Study On Ferroptosis Of SH-SY5Y Cells Induced By Atmospheric PM2.5 And Different Components

Objective:To study the regularity and mechanism of ferroptosis in human neuroblastoma cells?SH-SY5Y?induced by atmospheric fine particulate matter(PM_2.5)and different components,and provide a basis for studying the neurotoxicity mechanism of PM_2.5.Methods:SH-SY5Y cell lines were selected and divided into control group?deionized water?,positive control group?10?mol/L Erastin?,PM_2.5 poisoning group(40,80,160?g/mL PM_2.5),inhibitor group?20?mol/L apoptosis inhibitor Z-VAD-FMK,10?mol/L specific ferroptosis inhibitor Fer-1,100?mol/L specific ferroptosis inhibitor DFO.20?mol/L necrotizing apoptotic inhibitor Nec-1?,PM_2.5+inhibitor group(four inhibitors were added on the basis of the positive control group and each PM_2.5 infection group,and the dose was the same as that of the inhibitor group),and the dose relationship of ferroptosis induced by PM_2.5 in SH-SY5Y cells was observed for 24 hours..SH-SY5Y cells were selected into the control group?deionized water?,the exposure group at different times(40?g/mL PM_2.5 exposure for 24,48,and 72 hours,respectively)and the PM_2.5+inhibitor group at different times(Four inhibitors were added to each PM_2.5 exposure group at different times,with the same dosage as above).Four inhibitors were added on the basis of each exposure group,and the dose was the same as above),and the time-dependent relationship of ferroptosis induced by PM_2.5 in SH-SY5Y cells was observed.SH-SY5Y cells were selected and divided into a control group?deionized water?,a solvent control group?1.2%DMSO?,a PM_2.5 exposure group with different components(160?g/mL PM_2.5,86.4?g/mL preparation of the water-soluble extracts,24?g/mL organic extracts,49.6?g/mL carbon core component),inhibitor group?10?mol/L specific ferroptosis inhibitor Fer-1,100?mol/L specific ferroptosis inhibitor deferoxamine DFO?,PM_2.5 Different components+inhibitor groups?two ferroptosis inhibitors,Fer-1 and DFO,were added on the basis of different component exposures?.After 24 hours of exposure,observe that different components PM_2.5 induce ferroptosis in SH-SY5Y cells.CCK-8 method was used to detect the survival rate of SH-SY5Y cells.The kit was used to detect the intracellular MDA content,SOD activity,GSH content,GSH-PX activity,and Fe²⁺content.Flow cytometry was used to detect lipid ROS levels.Western blot was used to detect HO-1,Nrf2,GPX4,SLC7A11,FPN1 protein content.Results:With the increase of PM_2.5 exposure dose,the survival rate of SH-SY5Y cells,SOD activity,GSH content,and GSH-PX activity all showed a downward trend?P trend<0.001?,and the MDA content,Fe²⁺content,and lipid ROS levels showed a downward trend.Upward trend(P _trend<0.001);cell survival rates in the 40,80,and 160?g/mL PM_2.5-treated and Erastin-treated groups were significantly lower than those in the control group?P<0.01?;apoptosis inhibitor Z-VAD-FMK It can significantly increase the cell survival rate and SOD content of the PM_2.5-exposed group and Erastin-treated group at 40,80,and 160?g/mL?P<0.01?,and significantly decrease the MDA content?P<0.01?.The levels of lipid ROS in mL PM_2.5 exposure group and Erastin treatment group decreased significantly?P<0.01?;there was no significant change in GSH content and GSH-PX activity in each group?P>0.05?;Fer-1 and DFO,ferroptosis inhibitors It can significantly increase the cell survival rate,SOD,GSH content,and GSH-PX activity of 80,160?g/mL PM_2.5-treated group and Erastin-treated group?P<0.05?,and the levels of MDA,Fe²⁺,and lipid ROS decreased significantly.?P<0.01?;Nec-1,an inhibitor of necrotic apoptosis,had no significant change in