| Objective: To investigate the role and mechanism of ferroptosis in neuronal death induced by aluminum(Al),and to provide a theoretical basis for further study of neurotoxicity of aluminum.Methods: 1.Determination of aluminum maltolate(Al(mal)3)dose and time Rat adrenal pheochromocytoma(PC12)cells were treated with high-glucose DMEM medium with final concentrations of 0,50,100,200,and 400 ?mol/L aluminum maltolate(Al(mal)3),respectively,for 12 h,24 h,and 48 h,the relative cell viability was determined using the Cell Counting Kit-8(CCK-8),and the final dose and treated time of Al(mal)3 were determined.2.Experimental grouping and measurement of indexes PC12 cells were divided into the following 10 groups: 0 ?mol/L Al(mal)3 group(control group),200 ?mol/L Al(mal)3-treated group,0.5% DMSO group(solvent control group),5 ?mol/L Erastin group,5 ?mol/L Fer-1 group,200 ?mol/L DFO group,200 ?mol/L Al(mal)3+0.5% DMSO group,200 ?mol/L Al(mal)3+5 ?mol/L Erastin group,200 ?mol/L Al(mal)3+ 5 ?mol/L Fer-1 group and 200 ?mol/L Al(mal)3+200 ?mol/L DFO group.DMSO is a solvent for Erastin(a classic agonist of ferroptosis),Fer-1 and DFO(inhibitors of ferroptosis).In the co-treatment group,PC12 cells were pretreated with DMSO or interfering agent for 2 h,then Al(mal)3 was added to a final concentration of 200 ?mol/L for 24 h.The cells were collected for subsequent experiments.The morphology of PC12 cells was observed under an inverted microscope;the viability of PC12 cells was detected by the CCK-8 assay;the ultrastructure of PC12 cells was observed by transmission electron microscopy;the glutathione(GSH)content of PC12 cells was detected by the microplate method;glutathione peroxidase(GSH-PX)activity and intracellular iron content in PC12 cells were detected by colorimetric method;levels of reactive oxygen species(ROS)in cultured PC12 cells were examined using flow cytometry;the protein expression levels of cystine/glutamate transporter(System Xc-),glutathione peroxidase 4(GPX4),transferrin receptor 1(TFR1),divalent metal ion transporter metal transporter 1(DMT1),and ferritin heavy chain 1(FTH1)were measured using Western Blot;the m RNA expression levels of SLC7A11,GPX4,TFR1,DMT1,and FTH1 were measured using q RT-PCR.Results: 1.Determination of Al(mal)3 dose and time Compared with the control group,there were no significant change in the viability of PC12 cells in each group induced by Al(mal)3 exposure for 12 h at different concentrations(P<0.05);after 24 h and 48 h of Al(mal)3 exposure,the cell viability of PC12 cells decreased in a dose-dependent manner,and the cell viability of PC12 cells in 200 ?mol/L Al(mal)3 group and 400 ?mol/L Al(mal)3 group significantly decreased(P<0.05).In general,cytotoxicity experiments are performed at concentrations of 70 to 80% for cell viability.This study showed that the viability of PC12 cells was(74.93 ± 0.08)% after exposure to 200 ?mol/L Al(mal)3 for 24 h,so the final determined dose was 200 ?mol/L Al(mal)3,and the duration of exposure was 24 h.2.The role of ferroptosis in PC12 cell death triggered by aluminum Cell viability results showed that compared with the control group,the viability of PC12 cells in Al(mal)3 group(74.39±0.02)was significantly decreased(P<0.05).Compared with DMSO group,there was no significant change in PC12 cell viability in Fer-1 group,and the viability of PC12 cells in Erastin group,DFO group and Al(mal)3+DMSO group(66.70±0.02)decreased obviously(P<0.05).Compared with Al(mal)3+DMSO group,the viability of PC12 cells in Al(mal)3+Erastin group(47.24±0.05)decreased significantly(P<0.05),the viability of PC12 cells in Al(mal)3+Fer-1 group(91.10±0.02)and Al(mal)3+DFO group(95.85±0.08)increased significantly(P<0.05).Observation with an inverted microscope revealed that compared with the control group,the number of PC12 cells in Al(mal)3 group was clearly reduced,the cell morphology was rounded and intercellular connections decreased.Compared with DMSO group,the number of PC12 cells in Fer-1 group did not change significantly,and the cell state was good;the number of PC12 cells in Erastin group,DFO group,and Al(mal)3+DMSO group decreased obviously,and the cells shrunk and round.Compared with Al(mal)3+DMSO group,the number of PC12 cells in Al(mal)3+Erastin group was significantly reduced;the number of PC12 cells was increased and the cell state was obviously improved in Al(mal)3+Fer-1 group and Al(mal)3+DFO group.Transmission electron microscopy observations showed that Al(mal)3 exposure caused PC12 cells to exhibit characteristic features of ferroptosis such as shrunken mitochondria and increased bilayer membrane density;characteristics of apoptosis such as chromatin