Neuroprotective potential of nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element signaling modulator cucurbitacin I upon glucose and oxygen deprivation/reperfusion (OGD/RP).

ABSTRACT

This study aimed to investigate the inhibitory effect and mechanism of Cucurbitacin I (Cu I) on apoptosis, oxidative stress, and mitophagy in PC12 cells with glucose and oxygen deprivation/reperfusion (OGD/RP) injury. OGD/RP cell injury model was established by gas anoxic cell incubator and glucose-free medium. The cells were divided into the control group, OGD/RP group, OGD/RP + Cu I group, and OGD/RP + Cu I + 2 µM nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor ML385 group. The results showed that apoptotic rate and reactive oxygen species (ROS) production were significantly increased in OGD/RP group, which were reversed by Cu I pretreatment. Meanwhile, western blot analysis proved that Cu I inhibited OGD/RP-induced mitophagy, manifested as the decreased expression of PTEN-induced kinase 1 (PINK1) and parkin RBR E3 ubiquitin-protein ligase (Parkin), and light chain 3 (LC3) Ⅱ∕LC3 I, as well as the increased expression of P62. Furthermore, immunofluorescence (IF) staining showed that Cu I reduced the co-localized puncta of LC3 with TOM20 in OGD/RP-induced PC12 cells. Similarly, transmission electron microscope finding is consistent with the IF results. Mechanically, after Cu I and OGD/RP treatments, nuclear Nrf2 expression and the levels of downstream target genes were significantly upregulated compared with OGD/RP alone treatment. Nrf2 inhibition reversed the protective effects of Cu I on OGD/RP-induced injury in PC12 cells. The present study provides evidence of the neuroprotective effect of Cu I unraveling its potential as a potential therapeutic candidate for the treatment of ischemic stroke.