Mechanism Of Activating Nrf2/ARE Pathway And Antioxidant Function By PMID

The antioxidant response elements (ARE) is a cis-acting enhancer sequencelocated in regulatory regions of phase2antioxidant and detoxifying genes. Nuclearfactor (erythroid-derived2)-like2(Nrf2) is a member of the Cap n‘Collar (CNC)family of transcription factors that binds to the ARE and regulates the transcription ofspecific ARE-containing genes. Under oxidative stress, Nrf2/ARE induction isfundamental to defense against reactive oxygen species (ROS) and serves as a keyfactor in the protection against toxic xenobiotics.In our previous study, we established a high throughput screening model of AREinducers based on ARE-luciferase reporter. Using this model,188compounds werescreened and2-indolinones derivatives were found to be able to induce theARE-mediated transcription. In these2-indolinones compounds, PMID(3-(3-Pyridylmethylidene)-2-Indolinone) was the most potent inducer.2-Indolinoneswas reported to act as protein kinase inhibitors and show anti-tumor activity. However,the role of2-indolinones in the oxidative stress remains unknown. PMID is a designedderivantive based on the core chemical structure of2-Indolinone. In the present study,we showed that PMID induced the activation of ARE-mediated transcription,increased the DNA-binding activity of Nrf2and then up-regulated the expression ofantioxidant and detoxifying genes. The level of Nrf2protein was increased in cellstreated with PMID by enhancing the stability and decreasing the turnover rate of Nrf2.We showed that PMID reduced the ubiquitination of Nrf2and disrupted the Cullin3(Cul3)-Keap1interaction. p38MAPK and phosphorylation events might be involvedin the activation of Nrf2/ARE pathway by PMID. PMID also up-regulated SODactivity and GSH level in the liver and kidney in BALB/c mice.Furthermore, cells treated with PMID showed resistance to cytotoxicity bypro-oxidant6-hydroxydopamine (6-OHDA). In addition, PMID pretreatment couldprotect mice form ionizing radiation-induced body weight lost, lethality andhemopoietic injuries.Meanwhile, three truncated protein of Nrf2and Keap1were prepared, whichcould be used in kinetics research of Nrf2-Keap1interaction and drug screening. Taken together, the compound PMID induces the ARE-mediated genesexpression through stabilization of Nrf2protein and activation of Nrf2/ARE pathwayand then protects against oxidative stress-mediated cell death. These results imply thatPMID might be a potential chemopreventive agent.