Akademik Veri Yönetim Sistemi
Physiology 2023, York, İngiltere, 10 - 12 Temmuz 2023, ss.84
Valproic acid (VPA) is an environmental risk factor for Autism Spectrum Disorders (ASD), especially during the first trimester of pregnancy, that is widely used in experimental designs for its ability to model ASD both morphologically and behaviourally on animals but was not studied on cell lines. Cell lines provide cheaper, ethically less problematic and reusable models (Xicoy et al., 2017). The aim of this study was to use SH-SY5Y, a human neuroblastoma cell line, to develop an in vitro model for studying ASD and observing VPA’s effects directly on cells during different stages of differentiation.
SH-SY5Y cells were seeded to a 96-well plate with 2500 cells per well. 24hrs after seeding, culture media was changed to differentiation media and this was accepted as the first day of differentiation. The differentiation protocol was 7 days in total (Kovalevich and Langford, 2013). Cells were treated with VPA for 24 hours on different days of differentiation and each treatment day had three different concentrations (1mM, 5mM and 10mM of VPA). Experimental groups were the following: Control group (vehicle); VPA on the first day of differentiation; VPA on the third day of differentiation; VPA on the fifth day of differentiation. The MTT assay was performed as triplicates to determine cell viability. Statistical analysis was performed with One Way ANOVA and on Sigma Plot.
MTT assay showed that VPA affects cell proliferation/viability when compared to the control group on the first day of differentiation for each dose group (p<0,001; p<0,001; p<0,05). Cells exposed to VPA at the third day of differentiation also had reduced cell viability when compared to control cells except for the lower concentration of VPA (p<0,001; p<0,001). VPA treatment on the fifth day of differentiation significantly affected cell viability, when treated with concentrations of 5mM and 10mM (p<0,001; p<0,001). However, the lowest concentration of VPA had the opposite effect, increasing cell counting when compared to control cells (p<0,001). Effect of VPA exposure on different days of differentiation was compared within the same dose groups. In 5mM and 10mM doses, VPA exposures on third day of differentiation resulted in significantly lower cell count compared to first and fifth day of differentiation (p<0,05; p<0,05).
Consistent with literature, our results showed that 1mM of VPA does not have negative effects on cell viability whereas 10mM showed detrimental effects (Jang et al., 2021). Our results also showed that third day of differentiation is the most vulnerable time for VPA exposure in terms of cell viability. We suggest that 5mM VPA should be used for further analysis for cellular and morphological parameters that are disrupted in in vivo models of ASD.