Analysis Of The Mechanisms That Tetrachlorobenzoquinone Induced The Activation Of Nrf2/ARE Signaling In HepG2 Cells

Tetrachlorobenzoquinone(TCBQ), known as a metabolite of industrial herbicide pentachlorophenol, have shown hepatotoxicity and genotoxicity via reactive oxygen species(ROS) mechanism in vitro and in vivo models. Nuclear factor erythroid-derived 2-like 2(Nrf2)/antioxidant response element(ARE) signaling pathway is a cellular sensor of oxidative stress, which regulates the expression of antioxidant enzymes and defensive proteins. Kelch- like ECH-associated protein 1(Keap1), BTB and CNC homolog 1(Bach1) are both important negative regulator of Nrf2/ARE signaling pathway, which plays an important role in the activation of the Nrf2 pathway. The purpose of this study was to analyze whether TC BQ can activate Nrf2/ARE signaling and if so to explore the specific mechanism, which can be provide evidence for TC BQ induced toxicity therapeutic intervention.Part â… : The activation of Nrf2/ARE signaling needs Nrf2 released from the Keap1, this part analyzes whether TC BQ can activate Nrf2/ARE signaling pathway, and analyzed the important role of Keap1 in activating Nrf2. The expressions of Nrf2 and antioxidant protein heme oxygenase-1(HO-1), NADH quinone oxidoreductase subunit 1(NQO1) were up-regulated in concentration and time dependent manner after treated with TCBQ in human hepatoma HepG2 cells. Immunofluorescence experiments also demonstrated the upregulated exp ression of Nrf2. The q RT-PCR experiments showed that a significant enhancement of Nrf2 mRNA but not Keap1 following exposure to TCBQ. The cell lysates in nuclear and cytosolic fractions were detected by Western Blot demonstrated that Nrf2 accumulation into nucleus. Subsequently, Nrf2 bind to ARE was determined by Dual Luciferase Reporter Gene Assay, which may be considered as an anti-oxidant response to TC BQ-intoxication. In current research, Keap1-dependent the activation of Nrf2 is mainly focused. Redox Western Blotting showed that TC BQ induced the formation of Keap1 cross- linking dimer. Immunoprecipitation assay shown that TCBQ does not induce the dissociation of Keap1 and Cullin3. Ubiquitination experiments showed TCBQ induced ubiquitin switch from Nrf2 transfer to Keap1. Here, we demonstrated TC BQ-induced the activation of Nrf2 through the ubiquitin switch from Nrf2 to Keap1 and the formation of Keap1 cross- linking dimer, these mechanisms are both Keap1 dependent, which causes a change in its conformatio n that induced the release of Nrf2 from Keap1, and then activate the Nrf2/ARE signaling pathway.Part â…¡: Liking Keap1, Bach1 is another important repressor transcription factor of Nrf2 that can compete with Nrf2 leading to negative regulation of ARE and antioxidant proteins expression. The purpose of this part was to explore the role of Bach1 in TCBQ induced the activation of Nrf2/ARE signaling. Western Blot and immunofluorescence assay clearly demonstrated that Bach1 export from the nuclear within 1 h along with the import of Nrf2 into the nucleus after treated with TC BQ for the indicated times. Immunoprec ipitation experiment demonstrated that TCBQ induced the nuclear export of Bach1 is dependent on the interaction with chromosomal region maintenance 1(Crm1) and tyrosine phosphorylation. C hIP assay showed the competitive bindings of Nrf2 and Bach1 to the gene ARE under TC BQ treatment. Ubiquitination experiment showed that Bach1 was degraded after export from nuclear into the cytoplasm. The qRT-PCR assay demonstrated that TCBQ increased transcriptional levels of Bach1. The introduction of cycloheximide(CHX) showed that Bach1 after 3 h was newly synthesis, and then reentered into the nucleus. Bach1 as an important regulator gene of Nrf2/ARE signaling, the newly synthesis and the increased transcription levels made TC BQ-induced oxidative stress gradually restored to the normal levels. Moreover, we also illustrated that TCBQ- induced the regulation of Nrf2/ARE signaling pathway involves JNK-P62 signaling.