Studies On Bmal1 Regulation Of Cyp3a11 Expression And Drug Chronotoxicity

ObjectivesBmal1(Arntl)is the core clock factor,which can participate in the regulation of important physiological behaviors and lives processes.CYP3A4(murine Cyp3a11)is an essential member of the cytochrome P450 enzymes and plays an important role in the metabolism and detoxification of drugs.The expression of murine Cyp3a11 shows a circadian rhythmicity.However,whether Bmal1 has a regulatory effect on murine Cyp3a11 is still unclear.Therefore,our paper aims to explore the regulation of Bmal1 on Cyp3a11,and to elucidate the effect of Bmal1 on the regulation of Cyp3a11-mediated drug metabolism and chronotoxicity.Methods 1.Mouse genotyping were identified by PCR-gel electrophoresis.Real-time fluorescence quantification and Western blotting were used to detect the expression of Cyp3a11 and its related transcription factors in wild-type and Bmal1 knockout mice at six circadian time points.In addition,Cyp3a11 activity assay was determined using microsomal incubation experiments.2.The effects of Bmal1 on Cyp3a11 and its related transcription factors expressions were determined in mouse liver/intestinal cells through Bmal1 over-expression,siRNA knockout,real-time fluorescence quantification and Western blotting.The regulation mechanism of Bmal1 on Cyp3a11 was elucidated by luciferase reporter gene assay,electrophoretic mobility shift assay and chromatin immunoprecipitation experiments.3.Enzyme incubation experiments were used to obtain the major metabolic enzymes of aconitine and triptolide.Wild-type and Bmal1 knockout mice were used as experimental subjects.The analyses of cardiotoxicity indicators(creatine kinase and malondialdehyde)and hepatotoxicity indicators(alanine aminotransferase and aspartate aminotransferase)were evaluated the chronotoxicity of aconitine and triptolide.The diurnal differences in the liver metabolic capacity of aconitine and tripterygium were investigated through liver microsome incubation experiments.Results 1.Bmal1 knockout causes Cyp3a11 to lose its rhythm.Cyp3a11 expression is rhythmic.Bmal1 knockdown inhibited the expression of Cyp3a11 and blunted its circadian rhythm.2.Bmal1 indirectly regulates Cyp3a11 expression.Bmal1 activated the Cyp3a11 promoter activity.In addition,the 320 bp(-220 bp~+100 bp)sequence on the Cyp3a11 promoter is a key region for Bmal1 to regulate Cyp3a11.The analysis of the promoter sequence revealed that there was no E-box(Bmal1 site of action)in this region.This result indicated that Bmal1 indirectly regulated Cyp3a11 expression.We further found that Bmal1 knockdown reduced Dbp and Hnf4α(two Cyp3a11 regulatory factors)expressions in animal and cell models.In addition,there are binding sites for Dbp and Hnf4α(D-box and DR1sequence)in the region of Cyp3a11 promoter(-220 bp to +100 bp).Knockdown of Hnf4α and Dbp,or mutation of D-box and DR1 in Cyp3a11 promoter,caused disappearance of regulatory effects on Cyp3a11 by Bmal1.3.Bmal1 regulates Hnf4α.Bmal1 binded to E-box site of the Hnf4α P1 distal(-6100~-5900 bp)promoter and enhanced Hnf4α expression.4.Bmal1 regulates the toxicity of aconitine and triptolide.Cyp3a11 is a major metabolic enzyme of aconitine and triptolide.The toxicity of aconitine and triptolide is time-dependent.In the wild-type mice,the toxicity at ZT2 was higher compared to the toxicity at ZT14.However,in the Bmal1 knockout mice,the toxicity of aconitine and triptolide increased,and its toxicity was not significantly different between ZT2 and ZT14.Wild-type mice had a higher metabolic capacity for aconitine and triptolide at ZT2 than ZT14.There was no difference in metabolic capacity for aconitine and triptolide between two time points in the Bmal1 knockout mice.Conclusion 1.Bmal1 regulates Cyp3a11 circadian expression;2.Bmal1 regulates the expression of Dbp and Hnf4α at the transcriptional level,and indirectly regulates Cyp3a11;3.The toxicities of Aconitine and triptolide are time-dependent;4.Bmal1 modulates the chronotoxicity of aconitine and triptolide.