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).