Study On The Mechanism Of Mitochondrial Oxidative Damage And Dysfunction Of Nerve Cells By Silica Nanoparticles

Objective:An in vitro experimental model of SH-SY5Y cells exposed to Silica nanoparticles was established to study the toxic effect of silica nanoparticles on nerve cells,and it was clear that Silica nanoparticles exposure could cause oxidative damage and mitochondrial dysfunction in cells and mitochondria.To reveal the role and mechanism of VDAC1 channel protein in the oxidative damage and dysfunction of nerve cells induced by Silica nanoparticles,and provide experimental and theoretical basis for further elucidating the mechanism of Silica nanoparticles neurotoxicity.Method:Human neuroblastoma cells（SH-SY5Y）were used as the cell model,and Silica nanoparticles solutions with different concentrations of 0,3.125,6.25,12.5,25.0 and50.0μg/m L were used as the test substance,and they were exposed for 24h.The particle size of nanoparticles was characterized by transmission electron microscopy;particle dispersion and stability were detected by Zeta potential analyzer;cell viability was detected by MTT method;cell growth and intracellular reactive oxygen species（ROS）levels were observed by fluorescence microscopy;flow cytometry was used to detect Mitochondrial membrane potential and apoptosis;biochemical method to detect the activities of superoxide dismutase（SOD）,glutathione peroxidase（GSH-Px）and mitochondrial respiratory chain enzyme I in cells and mitochondria;Western Blot method The expression of oxidative damage and dysfunction-related proteins in cells and mitochondria was detected.Result:1.The effect of Silica nanoparticles on the viability and morphology of SH-SY5Y cells.The results of MTT showed that SH-SY5Y cells were exposed to Silica nanoparticles for 24 h.With the increase of exposure dose,the cell viability decreased,and the difference between each exposure dose group and the control group was statistically significant（P<0.05）.The growth status and morphological results showed that the cells in the control group grew well,the cell density was evenly distributed,and the cells were long-spindle.In the exposure group,with the increase of exposure dose,the cells shrank and became round,the cell antenna disappeared,and the number of cells decreased significantly.2.The effect of Silica nanoparticles on intracellular oxidative damage in SH-SY5Y cells.The intracellular ROS was observed at the live cell workstation.The results showed that Silica nanoparticles exposed SH-SY5Y cells for 3 h.The content of ROS increased gradually,and the results indicated that the generation of ROS was in the early stage of the action of Silica nanoparticles;biochemical methods were used to detect SOD and GSH-Px in cells 24 h after exposure,and the results showed that with the increase of exposure dose,the activity of SOD decreased,compared with the control group at each dose of exposure,the difference was significant（P<0.05）,and the intracellular GSH-Px activity increased first with the increase of exposure dose,and the highest activity was in the 25.0μg/m L dose group.The enzyme activity was inhibited when reaching 50.0μg/m L.Except for the 3.125μg/m L dose group,the difference between each exposure group and the control group was statistically significant（P<0.05）.With the increase of the exposure dose,the expression of SOD protein decreased,and the 25.0 and 50.0μg/m L dose groups were significantly lower than the control group（P<0.05）.The protein expression of glutathione peroxidase 1（GPx1）was up-regulated,and the protein expression in the 12.5,25.0 and 50.0μg/m L dose groups was significantly higher than that in the control group（P<0.05）.The protein expression of peroxireductase 3（PRDX3）increased with the exposure dose,and the protein expression of each dose group was significantly different from that of the control group（P<0.05）.3.The effect of Silica nanoparticles on mitochondrial oxidative damage and dysfunction in SH-SY5Y cells.Flow cytometry detection of Silica nanoparticles exposure to 3 h,the mitochondrial membrane potential decreased,12.5,25.0 and 50.0μg/m L dose groups compared with the control group significant（P<0.05）,indicating severe damage to mitochondrial membrane potential;Biochemical methods were used to detect mitochondrial superoxide dismutase（Mn-SOD）activity.The activity was significantly higher