Effect of Atorvastatin on the Rhythmicity of Circadian Clock Genes in Human Hepatocarcinoma Cell Line HepG2
Objective To investigate the effect of atorvastatin(AT)on the expression rhythm of circadian clock genes and proteins in human hepatocarcinoma cell line HepG2.Methods The virtual experiment for molecular docking between AT and circadian clock proteins was designed based on MOE software.The experimental groups(added with 101,102,103,104 nmol/L of AT),the control group(added with 0 nmol/L of AT),and the blank group(added with 0.1％dimethyl sulfoxide)were set in HepG2 and human osteosarcoma BMAL1::LUC-U2OS cells(hereinafter refferred to as U2OS).After 24 h of administration,CCK-8 assay was used to detect the changes of cell viability after different doses of AT treatment.The expression levels of brain and muscle ARNT-like protein 1,aryl hydrocarbon receptor nuclear translocator-like protein 1(BMAL1),period circadian protein 2(PER2),retinoic acid receptor-related orphan receptor γ(RORγ),and nuclear receptor subfamily 1 member D(NR1D1)protein were detected by the Western blot at concentrations that did not affect normal cell viability.The oscillations of the cellular circadian gene BMAL1 were detected by the LumiCycle experiment,and the rhythmic expression of other circadian genes BMAL1,PER2,NR1D1 and CRY2 was determined through serum shock assay.Results AT higher than 100 nmol/L induced cytotoxicity to HepG2 cells.In the context of avoiding the influence of AT on the activity of normal cells,a concentration of AT 100 nmol/L was selected for the follow-up experiment.After the stimulation of AT,the expression level of BMAL1 and PER2 protein decreased(P＜0.05).The LumiCycle experiment results showed that the phase in the experimental group(100 nmol/L of AT)lagged behind that in the control group(10 nmol/L)by 2.696 h(P＜0.01).The serum shock experiment showed that the expression of PER2 gene in the experimental group was significantly down-regulated compared to that in the control group(P＜0.05).Conclusion AT can regulate the expression of peripheral circadian clock proteins and genes,resulting in delayed expression of the core circadian clock gene BMAL1 and significant down-regulation of PER2 gene and protein expression.It has potential applications in the treatment of circadian clock disorders,sleep disorders,and other related diseases.
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