edge set and apoptotic body formation;characteristic of autophagy,such as the formation of bilayer membrane autophagosomes;and features of necrosis,such as nuclear chromosome degradation and nuclear membrane lysis.The mitochondrial ultrastructure showed that compared with the control group,the mitochondrial shrunk significantly,the double-layer membrane density was increased,the mitochondrial ridges were reduced or disappeared,and some of the outer mitochondrial membranes were ruptured of PC12 cells in Al(mal)3 group.Compared with DMSO group,Erastin group and Al(mal)3+DMSO group had small mitochondrial and increased bilayer membrane density;while Fer-1 group and DFO group had normal mitochondrial morphology and complete structure.Compared with Al(mal)3+DMSO group,mitochondrial damage was worsened,and the outer membrane was severely ruptured of PC12 cells in Al(mal)3+Erastin group;while the mitochondrial damage of PC12 cells in Al(mal)3+Fer-1 group and the Al(mal)3+DFO group was obviously relieved,and the mitochondrial morphology and structure tended to be normal.Biochemical indicators of ferroptosis showed that compared with the control group,the GSH content(22.41±2.86 ?mol/gprot)and GSH-PX activity(106.21±5.20 viability units/mgprot)of PC12 cells in Al(mal)3 group were significantly reduced(P<0.05).Compared with DMSO group,the GSH content and GSH-PX activity of PC12 cells in Erastin group were obviously reduced(P<0.05),and the GSH-PX activity of PC12 cells in Fer-1 group and DFO group was obviously reduced(P<0.05),the GSH content(18.96±2.00 ?mol/gprot)and GSH-PX activity(89.98±4.95 viability units/mgprot)of PC12 cells in Al(mal)3+DMSO group also decreased significantly(P<0.05).Compared with Al(mal)3+DMSO group,the GSH content(11.27±0.68 ?mol/gprot)and GSH-PX activity(66.92±1.87 viability units/mgprot)of PC12 cells in Al(mal)3+Erastin group reduced significantly(P<0.05),the GSH content(24.16±1.22 ?mol/gprot)and GSH-PX activity(102.64±4.05 viability units/mgprot)of PC12 cells in Al(mal)3+Fer-1 group were obviously increased(P<0.05),and the GSH content(22.71±1.79 ?mol/gprot)and GSH-PX activity(116.05±3.83±1.87 viability units/mgprot)of PC12 cells in Al(mal)3+Fer-1 group were distinctly improved(P<0.05).Compared with the control group,the iron content of PC12 cells in Al(mal)3 group(38.43±3.94 ?mol/gprot)was obviously increased(P<0.05).Compared with DMSO group,the iron content in PC12 cells of Erastin group,Fer-1 group and DFO group did not change significantly,the iron content in PC12 cells of Al(mal)3+DMSO group(35.71±12.00 ?mol/gprot)increased distinctly(P<0.05).Compared with Al(mal)3+DMSO group,the iron content of PC12 cells in Al(mal)3+Erastin group(31.69±15.24 ?mol/gprot)had no significant change(P>0.05),the iron content of PC12 cells in Al(mal)3+Fer-1 group(22.62±6.29 ?mol/gprot)decreased but there was no statistical difference(P>0.05),the iron content of PC12 cells in Al(mal)3+DFO group(19.72±4.39 ?mol/gprot)reduced clearly(P<0.05).Compared with the control group,the ROS fluorescence intensity of PC12 cells in Al(mal)3 group(81.45±6.20)was obviously improved(P<0.05).Compared with DMSO group,the ROS fluorescence intensity of PC12 cells in Erastin group was significantly increased(P<0.05),the ROS fluorescence intensity in Fer-1 group was clearly decreased(P<0.05),the ROS fluorescence intensity in DFO group had no significant change,the ROS fluorescence intensity of PC12 cells in Al(mal)3+DMSO group(76.00±5.98)increased,but there was no statistical significance(P>0.05).Compared with Al(mal)3+DMSO group,the ROS fluorescence intensity of PC12 cells in Al(mal)3+Erastin group(109.82±8.15)was distinctly increased(P<0.05),the ROS fluorescence intensity of PC12 cells in Al(mal)3+Fer-1 group(60.19±2.81)and Al(mal)3+DFO group(53.94±13.20)decreased obviously(P<0.05).3.Mechanisms of ferroptosis triggered by aluminum in PC12 cells The study of the mechanism of oxidative damage showed that compared with the control group,the protein and m RNA expression of SLC7A11 in PC12 cells of Al(mal)3 group decreased,there was no statistical significance(P>0.05).Compared with DMSO group,the protein expression of SLC7A11 in PC12 cells of Erastin group was significantly reduced(P<0.05),while the m RNA expression of SLC7A11 was significantly increased(P<0.05);the protein and m RNA expression of SLC7A11 in PC12 cells of Fer-1 group and DFO group were not statistically different;the protein expression of SLC7A11 in PC12 cells of Al(mal)3+DMSO group was decreased dramatically(P<0.05),and the m RNA expression of SLC7A11 also decreased,but there was no statistical difference(P>0.05).Compared with Al(mal)3+DMSO group,the m RNA expression