Effect of SA on N-methyl-4-phenylpyridinium
(MPP+)-induced cytotoxicity in human SH-SY5Y
neuroblastoma cells
First, we investigated the cytotoxicity of SA in SH-SY5Y cells using an
MTT assay. SA did not show any toxicity to cells at a variety of
concentrations (from 1 to 40 µM) (data not shown). To examine the effect
of SA on MPP+-induced cytotoxicity, we treated cells
were treated with SA up to a concentration of 40 µM with or without 2 mM
MPP+. Figure 1A shows that the cell survival rate was
significantly decreased by 2 mM MPP+. However, SA
treatment dose-dependently protected the inhibition of
MPP+ on the survival rate of SH-SY5Y cells. To further
investigate the protective effects of SA, we measured the level of
tyrosine hydroxylase (TH), which is an indicator of the abundance of
dopaminergic neurons. As shown in Figure 1B, the protein expression of
TH was significantly inhibited to approximately 51% in the
MPP+ group relative to the control group. This
decrease was attenuated by SA in a dose-dependent manner. cellular ATP
levels and glutamate dehydrogenase (GDH) activity are usually detected
for the measurement of mitochondrial function. GDH is located in the
mitochondrial matrix, and leakage of GDH from mitochondria indicates
disruption of mitochondrial membrane integrity. Several studies have
shown that the loss of GDH activity is associated with mitochondrial
function, as with a reduction in ATP levels (Holownia, Chwiecko &
Farbiszewski, 1994; Lee, Youn, Jang & Yang, 2019). After
MPP+ treatment, the cellular ATP level was
significantly decreased to approximately 36%
relative to the control group, while
the SA 40µM treatment group showed recovery of the cellular ATP level
(Figure 1C). The median GDH activity in the MPP+ group
markedly increased to 1.7 times that of the control group, and 40 µM SA
attenuated this increase (Figure 1D). To investigate how SA showed a
protective effect against MPP+-induced neurotoxicity,
we measured the protein expression of REV-ERB α, a circadian clock
component, as a potent regulatory mechanism of mitochondrial fission.
The protein expression of REV-ERB α was significantly decreased to 72%
in the MPTP group, while SA attenuated REV-ERB α protein expression
(Figure 2A). To determine whether the protective effect of SA against
MPP+-induced neurotoxicity is dependent on REV-ERB α,
we used the REV-ERB α agonist and antagonist with SA. As shown in Figure
2B and C, both REV-ERB α antagonist treatment and SA treatment
diminished the protein expression of TH and REV-ERB α, but agonist
treatment with SA elicited no change compared with SA only.