Study On The Effect And Mechanism Of PM_2.5 On The Expression Of Alzheimer’s-like Lesions Related Proteins In SH-SY5Y Cells

Objectives To observe the effect of fine particulate matter(PM_2.5)exposure on the expression of Alzheimer’s disease-like proteins in SH-SY5Y cells.To explore the possible mechanism of AD-like lesions in central nervous system induced by PM_2.5.To provide theoretical basis for the prevention and treatment of AD.Methods 1 Atmospheric PM_2.5 was collected to prepare the suspension,and SH-SY5Y cells were treated with PM_2.5 suspension（0,25,50,100,200,400μg/mL）for 24 h.Morphological characteristics of cells were observed under light microscope,cell viability was determined by CCK-8,the expression of total Aβ,p-Tau and Tau were detected by Western blot to determine the optimal concentration of PM_2.5.The control group and PM_2.5group（optimal concentration）were treated with blank medium and PM_2.5 suspension for 24h,respectively.SOD activity,MDA and ROS content in cells were detected using corresponding kits,and the contents of interleukin-1beta（IL-1β）,interleukin-6（IL-6）and tumor necrosis factor-α（TNF-α）were detected by ELISA.RT-q PCR was used to detect the mRNA expression of APP and Tau.2 Bioinformatics methods were used to analyze the public data set“micro RNA sequencing analysis of mouse hippocampal injury caused by PM_2.5 exposure”to compare the differences between the PM_2.5 group and the control group,and explore the possible mechanism of central nervous system injury caused by PM_2.5exposure.3 SH-SY5Y cells were treated with PI3K inhibitor LY294002（0,2.5,5,10,20,40μmol/L）for 24 h.Cell viability and PI3K phosphorylation were detected to determine the optimal concentration of LY294002.Then the groups were divided into control group,PM_2.5group,LY294002 group and LY294002+PM_2.5 group.Annexin V-FITC assay was used to detect cell apoptosis,SOD activity,MDA,ROS,IL-1β,IL-6 and TNF-αcontents were detected,mRNA expression of APP and Tau,Aβ_1-40 and Aβ_1-42 contents were detected by ELISA.Total Aβ,p-Tau,Tau,Phosphatidylinositol 3-kinase（PI3K）,Protein kinase B（PKB/Akt）,Glycogen Synthase kinase-3β（GSK-3β）and corresponding phosphorylated proteins were detected by Western blot.4 Cells were treated with ROS inhibitor n-acetyl-l-cysteine（NAC）（0,1.25,2.5,5,10,20 mmol/L）for 24 h to detect the cell viability and ROS content and determine the optimal concentration of NAC.Set groups:control group,PM_2.5group,NAC group and NAC+PM_2.5 group.Except ROS,IL-1β,IL-6 and TNF-α,all the indexes were the same as the LY294002 experiment.Results 1 With the increase of PM_2.5 exposure concentration,SH-SY5Y cells showed abnormal cell morphology,decreased number,decreased cell viability,increased expression of total Aβ,and increased Tau phosphorylation.The expression of total Aβ（r=0.817,P<0.01）and the phosphorylation of Tau protein（r=0.832,P<0.01）increased with the increase of PM_2.5 exposure concentration,set the optimal concentration of PM_2.5 was 100μg/mL.Compared with the control group,SOD activity in PM_2.5 group decreased（P<0.05）,MDA,ROS,IL-1β,IL-6 and TNF-αincreased,total APP and Tau mRNA expression increased（P<0.05）.2 Bioinformatics analysis showed that the PI3K/Akt signaling pathway was significantly enriched in the nervous system after PM_2.5 exposure.3 Compared with the control group,the expression of PI3K and Akt in PM_2.5 group was decreased（P<0.05）,the expression of GSK-3βwas increased（P<0.05）,and the phosphorylation of PI3K,Akt and GSK-3βwere decreased（P<0.05）.10μmol/L was selected as the optimal concentration of LY294002.Compared with the control group,apoptosis rate of cell,MDA,ROS,IL-1β,IL-6 and TNF-αcontents,mRNA expression of APP and Tau,Aβ_1-42/Aβ_1-40,total Aβ,Tau phosphorylation and GSK-3βexpression were increased in PM_2.5 group（P<0.05）.SOD activity,PI3K and Akt expression and phosphorylation degree,GSK-3βphosphorylation degree decreased（P<0.05）.Compared with LY294002 group,the results of LY294002+PM_2.5 group were consistent with those of the above two groups（P<0.05）.Compared with the PM_2.5 group,Apoptosis rate of cell,MDA content,IL-1β,IL-6 and TNF-αcontents,mRNA expression of APP and Tau,Aβ_1-42/Aβ_1-40,total Aβ,Tau phosphorylation and GSK-3βexpression were increased in LY294002+PM_2.5 group（P<0.05）,the expression of Akt,the expression of PI3K and phosphorylation were decreased（P<0.05）.4 The optimal concentration of NAC was 5 mmol/L.Compared with PM_2.5 group,apoptosis rate of cell,MDA content,mRNA expression of APP and Tau protein,expression of Aβ_1-42/Aβ_1-40,total Aβ,GSK-3βand phosphorylation of Tau and Akt were decreased in NAC+PM_2.5 group.SOD activity,Akt expression and GSK-3βphosphorylation were increased（P<0.05）.Conclusions 1 PM_2.5 can cause oxidative stress in SH-SY5Y cells and increase the expression of inflammatory factors,as well as the mRNA expression of APP（a precursor of AD-like pathologic protein Aβ）,protein expression of total Aβ,mRNA expression and protein phosphorylation of Tau.2 PM_2.5 reduces the protein activity of PI3K/Akt pathway by increasing the production of ROS in SH-SY5Y cells,and then activate GSK-3β,and promote the expression of AD-like disease-related proteins.ROS/PI3K/Akt/GSK-3βsignaling pathway is one of the mechanisms of PM_2.5 affecting the expression of AD-like pathologic proteins in SH-SY5Y cells.Figure 19;Table 23;Reference 125...