared with 0μmol/L Al（mal）₃group（372.89±3.61）,100μmol/L Al（mal）₃ group（339.16±7.97）,20μmol/L FINO₂group（296.06±9.95）,100μmol/L Al（mal）₃+DMSO group（297.05±7.68）and 10μmol/L Al（mal）₃+FINO₂ group（284.70±12.26）showed a significant decrease in fluorescence intensity（P<0.05）;compared with 0.5%DMSO group（320.08±15.46）,the fluorescence intensity of neurons in 100μmol/L Al（mal）₃ group（339.16±7.97）and 100μmol/L Al（mal）₃+FINO₂group（284.70±6.26）was reduced,and the difference was statistically significant（P<0.05）;the fluorescence intensity of 100μmol/L Al（mal）₃+FINO₂group（284.70±6.26）was even lower compared with 100μmol/L Al（mal）₃+DMSO group（297.05±7.68）（P<0.05）.3.Role of necrosis,apoptosis,and autophagy in aluminum-induced primary neuronal deathCell viability assays after 24 h of Al（mal）₃ alone and in combination with Z-VAD-FMK,Nec-1 and 3-MA staining of primary neurons showed that compared with 0μmol/L Al（mal）₃group（100±0.00）%,100μmol/L Al（mal）₃ group（72.81±0.04）%,100μmol/L Al（mal）₃+DMSO group（71.94±0.03）%,100μmol/L Al（mal）₃+Nec-1 group（59.26±0.04）%,100μmol/L Al（mal）₃+Z-VAD-FMK group（61.50±0.03）%and 100μmol/L Al（mal）₃+3-MA group（56.73±0.03）%had significantly lower cell survival（P<0.05）;compared with 0.5%DMSO group（102.59±0.05）%,100μmol/L Al（mal）₃ group（72.81±0.04）%,100μmol/L Al（mal）₃+DMSO group（71.94±0.03）%,100μmol/L Al（mal）₃+Nec-1 group（59.26±0.04）%,100μmol/L Al（mal）₃+Z-VAD-FMK group（61.50±0.03）%and 100μmol/L Al（mal）₃+3-MA group（56.73±0.03）%were also significantly lower（P<0.05）;the cell survival rates of different death mode inhibitors alone 50μmol/L Nec-1 group,20μmol/L Z-VAD-FMK group and 50μmol/L 3-MA group,were（110.87±0.03）%,（98.47±0.04）%,and（106.07±0.04）%were higher than those of 100μmol/L Al（mal）₃+DMSO group（71.94±0.03）%（P<0.05）,while the cell survival rates of the combined staining groups that 100μmol/L Al（mal）₃+Nec-1 group（59.26±0.04）%,100μmol/L Al（mal）₃+Z-VAD-FMK group（61.50±0.03）%,and 100μmol/L Al（mal）₃+3-MA group（56.73±0.03）%,were lower than those of 100μmol/L Al（mal）₃+DMSO group（71.94±0.03）%（P<0.05）.In conclusion,the different death mode inhibitor groups did not result in a significant increase in the cell survival rate caused by Al（mal）₃ staining.After 24 h Al（mal）₃ staining alone and its combined staining with Z-VAD-FMK,Nec-1 and 3-MA in primary neurons,the following qualitative results were observed on the morphology of fluorescently stained neurons.The 50μmol/L Nec-1,20μmol/L Z-VAD-FMK,and 50μmol/L 3-MA were applied to primary neurons alone,their morphology did not change significantly,with large and full cytosol,interconnected axons,and uniformly interwoven network;while when they were combined with Al（mal）₃ staining that 100μmol/L Al（mal）₃+Nec-1,100μmol/L Al（mal）₃+Z-VAD-FMK,100μmol/L Al（mal）₃+3-MA group,the reticulocyte structure was significantly reduced,with broken axons,wrinkled cytosol and poor neuronal status.Statistical analysis of the immunofluorescence intensity after 24 h of Al（mal）₃ staining alone and its co-staining with Z-VAD-FMK,Nec-1 and 3-MA in primary neurons yielded the following quantitative results.Statistical analysis of fluorescence intensity yielded a significant decrease（P<0.05）in fluorescence intensity in 100μmol/L Al（mal）₃group（372.89±3.61）,100μmol/L Al（mal）₃+DMSO group（297.05±7.68）and 100μmol/L Al（mal）₃+3-MA group（292.90±8.09）compared with 0μmol/L Al（mal）₃,and the co-stained group 100μmol/L Al（mal）₃+Nec-1（312.09±3.83）,100μmol/L Al（mal）₃+Z-VAD-FMK（314.52±3.80）,and 100μmol/L