Study On The Role And Mechanism Of AIF/PARP In Parthanatos Induced By Silica Nanoparticles In SH-SY5Y Cells

Objective:By establishing an experimental model in vitro of SiNPs exposed to SH-SY5Y cells,it was confirmed that SiNPs had toxic effects on cells,and it was clear that SiNPs exposure could induce oxidative stress and Parthanatos in SH-SY5Y cells,revealing the role and mechanism of AIF/PARP in SiNPs-induced oxidative stress response and Parthanatos,which provided new ideas for further elucidating the mechanism of SiNPs neurotoxicity.Methods:Human neuroblastoma cells SH-SY5Y were used as the cell model and SiNPs as the test substance,the exposure doses were 0μg/m L,3.125μg/m L,6.25μg/m L,12.5μg/m L,25.0μg/m L and 50.0μg/m L,and the action time was 24 h.The size of nanoparticles was characterized by transmission electron microscopy;Zeta potential analyzer detected particle dispersion and stability;Cell growth was observed by inverted fluorescence microscope;Cell viability was measured by MTT method;Intracellular ROS was observed by DCFH-DA labeling fluorescence microscope;The content of ROS and Ca²⁺in cells was detected by flow cytometry;Hoechst 33342staining and TUNEL method were used to observe apoptosis;The activities of SOD and ATPase and the content of ATP in cells were detected by biochemical method;The expression of Parthanatos-related and caspase-dependent apoptosis-related proteins were detected by Western blot;Immunofluorescence technique was used to observe the distribution of Parthanatos-related proteins induced by SiNPs.Results:1.Effects of SiNPs on growth status and viability of SH-SY5Y cells.The cell growth state was observed by light microscopy,and the cell growth status of the control group was good,the cell density was uniform,and the cells were long spindle type.With the increase of the dose of SiNPs exposure,the cells contracted and rounded,the cell antennae disappeared,and the number of cells decreased significantly.The MTT results showed that the cell survival rate decreased with the increase of the dose of SiNPs for 24 h.The difference between the 25.0μg/m L and50.0μg/m L dose groups was statistically significant compared with the control group（P<0.05）.2.Effects of SiNPs on Intracellular ROS and Ca²⁺Content of SH-SY5Y.Fluorescence microscopy and flow cytometry showed that the content of intracellular ROS.With the increase concentration of SiNPs,intracellular green fluorescence gradually increased,indicating that intracellular ROS production gradually increased,especially in the high-dose group（P<0.05）。The results of intracellular Ca²⁺showed that with the increase dose of SiNPs,the concentration of Ca²⁺in the cell gradually increases,especially in the 50.0μg/m L dose group（P<0.05）.3.Effect of SiNPs on oxidative damage of SH-SY5Y cells.With the increase dose of SiNPs,the activity of SOD enzyme decreased,and the difference between the dose group and the control group was statistically significant（P<0.05）,while the activity of ATPase and ATP content also showed an overall downward trend,and the highest dose group（50.0μg/m L）was significantly different from the control group（P<0.05）.4.Effect of SiNPs on apoptosis of SH-SY5Y cells.With the gradual increase dose of SiNPs,Hoechst33342 staining bright blue fluorescence and green fluorescence of TUNEL stain gradually increased,the degree of cell nucleus damage and DNA fragment damage increased,the apoptosis rate gradually increased.5.SiNPs induce the expression of Caspase-dependent apoptosis-related proteins in SH-SY5Y cells.Compared with the control group,the expression of intracellular proteins such as Cyt C,Caspase-3 and Caspase-9 increased significantly in the high-dose group（P<0.05）.Further,after the addition of the inhibitor Z-VAD,intracellular Cyt C expression was reduced compared with the infected group.6.SiNPs induced Parthanatos-related protein expression in SH-SY5Y cells.Compared with the control group,the expression of proteins such as PARP,AIF in the nucleus and in the cytoplasm increased significantly in the high-dose group（P<0.05）.The expression of AIF in mitochondria decreased significantly with the exposure dose increased（P<0.05）。Moreover,after the addition of PARP inhibitor Olaparib,the expression of PARP,AIF proteins decreased compared with that of the SiNPs group.The same results were observed by immunofluorescence test.7.SiNPs induce SH-SY5Y cell death by Caspase-dependent and Parthanatos.Western Blot showed that after adding Z-VAD,the expression of Cyt C decreased,and the expression of PARP and AIF proteins also decreased compare with that of SiNPs group（P<0.05）.Olaparib can inhibit the expression of PARP proteins,and also reduce the expression of Cyt C and Caspase-3,suggesting that Parthanatos and Caspase-dependent apoptosis pathways are not independent of each other,but related.Conclusions:1.SiNPs can reduce the survival rate of SH-SY5Y cells and change cell morphology.2.SiNPs cause oxidative damage and energy imbalance in SH-SY5Y cells.3.SiNPs induce Caspase-dependent apoptosis in SH-SY5Y cells.4.SiNPs can induce the occurrence of Parthanatos in SH-SY5Y cells.5.Olaparib can reduce the occurrence of Parthanatos by inhibiting PARP and improve cell viability.6.Parthanatos and Caspase dependent apoptosis pathways can coexist and interact with each other.