Study About Potential Mechanism Of Histone Deacetylase Inhibitor TSA Regulating Keap1-Nrf2 Signaling Pathway

Backgrounds:An important cellular defense mechanism is the activation of Keapl-Nrf2 signaling pathway. Under unstressed conditions, Keapl serves as a substrate adaptor protein for Cu13-dependent E3 ubiquitin ligase complex, and promotes Nrf2 degradation by 26S proteasome. Under conditions of oxidative stress or injury, Nrf2 transfers into nucleus and regulates its downstream target gene expression after uncoupling with Keap1. Hence, cytoplasmic protein Keap1 is significant for Nrf2 activation. Histone deacetylases (HDACs), which prevent acetylation of histone, have been thought to be potentially useful therapeutic targets for many human diseases, including central nervous system (CNS) diseases. Studies have shown that HDAC inhibition plays a neuroprotective role by activating Keapl-Nrf2 pathway, but the avtivation mechanism is not yet fully elucidated. This study aimed to investigate the possible mechanism of HDAC inhibitior TSA regulating Keapl-Nrf2 signalling pathway in RAW 264.7 cells.Objectives:1. Confirming that TSA could activate Keapl-Nrf2 pathway.2. Build Eukaryotic recombinant plasmid pcDNA3.1-6cmyc-Keapl to study the functional fragment of Keapl.3. Proving that TSA could promote the protein degradation of Keap1 directly, then to explore the possible mechanisms.Methods:1. The cultured RAW 264.7 cells were treated with different concentrations of TSA (10ng/ml,30ng/ml, 100ng/ml) for 16 hours. Western Blot and RT-PCR were performed to detect the intracellular Keapl and its transcription factor Nrf2.2. The cultured RAW 264.7 cells were treated with TSA (30ng/ml) for 4h. Observed whether Nrf2 into the nucleus by Immunofluorescence.3. The recombinant plasmids were identified by restriction enzyme and DNA sequencing.4. The cultured RAW 264.7 cells were co-treated with TSA (30ng/ml) and ActD (0.5ug/ml) for 12h. Western Blot and RT-PCR were used to detect the intracellular Keapl.5. To detect the protein levels of exogenous Keapl by Western Blot, protein was collected 16h after TSA (30ng/ml) treat and 48h after transfection by Lipo 2000.6. The cultured RAW 264.7 cells were co-treated with TSA (30ng/ml) and MG-132 (5umol/L) or 3-MA (5mmol/L) for 12h. Western Blot was performed to detect Keapl protein levels.Results:1. TSA reduced intracellular Keap1 protein and mRNA levels in a contentration-and time-dependent manner.2. TSA promoted Nrf2 nuclear translocation and upregulated its downstream proteins HO1 and NQO1.3.The recombinant plasmids were confirmed to be successfully constructed.4. After TSA and ActD co-treatment, Keapl protein levels were reduced significantly but not its mRNA levels.5. TSA could promote the degradation of exogenous Keapl protein directly.6. The degradation of Keap1 protein induced by TSA may not be dependent on the ubiquitin-proteasome or autophagy-lysosome pathway.Conclusions:We report here that HADC inhibitior TSA could activate Keapl-Nrf2 pathway by inhibiting the expression of Keapl. TSA not only reduces Keap1 mRNA levels also reduces Keap1 protein levels. TSA could promote the degradation of exogenous Keap1 protein directly, but the degradation of Keap1 protein induced by TSA may not be dependent on the ubiquitin- proteasome or autophagy- lysosome pathway, and the precise mechanism remains to be futher studied.