Al（mal）₃+3-MA（292.90±8.09）still did not increase the fluorescence intensity to the level of DMSO in the blank group as well as the solvent control group compared with 50μmol/L Nec-1（329.04±9.95）,20μmol/L Z-VAD-FMK（323.33±6.07）,and 50μmol/L 3-MA（340.36±8.59）,which were individually dyed.4.Possible mechanism of aluminum-induced Ferroptosis in primary neuronsThe results of protein expression level analysis of oxidative damage related protein SLC7A11 were as follows:in 100μmol/L Al（mal）₃ group（0.97±0.15）,20μmol/L FINO₂group（0.83±0.10）and 100μmol/L Al（mal）₃+FINO₂ group（0.85±0.06）compared with 0μmol/L Al（mal）₃ group（1.18±0.13）,but the difference was not statistically significant（P>0.05）;compared with 0.5%DMSO group（1.07±0.10）,SLC7A11 protein expression was reduced in both 20μmol/L FINO₂ group（0.83±0.10）and 100μmol/L Al（mal）₃+FINO₂group（0.85±0.06）,but the difference was not statistically significant（P>0.05）;compared with 100μmol/L Al（mal）₃+DMSO group（1.22±0.06）,SLC7A11 protein expression in 100μmol/L Al（mal）₃+FINO₂ group（0.85±0.06）was reduced,but the difference was not statistically significant（P>0.05）.The results of the protein expression level analysis of GPX4,a protein related to oxidative damage,were as follows:GPX4 protein expression was decreased in the 100μmol/L Al（mal）₃ group（0.80±0.04）,20μmol/L FINO₂ group（0.84±0.19）and 100μmol/L Al（mal）₃+DMSO group（0.89±0.17）that compared with 0μmol/L Al（mal）₃ group（1.25±0.78）and the difference was not statistically significant（P>0.05）,GPX4 protein expression was significantly lower in 100μmol/L Al（mal）₃+FINO₂ group（0.61±0.19）compared with 0μmol/L Al（mal）₃ group（1.25±0.78）;compared with 0.5%DMSO group（1.02±0.15）,GPX4protein expression in 20μmol/L FINO₂ group（0.84±0.19）and 100μmol/L Al（mal）₃+FINO₂group（0.61±0.19）showed a decreasing trend,but the difference was not statistically significant（P>0.05）;compared with 100μmol/L Al（mal）₃+DMSO group,the GPX4 protein expression of 100μmol/L Al（mal）₃+DFO group（1.37±0.07）was elevated,while the GPX4protein expression of 100μmol/L Al（mal）₃+FINO₂ group（0.61±0.19）was reduced,but the difference was not statistically significant（P>0.05）.The results of the analysis of GSH-PX content（viability units/mgprot）,a substance related to oxidative damage,were as follows:compared with 0μmol/L Al（mal）₃group（211.68±3.74）,the GSH-PX content in 100μmol/L Al（mal）₃ group（211.68±3.74）,20μmol/L FINO₂ group（146.91±6.44）and 100μmol/L Al（mal）₃+FINO₂ group（108.22±6.2）were significantly reduced（P<0.05）;compared with 0.5%DMSO group（205.37±1.36）,the GSH-PX content of 20μmol/L FINO₂ group（146.91±6.44）and 100μmol/L Al（mal）₃+FINO₂group（108.22±6.2）was significantly reduced（P<0.05）;compared with 100μmol/L Al（mal）₃+DMSO group（168.31±4.41）,the GSH-PX content of 100μmol/L Al（mal）₃+FINO₂group（108.22±6.2）was reduced（P<0.05）;the GSH-PX content of 50μmol/L DFO group（205.37±1.36）and 100μmol/L Al（mal）₃+DFO group（188.13±5.51）were increased（P>0.05）.The results of the analysis of the GSH content（μmol/gprot）of substances related to oxidative damage were as follows:compared with 0μmol/L Al（mal）₃group（80.40±6.37）μmol/gprot and 0.5%DMSO group（85.52±8.66）μmol/gprot,the GSH content was reduced in both 20μmol/L FINO₂ group（60.60±3.69）μmol/gprot and 100μmol/L Al（mal）₃+FINO₂ group（51.71±4.71）μmol/gprot（P<0.05）;compared with 100μmol/L Al（mal）₃+DMSO group（66.55±3.75）μmol/gprot,0.5%DMSO group（85.52±8.66）μmol/gprot and 50μmol/L DFO group（85.56±4.59）μmol/gprot had significantly higher GSH content（P<0.05）.The GSH