Transcriptional Regulation Of HNF1α And HNF4α On CYP450 Under High-Altitude Hypoxia

Objective:To investigate the effects of high-altitude hypoxic environment on CYP450 activity and expression as well as the expression of HNF1αand HNF4α.To screen for CYP450 that bind to HNF1αand HNF4α,and explore the transcriptional regulation of HNF1αand HNF4αon CYP450 under hypoxia.Methods:In this study,we systematically evaluated the changes of CYP1A2,CYP2C11,CYP2C22,CYP2E1 and CYP3A1 activities in rats by measuring the pharmacokinetic characteristics of five CYP450 probe substrate drugs and their metabolites in rats under the high-altitude hypoxic environment.Sprague Dawley rats were divided at random into five groups,caffeine,diclofenac,omeprazole,chlorzoxazone,and midazolam,which were further divided into plain control（Xi’an City,Shaanxi Province,altitude:390 m,Pa O₂:20.0 k Pa）,acute hypoxia（Maduo County,Qinghai Province,altitude:4300 m,Pa O₂:12.4 k Pa,exposure for 7 d）,and chronic hypoxia（Maduo County,Qinghai Province,altitude:4300 m,Pa O₂:12.4 k Pa,exposure for 30 d）groups.Blood sample was collected from the eye sockets of rats at the corresponding time points after drug administration.The concentration of caffeine,diclofenac,omeprazole,chlorzoxazone,midazolam and their metabolites1,7-dimethylxanthine,4’-OH diclofenac,5-OH omeprazole,6-OH chlorzoxazone and1’-OH midazolam were determined in the plasma of rats by HPLC.To investigate the effect of hypoxia on CYP450,HNF1αand HNF4αexpression,protein and m RNA expression assays were carried out in vivo and in vitro,respectively.Sprague Dawley rats were randomly divided into plain control group,acute hypoxia group and chronic hypoxia group,and liver tissues were collected for protein and m RNA expression measurements of five CYP450,HNF1αand HNF4αby Western blot and RT-q PCR.Hep G2 cells were treated with 21%oxygen concentration as the normoxia group,and2%oxygen concentration for 24 h and 48 h as hypoxia-24h and hypoxia-48h groups,respectively.Protein and m RNA expression of five CYP450,HNF1αand HNF4αin Hep G2 cells were examined by Western blot and RT-q PCR.To screen for CYP450that bind to HNF1αand HNF4α,Ch IP-seq was used to analyze the binding sites of HNF1αand HNF4αat a genome-wide scale of Hep G2 cells.After choosing a subtype of CYP450,exploring the transcriptional regulatory mechanism of HNF1αand HNF4αon CYP450.Protein-protein interactions of HNF1αand HNF4αwere detected using immunoprecipitation.The protein and m RNA expression levels of HNF4αand CYP450 were detected after silencing HNF1A using si RNA,and protein and m RNA expression levels of HNF1αand CYP450 were detected after silencing HNF4A.The activation of CYP450 promoter by HNF1αand HNF4αwere detected using luciferase reporter gene assay,and the binding sites of HNF1A/HNF4A to CYP450 promoter were identified by mutagenesis.Results:The pharmacokinetic parameters of CYP1A2 probe drug caffeine,CYP2C11 probe drug diclofenac,CYP2C22 probe drug omeprazole,CYP2E1 probe drug chlorzoxazone,CYP3A1 probe drug midazolam and their metabolites1,7-dimethylxanthine,4’-OH diclofenac,5-OH omeprazole,6-OH chlorzoxazone,and1’-OH midazolam were significantly altered in rats under the high-altitude hypoxic environment,suggesting that the activities of CYP1A2 and CYP2E1 were significantly increased and the activities of CYP2C11,CYP2C22 and CYP3A1 were significantly decreased in rats.Hypoxia significantly increased the protein and m RNA expression of CYP1A2（P<0.05）and the m RNA expression of CYP2C11 in rats（P<0.05）,and decreased the protein and m RNA expression of CYP2C22 and CYP3A1 in rats（P<0.05）.The protein expression of CYP2C11 were not statistically significant in rats.The m RNA expression of CYP2E1 was significantly higher and the protein