Mechanism Of Arsenic Regulation Of Mitochondrial Damage And Autophagy Induced Synaptic Damage Through SIRT1 And Protective Effect Of Melatonin

Objective:In this study,we investigate the role of SIRT1-mediated mitochondrial damage and autophagy in the alteration of synaptic damage induced by sodium arsenite（Na As O₂）,which provides a scientific basis for the study of the neurotoxic mechanism of arsenic（As）.After the intervention of SIRT1 activator,the changes of mitochondrial damage,autophagy and related synaptic damage in cells were observed,and the regulation of SIRT1 on the expression of synaptic functional proteins was discussed.Meanwhile,the use of Melatonin（Mel）as a neuroprotector is demonstrated to regulate Na As O₂ induced synaptic damage through the SIRT1 signaling pathway.Methods:HT22 cells were stained with different concentrations of sodium arsenite（Na As O₂）or SRT1720 or Mel for 24 h.The cell survival rate was detected by CCK-8method.According to the cell survival rate,HT22 cells were divided into control group,low-dose As（2 μM）group,medium-dose As（4 μM）group,high-dose As（8μM）group,DMSO control group,SRT1720 group,high-dose As（8 μM）+ Sirt1activator（SRT1720）group,Mel group and high-dose As（8 μM）+ melatonin group,and observed under inverted microscope.The morphological changes of HT22 cells were observed under inverted microscope,and cell viability was detected by CCK-8method;SIRT1,C-FOS and LC3 were detected by immunofluorescence method;the levels of SIRT1,mitochondrial proteins（PINK1 and Parkin）,autophagy proteins（LC3B-I,LC3B-II,P62）,synaptic proteins（C-FOS,α-SYN）,and SIRT1 were detected by immunoblotting（WB）method in HT22 cells,α-SYN)protein levels,transmission electron microscopy observation of mitochondria and autophagic vesicles,etc.Results:1.Cell survival rate: The survival rate of HT22 cells gradually decreased with the increase of Na As O₂ exposure dose.After 24 h of Na As O₂ staining,the overall cell survival rate showed a decreasing trend,and with the increase of Na As O₂ staining dose,the cell survival rate was lower,and 16 μM decreased to 52%,and the difference was statistically significant（P<0.05）.2.Cellular and synaptic morphological changes: The neurite length and the number of neurites per cell were significantly reduced in HT22 cells,as well as the cell body size was reduced after Na As O₂ treatment.After treated with sodium arsenite for 24 hours,the growth and development of neuronal processes were significantly inhibited,which showed that the Neuron longest protrusion length was significantly decreased,the number of primary processes（PDN）was significantly reduced,and neuronal process growth index（NOI）were significantly reduced.SRT1720 and melatonin could reduce the injurious changes of hippocampal neurons induced by Na As O₂ after intervention.3.Pathological structural changes of cells（electron microscopic structure）: In the control group,the morphology was normal and the mitochondrial structure was clear;in the arsenic group,the mitochondrial structure was extremely disturbed and the membrane-like structure of autophagosomes increased,and mitochondrial autophagy and residual melanin after autophagy were visible;in the melatonin combined with staining group and SRT1720 combined group,the membrane-like structure of autophagosomes was less and the mitochondrial structure was disturbed to some extent.4.Change in protein content of SIRT1 pathway: SIRT1 protein expression levels tended to decrease in the exposed group and were statistically different.The intracellular SIRT1 content of HT22 cells in 2μM,4μM As and 8μM As groups gradually decreased with increasing exposure concentration.After combined treatment with Na As O₂ and SRT1720,SIRT1 protein expression levels were increased in the combined exposure group compared with the Na As O₂ exposure group.Pretreatment with melatonin increased SIRT1 protein expression.After Na As O₂exposure,the SIRT1 fluorescence intensity in the exposed group was attenuated compared with the control group,especially in the 8 μ M Na As O₂ group.The combined treatment with activator SRT1720 and melatonin increased SIRT1 fluorescence intensity compared with the 8μM Na As O₂-exposed group.5.Changes in intracellular mitochondrial function: PINK1 and Parkin contents in HT22 cells were lower in the 2 μM As,4 μM As,and 8 μM As groups than in the control group（P < 0.05）.PINK1 and Parkin contents were higher in the 8 μM As +SRT1720 and 8 M As + melatonin groups than in the control group.6.Change of autophagy index: p62 and LC3 B contents in HT22 cells were higher in the 2 μM As,4 μM As,and 8 μM As groups than in the control group（P< 0.05）.The contents of p62 and LC3 B in the 8 μM As + SRT1720 and 8 μM As +melatonin groups were lower than those in the control group（P < 0.05）.Melatonin pretreatment alleviated Na As O₂-induced autophagy（P < 0.05）.After 24 h of exposure,the LC3 fluorescence intensity in the exposed group increased compared with the control group,especially in the 8 M Na As O₂ group.Compared with 8M Na As O₂ group,the fluorescence intensity of LC3 decreased in SRT1720 combined with Na As O₂ group.Combined treatment with melatonin similarly restored LC3 fluorescence intensity.7.Change in relative expression of synaptic related proteins: The expression levels of C-FOS and α-SYN decreased with increasing Na As O₂ dose.The fluorescence intensity of C-FOS decreased after Na As O₂ poisoning.SRT1720 and melatonin reduced the expression of synaptic functional proteins induced by Na As O₂,and the fluorescence intensity of C-FOS was significantly restored.Conclusion:1.SIRT1 pathway mediates mitochondrial autophagy changes in arsenic-induced synaptic damage.2.The neuroprotective effect of melatonin may be mediated by activation of the SIRT1 pathway.