| Objective:To explore the effect and mechanism of ferroptosis on neuronal death induced by aluminum-maltolate[Al?mal?3]in vivo and in vitro,and to provide theoretical reference for further study of the neurotoxic mechanism of aluminum.Methods:In vivo experiment:18 healthy male Specific Pathogen Free?SPF?Sprague Dawley?SD?rats were randomly divided into three groups according to their body weights:the saline group?control group?,the 9?M/kg Al?mal?3 exposed group,and 18?M/kg Al?mal?3 exposed group,6 rats in each group.Rats in 9?M/kg Al?mal?3 and 18?M/kg Al?mal?3 exposed groups received intraperitoneal injections of Al?mal?3 at doses of 9 and18?M respectively and rats in the control group received intraperitoneal injections of 0.9%saline,1mL/kg body weight once a day for 90 days.At the end of 90th administration,the learning and memory ability and the general activity of the rats were tested with the Morris water maze?MWM?test,the step-down test?SDT?,and the open field test?OFT?.The changes of mitochondria ultrastructure in hippocampal neurons of rats were observed by transmission electron microscopy?TEM?and morphometric quantitative analysis of mitochondrial membrane density and mitochondrial cristae.The content of total iron ions in cerebral cortex of rats was determined by spectrophotometer.The content of glutathione?GSH?and the activity of glutathione peroxidase?GSH-Px?in cerebral cortex of rats were determined by 5,5'-dithiobis-?2-nitrobenzoic acid??DTNB?method,and the content of malonaldehyde?MDA?in cerebral cortex of rats was determined by thiobarbituric acid?TBA?method.In vitro experiment:The differentiated rat pheochromocytoma?PC12?cells in logarithmic growth phase were divided into five groups:0?M Al?mal?3 group?control group?,50?M Al?mal?3 group,100?M Al?mal?3 group,200?M Al?mal?3 group and 400?M Al?mal?3 group.The PC12 cells were cultured in Dulbecco's Modified Eagle Medium?DMEM?high-sugar medium with final concentrations of 0,50,100,200 and 400?M Al?mal?3 for 12 h,24 h,and 48 h respectively.The morphology of PC12 cells was observed by inverted microscope,and the survival rate of PC12 cells was detected by Cell Counting Kit 8?CCK-8?method.The changes of mitochondria ultrastructure in PC12 cells were observed by TEM and morphometric quantitative analysis of mitochondrial membrane density and mitochondrial cristae.The content of total iron ions in PC12 cells was determined by Ferrous Benzoxazines Colorimetry.The content of GSH and the activity of GSH-Px in PC12 cells were determined by DTNB method.The content of MDA in PC12 cells was determined by TBA method.Results:In vivo experiment:In the MWM test,the escape latencies in the place navigation test of the rats in each group were significantly shortened with the extension of training time?P<0.05?.In the spatial exploration experiment,compared with the control group,the time of finding platform site of Al?mal?3 groups was significantly gradually prolonged with the increase of Al?mal?3 dosage?P<0.05?.The average number of crossings of the platform site were relatively reduced when compared to those of the control group,but there was no statistically significant difference?P>0.05?.In the SDT test,with the increase of Al?mal?3 dosage,the latency time of each Al?mal?3 group was gradually shortened,and the error number l and error number 2 of the 18?M/kg Al?mal?3 group increased significantly compared to those of the control group,the difference was statistically significant?P<0.05?.In the OFT test,with the increase of Al?mal?3 dosage,the time in central grid of the 18?M/kg Al?mal?3 group was significantly prolonged,and the number of standing was significantly increased when compared with the control group,while the number of grooming in the exposed groups was relatively lower than those in the control group,but the difference was not statistically significant?P>0.05?.TEM observation showed that the mitochondria of hippocampal neurons in the control group were full,the membrane structure was intact,the membrane density was normal,and the mitochondrial cristae were clearly visible,and the mitochondria of hippocampal neurons in the exposed groups showed specific characteristic changes of ferroptosis:shrinkage,membrane rupture,increase of membrane density and disappearance of cristae.Compared with the control group,the mitochondrial mean membrane density increased significantly,and the average area of mitochondrial cristae of the 18?M/kg Al?mal?3 group decreased significantly?P<0.05?.The content of total iron ions in cerebral cortex of rats in the 18?M/kg Al?mal?3 group increased significantly with the increase of Al?mal?3 dosage?P<0.05?.With the increase of Al?mal?3 dosage,the content of GSH decreased gradually,the activity of GSH-Px decreased gradually,and the content of MDA increased gradually in cerebral cortex of rats,which were statistically significant compared with the control group?P<0.05?.In vitro experiment:Compared with 0?M Al?mal?3 group,PC12 cells exposed to 400?M Al?mal?3 for 12 h,200,400?M Al?mal?3 for 24 h,and 100,200,400?M Al?mal?3 for48 h all showed significant decrease in number,and the cells became round,the axonal mutation was short,the cell junction decreased and disappeared,etc.With the increase of Al?mal?3 dosage and exposure time,the overall survival rate of PC12 cells showed a decreasing trend.Compared with 0?M Al?mal?3 group at the same time,The survival rate of PC12 cells in 400?M Al?mal?3 for 12 h,200,400?M Al?mal?3 for 24 h,and 100,200,400?M Al?mal?3 for 48 h were all significantly decreased?P<0.05?.In this study,four dosages of 0,50,100,and 200?M Al?mal?3 were selected for subsequent experiments according to the results of 50%inhibiting concentration?IC50?and preliminary test results of the research group.The results of TEM showed that the mitochondria of PC12 cells in 0?M Al?mal?3 group were plump,the membrane structure was intact,the membrane density was normal,the mitochondrial ridge was clearly visible,and with the increase of Al?mal?3exposure time and dosage,the specific changes of ferroptosis in the mitochondria of PC12cells in each treated group such as shrinkage,rupture of membrane,increase of membrane density and disappearance of ridge were more obvious.Compared with those of 0?M Al?mal?3 group at the same time point,the mitochondrial mean membrane density increased significantly,and the average area of mitochondrial crista of PC12 cells in 100,200?M Al?mal?3 group at 24,and 48 h decreased significantly?P<0.05?.Compared with 0?M Al?mal?3 group at the same exposure time,the content of total iron ions of PC12 cells in each Al?mal?3 treated group for 12 h,200?M Al?mal?3 for 24 h,and 100,200?M Al?mal?3 for 48 h all increased significantly?P<0.05?.The contents of GSH and the activity of GSH-Px of PC12 cells of Al?mal?3 treated group at each exposure time all increased with the increase of Al?mal?3 dosage,which had statistical significance compared with 0?M Al?mal?3 group at the same time?P<0.05?.The MDA content of PC12 cells in each Al?mal?3 treated group for 12 h,100,200?M Al?mal?3 for 24 h,and 100,200?M Al?mal?3 for 48 h all increased significantly compared with 0?M Al?mal?3 group at the same exposure time?P<0.05?.Conclusion:1.Exposure to Al?mal?3 can lead to ferroptosis in rat neurons and PC12 cells.2.The increase of iron content in neurons,lipid peroxidation damage due to the decrease of antioxidant capacity in neurons may be one of the mechanisms of neuron ferroptosis induced by Al?mal?3. |
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