Curcumin Antagonizes The Effect Of Acrylamide On Learning-memory-related Pathways And Proteins In SH-SY5Y Cells

Acrylamide（Acrylamide,ACR）is a water-soluble unsaturated amide,widely used in production and life,can affect the cognitive function of humans and experimental animals,but because its toxicity mechanism is not yet clear,there is still no effective intervention.Curcumin（Curcumin,Cur）has anti-inflammatory,anti-oxidant,inhibits the formation and aggregation of Aβ,and shows good application prospects in the diagnosis and prevention of neurodegenerative diseases,especially AD.In this paper,we use in vitro experimental methods to observe whether Cur can improve the impact of ACR on learning and memory related pathways and proteins,and provide new ideas for exploring intervention methods to alleviate ACR learning and memory impairment.Objective:Using SH-SY5Y cell as the experimental material,the effects of ACR on the PERK-eIF2αand CREB-BDNF pathways,tau protein and synaptic vesicle protein before and after Cur pretreatment were observed and compared.Provide evidence of cell biology.Methods:The dosage and time of ACR and Cur were screened by CCK8 test.The intervention study was divided into four groups:control group,2.5 mmol/L ACR group,6μmol/L Cur group,Cur pretreatment group(6μmol/L Cur pretreatment for 2 h After treatment with 2.5 mmol/L ACR for 24h,the cell morphology was observed by phase contrast microscopy;DCFH-DA fluorescent probe method was used to detect the ROS index of each group of cells;Enzyme labeling method was used to detect the MDA level and GSH content of cells;immunofluorescence technique was used to observe the distribution of phosphorylated tau protein in cells;Western Blot method was used to detect the expression levels of tau protein,synaptic vesicle proteins Sy-1 and Syn,PERK-eIF2αsignaling pathway proteins GRP78,P-PERK,P-eIF2α,ATF4 and CREB signaling pathway proteins P-CREB and BDNF.Results:（1）CCK8 method was used to detect cell survival rate:after 0～10mmol/LACR treatment for 24 h,the cell survival rate of 2.5～10 mmol/L ACR group decreased to 87%,66%,57%,38%of the control group,respectively;The differences were statistically significant（all P<0.001）;After 2～30μmol/L Cur treatment for 2h,there was no significant decrease in cell survival rate in 2,4,and 6μmol/L Cur groups（P>0.05）;2～6μmol/L Cur pretreatment for 2h,and then treated with 2.5 mmol/L ACR for 24 hours,the phase contrast microscope observed that the cells in the control group and the Cur group grew well,the cell adhesion ability of the ACR group was reduced,the cell body was spherical,and the nerve process Contraction,the fusiform and polygonal cells in the pretreatment group increased;Compared with the ACR group,the cell survival rate of the 6μmol/L Cur pretreatment group increased significantly（P<0.01）.In subsequent experiments,the doses of ACR and Cur were 2.5 mmol/L and 6μmol/L,respectively.（2）Oxidative stress:Compared with the control group,the levels of MDA and ROS in the ACR treatment group increased significantly（both P<0.001）;GSH content was significantly reduced（P<0.001）;compared with the ACR treatment group,the ROS level of the 6μmol/L Cur pretreatment group was significantly reduced（P<0.05）,and the MDA content was significantly reduced（P<0.001）,The GSH content increased significantly（P<0.001）.It is suggested that Cur can reduce the level of oxidative stress caused by ACR.（3）PERK-eIF2αsignaling pathway:Compared with the control group,the protein expression levels of GRP78,P-PERK,and P-eIF2αin the ACR group were significantly increased（all P<0.001）.Compared with ACR group,the protein expression of GRP78and P-PERK in SH-SY5Y cells of Cur pretreatment group was significantly decreased（both P<0.01）,and the protein expression of P-eIF2αwas significantly reduced（P<0.05）.There was no significant change in the expression of T-PERK and T-eIF2αprotein in each group（both P>0.05）.It is suggested that Cur can inhibit the excessive activation of PERK-eIF2αsignaling pathway caused by ACR.（4）CREB signaling pathway:Compared with the control group,the expression of ATF4 protein in ACR group was significantly increased（P<0.001）,P-CREB（Ser133）and BDNF protein expression was significantly reduced（P<0.001）;Compared with the ACR group,the ATF4 protein expression in the Cur intervention group was significantly reduced（P<0.01）,the P-CREB（Ser133）protein expression was significantly increased（P<0.05）,and the BDNF protein expression was significantly increased（P<0.01）;There was no significant change in T-CREB protein expression in each group（P>0.05）.It is suggested that Cur can antagonize the increase of ATF4expression caused by ACR and the inhibition of CREB-BDNF pathway downstream.（5）Changes in synaptic vesicle protein:Compared with the control group,the expression of Sy-1 and Syn protein in hippocampus tissue of ACR group was significantly reduced（both P<0.001）;Compared with the ACR group,the amount of Sy-1 expression levels in the Cur intervention group increased significantly（P<0.05）,but there was no significant difference in the expression levels of Syn（P>0.05）.（6）Changes in phosphorylated tau protein:Immunofluorescence results showed that compared with the control group,the green fluorescence of the ACR group was enhanced（P<0.001）;Compared with the ACR group,the green fluorescence of the Cur intervention group decreased（P<0.01）;Western blot analysis showed that compared with the control group,the expression of inhibitory P-GSK-3β（Ser⁹）in the ACR group was significantly reduced and the expression of P-tau Ser²⁶²was significantly increased（both P<0.001）;Compared with the ACR group,the expression of inhibitory P-GSK-3β（Ser⁹）in the Cur intervention group was significantly increased,and the expression of P-tau Ser²⁶²was significantly reduced（both P<0.01）;There was no significant difference in T-GSK-3βand tau-5（reflecting the total tau level）in each group（both P>0.05）.Conclusions:Cur pretreatment can antagonize the excessive activation of PERK-eIF2αsignaling pathway and the inhibition of CREB pathway by ACR in SH-SY5Y cells,and improve the reduction of synaptic vesicle protein Sy-1 and the increase of tau protein phosphorylation caused by ACR.