various indicators in each exposure group?P>0.05?;Western blot results showed that with the increase of PM_2.5 exposure dose,HO-1,Nrf2 The protein content of GPX4 and SLC7A11 showed a downward trend?P trend<0.001?;compared with the control group,the expression levels of HO-1,Nrf2,GPX4,and SLC7A11 protein in the 80?g/mL and 160?g/mL PM_2.5-treated groups were significantly reduced.?P<0.01?;there was no significant change in the expression of FPN1 protein in each dose group?P>0.05?.With the extension of PM_2.5 exposure time,the survival rate of SH-SY5Y cells,SOD activity,GSH content,and GSH-PX activity all showed a downward trend?P trend<0.001?,MDA content,Fe²⁺content,and lipid ROS levels.It showed an upward trend?P trend<0.001?;Z-VAD-FMK intervention only increased the cell survival rate and SOD content of the PM_2.5-exposed group at 24h,48h,and 72h?P<0.01?,and the MDA content decreased significantly?P<0.01?,lipid ROS levels in the PM_2.5-exposed group decreased at 72h?P<0.01?;Fer-1,DFO intervention,48h,72h PM_2.5-exposed group,cell survival rate,SOD activity,GSH content,GSH-PX activity increased significantly?P<0.05?,MDA,Fe²⁺content,and lipid ROS levels decreased significantly?P<0.01?;Nec-1,a necrotic apoptosis inhibitor,interfered with each exposure group.There were no significant changes in the indicators?P>0.05?;Western blot results showed that with the extension of PM_2.5 exposure time,HO-1,Nrf2,GPX4,and SLC7A11 protein contents showed a downward trend(P _trend<0.001);compared with the control Compared with the control group,the expression levels of HO-1,Nrf2,GPX4,and SLC7A11 proteins in the PM_2.5-exposed group at 48h and 72h were significantly decreased;there was no significant change in the expression of FPN1 protein in the exposed group at different times?P>0.05?.PM_2.5 fat-soluble component?Po?,water-soluble component?Pw?and carbon particle component?Pc?exposure group,the cell survival rate was significantly lower than the corresponding control group?P>0.05?;ferroptosis inhibitor Fer-1,DFO intervention significantly increased the cell survival rate,SOD activity,GSH content,and GSH-PX activity of PM_2.5 and Po-exposed groups?P<0.05?,and the levels of MDA,Fe²⁺,and lipid ROS decreased significantly?P<0.01?.),There were no significant changes in the Pw and Pc-exposed groups?P>0.05?;Western blot results showed that PM_2.5 and Po-exposed groups could make HO-1,Nrf2,SLC7A11,and GPX4 protein expression levels significantly lower than In the corresponding control group?P<0.01?,there was no significant change in the protein content of FPN1?P>0.05?;neither of the Pw and Pc exposure groups caused the above protein content changes?P>0.05?.PM_2.5 organic extracts?Po?,preparation of the water-soluble extracts?Pw?and carbon core component?Pc?exposure group,the cell survival rate was significantly lower than the corresponding control group?P>0.05?;ferroptosis inhibitor Fer-1,The cell survival rate,SOD activity,GSH content,and GSH-PX activity of PM_2.5 and Po exposure groups were significantly increased by DFO intervention?P<0.05?,and there were no significant changes in intervention groups of Pw and Pc exposure?P>0.05?;The PM_2.5and Po exposure groups can significantly reduce the HO-1,Nrf2,SLC7A11,and GPX4protein levels?P<0.01?,and the FPN1 protein content has no significant change?P>0.05?;both of the Pw and Pc exposure groups Does not cause the above protein content changes?P>0.05?.ConcLusion:PM_2.5 exposure can induce the apoptosis of SH-SY5Y cells,and with the increase of dose and time,it can induce ferroptosis,which may be related to its fat-soluble components.Fer-1 and DFO can inhibit ferroptosis in SH-SY5Y cells induced by PM_2.5.