than that of the control group（P<0.05）.Compared with the control group,the GSH-Px activity in mitochondria was significantly increased（P<0.05）.The activity of mitochondrial respiratory chain enzyme I in each exposure group was significantly higher than that in the control group（P<0.05）;The results of mitochondrial protein detection by Western Blot showed that,except for the 3.125μg/m L dose group,SOD protein in each dose group was significantly higher at 24 h after exposure.The expression was significantly higher than that in the control group（P<0.05）.The expression level of PRDX3 protein in each dose group was significantly higher than that in the control group（P<0.05）.The expression of intracellular COX I protein was significantly increased in each dose group compared with the control group（P<0.05）.The expression of COX I protein in mitochondria was decreased compared with the control group in the 6.25μg/m L dose group（P<0.05）,and was significantly increased in the 25.0 and 50.0μg/m L dose groups compared with the control group（P<0.05）.The expression of intracellular and mitochondrial uncoupling protein 2（UCP2）protein was up-regulated with the increase of exposure dose,and the difference between each dose group and control group was significant（P<0.05）.Intracellular voltage-dependent anion channel 1（VDAC1）protein expression was significantly higher in each dose group than in the control group except for the 3.125μg/m L group（P<0.05）.The mitochondrial VDAC1 protein expression was significantly higher in the6.25,25.0 and 50.0μg/m L dose groups than in the control group,and the difference was statistically significant（P<0.05）.4.The effect of Silica nanoparticles on the apoptosis of SH-SY5Y cells.The results of flow cytometry showed that the apoptosis rate of cells increased with the exposure dose for 24 h after exposure to the cells.Compared with the control group,the difference between each exposure group was statistically significant（P<0.05）,when the Silica nanoparticles concentration reached 50.0μg/m L,the apoptosis rate of cells increased significantly.The early apoptosis rate increased with the increase of the exposure dose,and the difference was statistically significant between the 12.5,25.0and 50.0μg/m L exposure groups and the control group（P<0.05）.5.The role of VDAC1 in the mitochondrial dysfunction of SH-SY5Y cells by Silica nanoparticles.MTT results showed that cells were exposed to 24 h,1.0 m M for24 h,1.0 m M 4,4’-diisothiocyanatostilbene-2,2’-disulfonate（DIDS）compared with the control group,there was no difference in the survival rate of cells.Compared with the control group,the survival rate of the 50.0μg/m L Silica nanoparticles group was significantly decreased（P<0.05）.However,the cell viability of the 50.0μg/m L Silica nanoparticles exposure group pretreated with DIDS for 1 h was significantly higher than that of the non-DIDS pretreated group,and the difference was statistically significant（P<0.05）.Compared with the Silica nanoparticles exposure group,the generation of intracellular ROS in the DIDS+Si O₂ group exposed to 3 h exposure was significantly reduced;The results of flow cytometry showed that the mitochondrial membrane potential changes in the DIDS+Si O₂ exposure group for 3 h were compared with that in the Si O₂ exposure group.The difference was statistically significant（P<0.05）,indicating that mitochondrial membrane potential damage had recovered;Western Blot results showed that the expression level of SOD protein in DIDS+Si O₂group was increased compared with Si O₂ exposure group at 24 h（P<0.05）,the protein expression levels of PRDX3,VDAC1,and COX I decreased（P<0.05）,and the protein expression level of UCP2 decreased,but the differences were no statistically significant.The results suggest that inhibiting the channel protein VDAC1 can restore some mitochondrial functions and has a certain protective effect on cells.Conclusion:1.Silica nanoparticles can reduce SH-SY5Y cell viability and has cytotoxicity;2.Silica nanoparticles can cause oxidative damage to SH-SY5Y cells;3.Silica nanoparticles can cause oxidative damage and dysfunction of SH-SY5Y cell mitochondria;4.By inhibiting the channel protein VDAC1,the antioxidant response of cells and mitochondria after the action of silica nanoparticles can be improved,and the function of mitochondria can be restored to a certain extent.