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.