of SLC7A11 of PC12 cells in Al(mal)3+Erastin group was obviously elevated(P<0.05);the protein expression of SLC7A11 in Al(mal)3+Fer-1 group was obviously increased(P<0.05);the protein and m RNA expression of SLC7A11 in Al(mal)3+DFO group were distinctly increased(P<0.05).Compared with the control group,the protein expression of GPX4 of PC12 cells in Al(mal)3 group was significantly reduced,but the difference was not statistically significant(P>0.05),the m RNA expression of GPX4 was obviously increased(P<0.05).Compared with DMSO group,the m RNA expression of GPX4 in PC12 cells of Erastin group was significantly decreased(P<0.05),and the protein and m RNA expression of GPX4 of PC12 cells in Fer-1 group and the DFO group had no significant change,the protein expression of GPX4 in Al(mal)3+DMSO group decreased,but the difference was not statistically significant(P>0.05),while the m RNA expression of GPX4 increased obviously(P<0.05).Compared with Al(mal)3+DMSO group,the protein expression of GPX4 in PC12 cells in Al(mal)3+Erastin group reduced,but the difference was not statistically significant(P>0.05);the protein expression of GPX4 in Al(mal)3+Fer-1 group and Al(mal)3+DFO group increased,but the difference was not statistically significant(P>0.05);the expression of GPX4 m RNA was clearly decreased(P<0.05).Studies on the mechanism of iron metabolism showed that compared with the control group,the protein expression of TFR1 of PC12 cells in Al(mal)3 group was improved,but there was no statistical difference(P>0.05),and the m RNA expression of TFR1 was significantly increased(P<0.05).Compared with DMSO group,the m RNA expression of TFR1 in PC12 cells of Erastin group was clearly increased(P<0.05),and the protein and m RNA expression of TFR1 in PC12 cells of Fer-1 group and DFO group were not significantly changed,the protein and m RNA expression of TFR1 in PC12 cells in Al(mal)3+DMSO group were obviously increased(P<0.05).Compared with Al(mal)3+DMSO group,the protein and m RNA expression of TFR1 in PC12 cells of Al(mal)3+Erastin group was significantly improved(P<0.05);the protein and m RNA expressions of TFR1 in Al(mal)3+Fer-1 group and Al(mal)3+DFO group were not obviously changed(P>0.05).Compared with the control group,the protein expression of DMT1 in PC12 cells in Al(mal)3 group was clearly increased(P<0.05),and the m RNA expression of DMT1 was also increased,but there was no statistical difference(P> 0.05).Compared with DMSO group,the protein and m RNA expression of DMT1 in PC12 cells of Erastin group was significantly increased(P<0.05),the protein and m RNA expression of DMT1 was not obviously changed in Fer-1 group,and the protein expression of DMT1 was significantly increased in DFO group(P<0.05),the protein expression of DMT1 in Al(mal)3+DMSO group was obviously increased(P<0.05),and m RNA expression of DMT1 was also increased,but there was no statistical difference(P>0.05).Compared with Al(mal)3+DMSO group,the m RNA expression of DMT1 in PC12 cells of Al(mal)3+Erastin group improved,but the difference was not statistically significant(P>0.05),the protein and m RNA expression of DMT1 in Al(mal)3+Fer-1 group was obviously reduced(P<0.05),and the m RNA expression of DMT1 in Al(mal)3+DFO group increased,but there was no statistical difference(P>0.05).Compared with the control group,the protein expression of FTH in PC12 cells in Al(mal)3 group was increased,but the difference was not statistically significant(P>0.05),the m RNA expression of FTH was obviously increased(P<0.05).Compared with DMSO group,the protein and m RNA expression of FTH of PC12 cells in Erastin group was distinctly improved(P<0.05),and the protein and m RNA expression of FTH of PC12 cells in Fer-1 group and DFO group were not significantly changed,the protein expression of FTH in PC12 cells in Al(mal)3+DMSO group was increased,but there was no statistical difference(P>0.05),the m RNA expression of FTH was obviously increased(P<0.05).Compared with Al(mal)3+DMSO group,the m RNA expression of FTH in PC12 cells in Al(mal)3+Erastin group was clearly increased(P<0.05);the protein expression of FTH in Al(mal)3+Fer-1 group was significantly reduced(P<0.05);the protein and m RNA expression of FTH in Al(mal)3+DFO group were obviously decreased(P<0.05).Conclusion: 1.Ferroptosis can be induced by aluminum exposure in neurons;2.Oxidative damage and iron metabolism imbalance may be the underlying mechanism of neuronal ferroptosis induced by aluminum;3.Ferroptosis-specific inhibitor ferrostatin-1(Fer-1)and iron chelator deferoxamine(DFO)can inhibit aluminum-induced ferroptosis in neurons. |
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