Role And Mechanism Of DNA Methylation And PI3K/Akt Signaling Pathway In Schisandrin B Against Alzheimer’s Disease

Objective To investigate the therapeutic effect of Schisandrin B（Sch B）on Aβ1-42-injured human neuroblastoma cells（SH-SY5Y cells）and the role of DNA methyltransferase and PI3K/Akt signaling pathway in each experimental group,and to explore what mechanisms they participate in.Method The AD models of SH-SY5Y cells incubated with Aβ_1-42were divided into 7 groups:normal control group（C）,AD cell model group,Sch B high,medium and low dose group,LY294002 group（LY294002 as PI3K/Akt signal pathway inhibitor）,and LY294002+Sch B high dose group.The cell viability was measured by observing cell morphology and MTT assay.The m RNA expressions of DNMT1,DNMT3A,DNMT3B and Akt in each experimental group were detected by Real-time PCR.Western blot was used to detect the protein expression of DNMT3A,Akt and p-Akt in each experimental group.The expression of DNMT3B and DNMT1 protein in each group was detected by immunocytochemistry.The methylation profiles of normal group,AD model group and Sch B high dose group were detected by using Infinium Human Methylation 850K Bead Chip,and the biological analysis of the test results was carried out.Results（1）Sch B on Aβ_1-42injury in SH-SY5Y cells have a significant improvement on the cell morphology and activity.（2）Real-time PCR results showed that compared with the normal control group,m RNA expression of DNMT1,DNMT3A,DNMT3B and Akt in AD cell model group was significantly decreased（P<0.05）,m RNA of DNMT1,DNMT3A,DNMT3B and Akt（P<0.05）.The m RNA expression of DNMT1,DNMT3A,DNMT3B and Akt gradually increased with the increase of Sch B dose（P<0.05）.The m RNA expression of DNMT1,DNMT3A,DNMT3B and Akt decreased after pretreatment of LY294002 for 1 h（P<0.05）.Compared with high dose Sch B cells,the expression level of DNMT1,DNMT3A,DNMT3B and Akt decreased significantly in LY294002+high dose group（P<0.05）.（3）Western blot results showed that Aβ_1-42decreased the protein expression of DNMT3A and p-Akt.After Sch B treatment,the protein expression of DNMT3A and p-Akt was significantly increased（P<0.05）,and in a dose-dependent manner.The expression of DNMT3A and p-Akt protein in LY294002+Sch B group were significantly higher than that in LY294002+Sch B group after pre-treatment with LY294002 for 1h reduce;（4）Immunocytochemical staining showed that the protein expression of DNMT3B and DNMT1 decreased after Aβ_1-42treatment,and the protein expression of DNMT3B and DNMT1 increased significantly after Sch B treatment（P<0.05）.After pretreatment with LY294002 for 1 h,compared with AD model group,the expression of DNMT3B and DNMT1 protein were significantly decreased,compared with high-dose Sch B,LY294002+high-dose Sch B group DNMT3B and DNMT1 protein expression decreased;（5）Illumina methylation chip test results showed that in all the methylated sites with gene annotation,the pathways of differential methylation sites are Ras,c AMP,PI3K/Akt,etc.Aberrant DNA methylation can affect these pathways.Conclusions（1）Sch B can improve the morphology and viability of SH-SY5Y cell model injured by Aβ_1-42.（2）Sch B may be used to treat Alzheimer’s disease by regulating the m RNA and protein expression of DNMT3A,DNMT1,DNMT3B and p-Akt.（3）After the PI3K/Akt signaling pathway inhibitor LY294002,Sch B up-regulated the expression of the above protein was inhibited,which may be Sch B through the PI3K/Akt signaling pathway,up-regulation of the expression of these proteins play a role.（4）There are a large number of differentially methylated genes in the cells of the experimental groups,suggesting that the abnormal methylation of DNA is closely related to the development of AD.