expression was significantly lower in rats under hypoxia（P<0.05）.The protein expression of HNF1αwas significantly increased in the acute hypoxia group and significantly decreased in the chronic hypoxia group（P<0.05）,and the m RNA expression of HNF1αwas not statistically significant.The protein expression of HNF4αwas significantly lower in rats under hypoxia（P<0.05）.The m RNA expression of HNF4αwas significantly increased in rats under hypoxia（P<0.05）.The protein and m RNA expression of CYP1A2,CYP2C9,CYP2E1,CYP3A4,HNF1αand HNF4αwere significantly reduced in Hep G2 cells under hypoxic conditions（P<0.05）,and the differences in the protein and m RNA expression of CYP2C19 were not statistically significant.The above results suggested that significant changes in protein and m RNA expression of CYP450,HNF1αand HNF4αin rat liver and Hep G2 cells under hypoxic conditions.Ch IP-seq results showed that HNF1αand HNF4α-enriched target genes are involved in the metabolism of endogenous and exogenous substances in Hep G2 cells.58%of the total HNF1αbinding sites co-localized with HNF4αat a genome-wide scale,with 16 CYP450genes binding to HNF4α,8 CYP450 genes binding to HNF1α,of which 3 CYP450genes（CYP1A2,CYP2B6,and CYP26B1）bind to HNF1αand HNF4α.The HNF1αbinding site co-localized with the HNF4αbinding site in the promoter and downstream of CYP1A2.Therefore,subsequent experiments were conducted to explore the transcriptional regulatory mechanism of CYP1A2 by HNF1αand HNF4α.The results indicated that protein-protein interaction exists between HNF1αand HNF4α.The effect of hypoxia in reducing HNF4αand CYP1A2 protein and m RNA expression was diminished after silencing HNF1A,and the effect of hypoxia in reducing HNF1αand CYP1A2 protein and m RNA expression was similarly diminished after silencing HNF4A.CYP1A2 promoter activity in Hep G2 cells increased following transfection of HNF1A expression plasmid under normoxia and hypoxia（P<0.05）,while no change was observed after transfection with the HNF4A expression plasmid.CYP1A2 promoter activity was significantly reduced upon transfection with the HNF1A expression plasmid at-3500 bp and-3000 bp.Mutation of HNF1A binding site 2（-2892 bp to-2886 bp）deactivates CYP1A2 activity.There was no significant change in the activity of CYP1A2 after transfection of HNF4A expression plasmid and mutation of the assumed HNF4A binding sites.Conclusion:The study revealed that HNF1αand HNF4αcooperatively downregulated CYP1A2 by inhibiting each other’s expression under hypoxia,and the regulatory mechanism of CYP450 under hypoxic conditions may be different from thatofnormoxia,elucidatinganewmechanismof"hypoxia-HNF1α-HNF4α-CYP450"interaction.High-altitude hypoxic environment significantly affected the activities of CYP1A2,CYP2C11,CYP2C22,CYP2E1 and CYP3A1 in rats.Hypoxia significantly affected the expression of CYP1A2,CYP2C9,CYP2C19,CYP2E1,CYP3A4,HNF1αand HNF4αin rat liver and Hep G2 cells.HNF1αand HNF4αsynergistically downregulated CYP1A2 by inhibiting each other’s expression under hypoxia,and protein-protein interaction existed between HNF1αand HNF4α.HNF1A binds to the binding sites of HNF1A in the CYP1A2promoter（-2892 bp to-2886 bp）to activate CYP1A2.The effect of HNF4A on CYP1A2 is not in the promoter region.This study elucidates a new idea for the rational interpretation of the drug metabolizing enzymes changes in the high-altitude hypoxic environment,and provides an important theoretical basis for future studies on the regulation of CYP450 by HNF1αand HNF4α,and further emphasizes the necessity of studying the regulatory mechanism of drug metabolism under hypoxia.It also provides theoretical support for clinical rational drug use in the plateau areas and the prevention and treatment of mountain sicknesses.