3.5 DACA attenuates oxidative stress by regulating Nrf2 in cells.
We have previously shown that DACA enhances the MPP+-induced decrease in cell viability. The measurement of reactive oxygen species (ROS) serves as the most direct evaluation index for detecting oxidative free radicals. A reduction in superoxide dismutase (SOD) activity amplifies the increase in free radicals. When cells are exposed to high levels of oxidative stress, oxidized glutathione (GSSG) accumulates and leads to a decrease in the glutathione (GSH)/GSSG ratio, which has become a valuable tool for assessing cellular damage caused by free radicals and evaluating oxidative stress in cells and tissues. According to the results shown in Fig. 5A-C, it is evident that SH-SY5Y cells and primary neurons treated with MPP+ exhibited a significant elevation in ROS levels compared to the control group, accompanied by decreased SOD levels and a reduced GSH/GSSG ratio. However, DACA effectively lowered intracellular ROS levels, prevented SOD dissipation, and increased the GSH/GSSG ratio.
Western blot analysis revealed that treatment with DACA at concentrations of 5μM, 10μM, and 20μM significantly augmented the translocation of Nrf2 to the nucleus and upregulated the expression of its downstream proteins HO-1, GCLC, and GCLM (Fig. 5D-G) in both SH-SY5Y cells and primary neurons. Immunofluorescence staining corroborated the western blot results by demonstrating that DACA facilitated the translocation of cytosolic Nrf2 into the nucleus (Fig. 5H). These findings suggest that activation of Nrf2 leads to an enhancement in cellular antioxidants, which may be responsible for mitigating the toxic effects induced by MPP+. Furthermore, immunofluorescence staining using tuj1 demonstrated that MPP+ significantly altered neuronal cell morphology, synaptic shortening, and induced round-shaped cells. However, these changes were markedly reversed upon treatment with DACA (Fig. 5H).