content in the 100μmol/L Al（mal）₃+DFO group（75.32±4.76）μmol/gprot was higher than that in the 100μmol/L Al（mal）₃+DMSO group（66.55±3.75）μmol/gprot,but the difference was not statistically significant.The results of the statistical analysis of the total intracellular iron content were as follows:iron content was significantly increased in 100μmol/L Al（mal）₃ group（16.73±2.09）μmol/gprot,20μmol/L FINO₂ group（19.12±2.79）μmol/gprot and 100μmol/L Al（mal）₃+FINO₂ group（21.97±3.89）μmol/gprot compared with the 0μmol/L Al（mal）₃group（9.31±2.54）μmol/gprot and 0.5%DMSO group（7.08±1.48）μmol/gprot（P<0.05）.Compared with 100μmol/L Al（mal）₃+DMSO group（13.39±1.95）μmol/gprot,100μmol/L Al（mal）₃+FINO₂ group（21.97±3.89）μmol/gprot was significantly increased（P<0.05）.The results of the analysis of the expression levels of the iron ion storage related protein FTH1 were as follows:the difference in FTH1 expression between different intervention groups showed that compared with 0μmol/L Al（mal）₃ group（1.37±0.46）and 0.5%DMSO group（0.74±0.11）,100μmol/L Al（mal）₃ group（6.96±1.08）,20μmol/L FINO₂group（5.42±1.53）,and 100μmol/L Al（mal）₃+FINO₂ group（9.42±2.89）had a significant increase in FTH1 protein expression（P<0.05）.The FTH1 protein expression in 100μmol/L Al（mal）₃+FINO₂ group（9.42±2.89）was significantly higher（P<0.05）compared to 100μmol/L Al（mal）₃+DMSO group（3.07±0.30）,while there was no significant difference in protein expression between 100μmol/L Al（mal）₃+DFO group（3.43±0.71）and 100μmol/L Al（mal）₃+DMSO group（3.07±0.30）（P>0.05）.The results of the analysis of the expression levels of TFR1,a protein related to iron metabolism,were as follows:with elevated TFR1 protein expression in 100μmol/L Al（mal）₃group（1.77±0.14）and 100μmol/L Al（mal）₃+FINO₂ group（2.33±0.14）compared with 0μmol/L Al（mal）₃ group（1.35±0.25）as well as 0.5%DMSO group（1.44±0.12）（P<0.05）;compared with TFR1 protein expression in 100μmol/L Al（mal）₃+DMSO group（1.66±0.40）,100μmol/L Al（mal）₃+FINO₂ group（2.33±0.14）showed increased（P<0.05）,while 100μmol/L Al（mal）₃+DFO group（1.58±0.26）showed a slight decrease（P>0.05）.The results of the analysis of the expression levels of DMT1,a protein related to iron metabolism,were as follows:the expression of DMT1 was elevated in the 100μmol/L Al（mal）₃ group,100μmol/L Al（mal）₃+DMSO group,and 20μmol/L FINO₂group（0.81±0.14）,（0.93±0.47）,and（1.29±0.49）,respectively,compared with that in 0μmol/L Al（mal）₃ group（0.42±0.11）,the difference was statistically significant（P<0.05）;compared with 0.5%DMSO group（0.58±0.37）,DMT1 protein expression was elevated in100μmol/L Al（mal）₃+DMSO group（0.93±0.47）and 100μmol/L Al（mal）₃+FINO₂group（0.58±0.26）（P<0.05）.While DMT1 protein expression was no significantly different in 50μmol/L DFO group（0.50±0.36）.Compared with 100μmol/L Al（mal）₃+DMSO group（0.93±0.47）,DMT1 expression was significantly higher in 100μmol/L Al（mal）₃+FINO₂ group（1.29±0.49）（P<0.05）.The results of the analysis of TFR1 m RNA expression levels of iron metabolism-related substances were as follows:the difference in TFR1 m RNA expression was elevated in 20μmol/L FINO₂ group（1.87±0.59）and 100μmol/L Al（mal）₃+FINO₂ group（2.11±0.21）compared with 0μmol/L Al（mal）₃ group（1.00±0.00）（P<0.05）;compared with 0.5%DMSO group（1.23±0.21）,the 20μmol/L FINO₂ group（1.87±0.59）TFR1m RNA expression was significantly higher（P<0.05）.While TFR1m RNA expression was significantly lower（P<0.05）in 100μmol/L Al（mal）₃+DFO group（0.82±0.18）;compared with 100μmol/L Al（mal）₃+DMSO group（1.55±0.60）,100μmol/L