A Study Of EGCG Inhibits Aβ₄₂ Aggregation And Affects The Cytotoxicity In SH-SY5Y Cells

Alzheimer’s disease（AD）is a neurodegenerative disease caused by the interaction of genetic and environmental factors,which is characterized by progressive cognitive dysfunction and memory loss.（-）-Epigallocatechin-3-gallate（EGCG）is a kind of polyphenol with diversified biological activities such as antioxidant,anti-inflammatory and epigenetic regulation properties,which may also have a risk-reducing effect in AD.However,whether EGCG prevents the development of AD through affecting key pathophysiological processes in AD development is still unclear.Amyloidβpeptides（Aβ）are 38-43 amino acid residues peptides,of which Aβ₄₀and Aβ₄₂account for a larger proportion.Aβpeptides tend to abnormally aggregate to form amyloid plaques in the brain which has been deemed as one of the typical events in the early stage of AD.Amongst,Aβ₄₂is the most abundant peptide in formed amyloid plaques,more than Aβ₄₀.In this study,starting with the phenomenon of Aβ₄₂abnormal aggregation,we try to investigate the following points from two levels,cell-free system and isolated human neuroblastoma cell model SH-SY5Y:（1）the interactions between EGCG and Aβ₄₂monomer molecules and their effects on Aβ₄₂aggregation;（2）the effects of EGCG on oxidative stress,DNA methylation and genomic stability in Aβ₄₂-exposed cells.Our study aims to reveal the effects of EGCG on Aβ₄₂monomers aggregation,based on this,we further analyze the effects of EGCG on the cytotoxicity of Aβ₄₂-exposed cells and its potential mechanism.Firstly,the molecular docking technique was adopted to predict the interaction between EGCG and Aβ₄₂monomers,and thiosulfate-T（Th T）fluorescence method was adopted to investigate the effect of EGCG at different concentrations（0,5,10,20,and 40μmol/L）on the aggregation of Aβ₄₂monomers at low（5μmol/L）and high（50μmol/L）concentrations in the cell-free system.The results of molecular docking revealed that EGCG can interact with multiple amino acids（His 6,His 14,Gln 15,and Lys 16）at the N-terminalα-helix of Aβ₄₂monomers,which may inhibit the formation of toxicβ-fold conformation;the results of fluorescence experiment showed that the aggregation degree of Aβ₄₂monomers was significantly lower in EGCG-treated group than only Aβ₄₂group（P<0.05）,suggesting that EGCG can effectively inhibit the aggregation of Aβ₄₂.To further investigate the effect of EGCG on the cytotoxicity towards Aβ₄₂-exposed cells,the SH-SY5Y cells were divided into several groups,and treated with 20μmol/L EGCG（EG）,5μmol/L Aβ₄₂（AL）,50μmol/L Aβ₄₂（AH）,20μmol/L EGCG accompany with 5μmol/L Aβ₄₂（ALEG）,and 20μmol/L EGCG accompany with 50μmol/L Aβ₄₂（AHEG）,respectively,for 24-72h.Subsequently,（1）intracellular reactive oxygen species（ROS）levels,and malondialdehyde（MDA）levels and total antioxidant capacity of cells（TAOC）were detected to analyze the transcription of two key genes of oxidative stress,Nrf-2 and HO-1;（2）detected genome-wide 5-mC and 5-hmC levels,and evaluated the transcription level of key methylation genes DNMT1 and TET1;（3）evaluated cell genomic instability（GIN）and apoptosis levels by CBMN-Cyt.The results showed that（1）after treatment for 24h,the intracellular ROS and MDA levels were significantly increased while TAOC was dramatically decreased in all subject groups,compared to the control group（P<0.001）;after treatment for 72h,ROS and MDA levels were still significantly increased（P<0.001）and TAOC was dramatically decreased（P<0.001）in the AL and AH groups,while the intracellular oxidative stress in the ALEG and AHEG groups was significantly attenuated（P<0.01）;after treatment for 24h,the transcript levels of Nrf-2 and HO-1 were significantly upregulated in both EG and AH groups（P<0.05）,while after treatment for 72h,the transcript levels of Nrf-2 and HO-1 were significantly higher in the EG group than that in the AL and AH groups（P<0.05）.It was suggested that EGCG could induce intracellular oxidative stress through autoxidation within 24h,and subsequently upregulate Nrf-2 and HO-1 expression in response to cellular oxidative stress under Aβ₄₂exposure.（2）After treatment for 72h,genome-wide 5-mC and 5-hmC levels were significantly increased（P<0.01）in the ALEG group and significantly decreased（P<0.01）in the AH group.After treatment for 24h,the transcript levels of DNMT1and TET1 were significantly upregulated（P<0.001）in the EG and AH groups,while the transcript levels of DNMT1 and TET1 in the AL group were significantly down-regulated（P<0.001）.After treatment for 72h,the transcript levels of DNMT1and TET1 in the ALEG and AHEG groups were significantly up-regulated（P<0.001）.The above results suggested that EGCG may affect the epigenetic genome modifications by regulating the transcription of DNMT1 and TET1 under Aβ₄₂exposure.（3）After treatment for 24h,high levels of GIN and apoptosis were observed in AL and AH groups（P<0.01）;at 72h,GIN and apoptosis were significantly reduced in ALEG and AHEG groups（P<0.01）,which suggested that EGCG can suppress Aβ₄₂-induced GIN and apoptosis.In summary,extracellular EGCG may prevent the aggregation of Aβ₄₂by inhibiting the formation of toxicβ-fold conformation of Aβ₄₂.Intracellular EGCG first undergoes autoxidation,which induces cellular oxidative stress,and activates the Nrf-2/HO-1 pathway,thus the ROS levels rise briefly and then decrease,finally resulting antioxidant effects against Aβ₄₂-induced intracellular oxidative stress.Meanwhile,the antioxidant effect of EGCG could up-regulate the expression of TET1and regulate the methylation modification in the genome under Aβ₄₂exposure,which played a positive role in the maintenance of genome stability.In this study,the molecular mechanism of EGCG inhibition of Aβ₄₂toxicity was analyzed at the levels of oxidative stress,DNA methylation modification and genomic stability,which may provide some scientific basis for EGCG to reduce the risk of AD.