Investigation of the effectiveness of polyphenolic compounds on amyloid beta plaque dissociation in differentiated-SH-SY5Y cells and the effects of the differentiated cells on blood-brain barrier integrity in vitro

Introduction: Alzheimer's disease (AD) is a neurodegenerative disease that affects the hippocampal and cortical regions of the brain. Due to the complex pathogenesis of AD, currently, there is no treatment. In recent years, studies have focused on preventing or dissolving amyloid plaques that play an important role in AD. Due to their various properties, natural polyphenols might be used as potential agents in this field. Objective: •Determination of the time and administration using different agents the best neuronal differentiation in SH-SY5Y cells. •Determination of effect on Aβ aggregation dissociation of EGCG, Punicalagin, Ellagic acid, Gastrodin / combinations. •Forming and comparing blood-brain barrier (BBB) models with in vitro co-, and tri- cell culture with differentiated neuronal cells. •Investigation of the dissociation efficacy on Aβ1-42 plaques of polyphenols in the tri-cell culture BBB model. Method: The differentiation of SH-SY5Y cells was confirmed by morphologic changes with light microscopy, neurite length using neuron j program, and neuronal markers with immunofluorescence assay. The effect of polyphenols and Aβ1-42 plaques on differentiated SH-SY5Y cell viability was determined by WST-1 analysis. EGCG, Punicalagin, Ellagic acid, Gastrodin or combinations to dissociation efficiencies of Aβ aggregation were determined by Field emission scanning electron microscopy, Thioflavin T and aggregated Aβ ELISA methods. BBB model was constructed using tri- and co-cell culture, and transendothelial electrical resistance (TEER) values were compared to each other. Transition of polyphenols from the formed BBB was evaluated by Folin Ciocalteu methods and the effects of polyphenols on BBB model with Aβ toxicity were examined by ELISA and TEER values. Results: It was observed that the treatment time of differentiation of SH-SY5Y cells was 10 days and using the content of the media consisting of RA + BDNF + dc-AMP + KCI + B27 was similar and closer to the in vivo results. Aβ1-42 plaque dissolving efficacies of polyphenols whose doses resulted in 90% viability of the cells were evaluated at different time periods. All polyphenols were found to dissolve Aβ plaques at different doses. Combination-1 and Combination-2 were found to have more than 24 hours of Aβ dissolution effect (75% and 64%, respectively) than single polyphenol treatment (p <0.05). The triple culture model was found to have higher TEER value than the co-culture models and it was found to be more suitable for the BBB model (p <0.05). In the BBB model with Aβ1-42 toxicity, it was determined that TEER values increased with polyphenols and membrane integrity improved. Conclusion: As a result of the studies, reflecting the in vitro AH model BBB was created by using a triple culture system with differentiated SH-SY5Y cells. Kom 1 and Kom 2 polyphenol treatments were found to dissolve Aβ1-42 plaques through the triple BBB model. According to the results obtained, the first triple BBB model and polyphenol combinations created with differentiated cells and confirmed for the first time will contribute to in vitro research and development of AD treatment strategies that might be yield closer in vivo results.