Al（mal）₃+FINO₂ group（2.11±0.21）had significantly higher TFR1 m RNA expression（P<0.05）,but 50μmol/L DFO group（1.09±0.20）and 100μmol/L Al（mal）₃+DFO group（0.82±0.18）had lower TFR1 m RNA expression,but the differences were not statistically significant.The results of the analysis of DMT1 m RNA expression levels of iron metabolism-related substances were as follows:compared with 0μmol/L Al（mal）₃ group（1.00±0.00）,primary neuronal DMT1 m RNA expression was significantly higher（P<0.05）in 100μmol/L Al（mal）₃+DMSO group（1.45±1.06）and 100μmol/L Al（mal）₃+FINO₂ group（2.52±2.29）.After DFO treatment,100μmol/L Al（mal）₃+DFO group（1.05±0.22）showed no significant difference in DMT1 m RNA expression from 0μmol/L Al（mal）₃ group（1.00±0.00）（P>0.05）;compared with 0.5%DMSO group（1.12±0.6）,100μmol/L Al（mal）₃+DMSO group（1.45±1.06）,20μmol/L FINO₂ group（1.84±0.6）and 100μmol/L Al（mal）₃+FINO₂group（2.52±0.29）were elevated（P<0.05）,and DMT1 m RNA expression in 100μmol/L Al（mal）₃+DFO group（1.05±0.22）was not significantly different from that in 0.5%DMSO group（1.12±0.6）（P>0.05）;the DMT1 m RNA expression was elevated in 100μmol/L Al（mal）₃+FINO₂ group（2.52±2.29）compared to 100μmol/L Al（mal）₃+DMSO group（1.45±1.06）（P<0.05）.The results of the analysis of FPN1 m RNA expression levels of iron metabolism-related substances were as follows:FPN1 m RNA expression was decreased in 100μmol/L Al（mal）₃group（0.90±0.13）and 20μmol/L FINO₂ group（0.77±0.44）compared with 0μmol/L Al（mal）₃ group（1.00±0.00）,but the differences were not statistically significant（P>0.05）.Compared with 0μmol/L Al（mal）₃ group（1.00±0.00）,.Compared with 0μmol/L Al（mal）₃group（1.00±0.00）and 0.5%DMSO group（1.26±0.15）,100μmol/L Al（mal）₃+FINO₂group（0.45±0.23）was significantly decrease（P<0.05）.while there was no significant difference（P>0.05）that FPN1 m RNA expression between 0.5%DMSO group（1.26±0.15）and 50μmol/L DFO group（1.30±0.23）.Compared with 100μmol/L Al（mal）₃+DMSO group（1.07±0.53）,100μmol/L Al（mal）₃+FINO₂ group（0.45±0.23）FPN1m RNA expression was significantly lower（P<0.05）.The results of the analysis of FTH1 m RNA expression levels of iron metabolism-related substances were as follows:compared with 0μmol/L Al（mal）₃ group（1.00±0.00）,0.5%DMSO group（1.03±0.25）and 100μmol/L Al（mal）₃+DMSO group（1.12±0.40）,FTH1m RNA expression elevated expression in 100μmol/L Al（mal）₃+DFO group（2.06±0.27）and100μmol/L Al（mal）₃+FINO₂ group（4.51±0.94）,the difference was statistically significant（P<0.05）.Conclusions:Al（mal）₃ treating can lead to ferroptosis,apoptosis,necrosis and autophagy in primary neurons,and ferroptosis was an important mode of death.Oxidative damage was one of the mechanisms by which aluminum causes ferroptosis in primary neurons,and the specific mechanism is that aluminum can reduce the amount of cysteine entering the cell by inhibiting the expression of SLC7A11 in primary neurons,which in turn reduces the synthesis of GSH.The combination of GSH reduction and GPX4inactivation leads to the disruption of the balanced redox state in the cell,further inducing the development of ferroptosis.Aluminum increases the amount of iron ions entering the primary neurons by altering the expression of TFR1 and DMT1,and conversely decreases the amount of iron ions exiting the primary neurons by decreasing the expression of FPN1,resulting in the disruption of intracellular iron ions content and the increase of FTH1 content,which in turn causes the imbalance of iron metabolism and induces ferroptosis in the primary neurons.