Bavachalcone Enhances RORα Expression, Controls Bmal1 Circadian Transcription, and Depresses Cellular Senescence in Human Endothelial Cells

The circadian clock regulates many aspects of (patho)physiology in the central nervous system and in the peripheral tissues. RAR-related orphan receptor α (RORα), an orphan nuclear receptor, is involved in circadian rhythm regulation, including regulation of cardiovascular function. Bavachalcone, a prenylchalcone, is a major bioactive chalcone isolated from Psoralea corylifolia. This natural ingredient activated RORα1 luciferase reporter activity on drug screening. In addition, bavachalcone induced RORα1 expression in mRNA and protein levels in a dose-dependent manner and enhanced the circadian amplitude of Bmal1 mRNA expression after serum shock. Moreover, bavachalcone suppressed senescence in human endothelial cells and mRNA expression of p16ink4a (a marker of replicative senescence) and IL-1α (a proinflammatory cytokine of the senescence-associated secretory phenotype). These inhibitory effects were partially reversed by the RORα inhibitor VPR-66. Our results demonstrate that bavachalcone, as a natural medicine ingredient, has a pharmacological function in regulating RORα1.


Introduction
The circadian clock not only controls the central nervous system, but also regulates many aspects of (patho)physiology in the peripheral tissues, including cardiovascular function. ROR , an orphan nuclear receptor, is involved in the circadian rhythm regulation. ROR 1 and ROR 4, not ROR 2 and ROR 3, are the predominant forms of ROR expression in vascular endothelial cells, smooth muscle cells, and fibroblasts [1,2]. ROR directly activates transcription of the circadian gene Bmal1 through conserved ROR response elements [3,4]. Homozygous Rora (sg/sg) mutant mice exhibited an enhanced susceptibility to atherosclerosis and hypoalphalipoproteinemia [5]. In addition, ROR has been identified as a regulator of human apo A-1, A-5, and C-3 gene expression [5][6][7]. ROR not only suppressed TNF--induced VCAM-1 and ICAM-1 expression in human endothelial cells [8], but also reduced oxidative stress through the induction of SOD2 and GPx1 expression [9]. Moreover, reduced circadian clock expression was closely associated with age in genetic hypertensive rats [10].
Bavachalcone, a prenylchalcone, is a major bioactive chalcone isolated from Psoralea corylifolia; it exhibits many biological activities. For example, Lee et al. reported dosedependent reduction of inducible nitric oxide synthase activity in activated microglial cells treated with bavachalcone [11]. In osteoclasts treated with bavachalcone, Park et al. observed reduced activation of MEK, ERK, and Akt and suppression of osteoclast differentiation through NFATc1 [12]. In addition, a strong inhibitory effect of bavachalcone against 2 important isoforms of UDP-glucuronosyltransferases (UGT), UGT1A1, and UGT1A7, was observed [13]. In a previous study, we found that bavachalcone activates AMPK kinase activity, promotes the expression of MnSOD, and reduces mitochondrial superoxide anion [14]. On drug screening, bavachalcone enhances ROR 1 expression. Therefore, this study investigated the pharmacological effects of bavachalcone on ROR 1.

Cell Cultures.
Human umbilical vein endothelial cells (HUVECs) (CRL-1730) were ordered from ATCC (Manassas, USA), and human embryonic kidney 293 (HEK-293) cells were purchased from the Institute of the Chinese Academy of Medical Sciences Cell Bank (Shanghai, China). HUVECs were maintained in a 37 ∘ C incubator supplemented with 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. The medium was changed every 2 days and the cells were passaged with trypsin-EDTA. The population-doubling level (PDL) was calculated after each passage as PDL = (log 10 − log 10 )/ log 10 2 , where is the number of cells counted at the end of the passage and is the number of cells seeded. Cumulative population doubling was calculated as the sum of all PDL the changes [15].

Serum Shock Procedures.
Serum shock was performed as follows: approximately 1 × 10 6 cells per 60-mm petri dish were plated 1 day prior to the experiment so that the cells would be approximately 80% confluent on the day of the experiment. The medium was exchanged with 0.3% serum DMEM medium for 16 h. At time = 0, the medium was exchanged with serum-rich DMEM containing 50% new born bovine serum with or without 20 mol/L bavachalcone; after 2 h, this medium was replaced with 0.3% serum DMEM with or without 20 mol/L bavachalcone. At the indicated times, the petri dishes were washed twice with ice-cold phosphate buffered saline (PBS) and frozen on a liquid nitrogen layer or stored at −70 ∘ C until the extraction of whole cell RNA.

ROR 1 Reporter Luciferase Plasmid Construct and Assay.
The thymidine kinase (−83 to +91) plus 3× RORE [TCG ACT CGT ATA ACT AGG TCA AGC GCT G] sequence was generated by inserting the corresponding annealed oligonucleotides into the Luc pGL3-basic plasmid. HEK293 cells were cultured in 24-well plates at a density of 2 × 10 5 cells/well. When the cell density reached 70%-80%, the cells were used for transfection. In each well, 0.8 g of a reporter vector of the pGL3 ROR reporter firefly luciferase gene and 0.016 g of a reporter pRL-SV40 plasmid of the Renilla luciferase gene as normalization control were transfected and diluted with the FuGENE HD transfection reagent in the Opti-MEM transfection medium. The medium was replaced with normal DMEM 6 h after transfection. After incubation with bavachalcone (Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China) for 16 h, the activity of the luciferase reporter gene was assayed using the dual-luciferase reporter 1000 assay system and detected using a Varioskan Flash microplate spectrophotometer (Thermo Scientific, USA).

Statistical
Analysis. Data are expressed as the mean ± standard deviation (SD). Paired t-test analysis and one-way ANOVA were used to compare the differences between and within the groups. All statistical analyses were performed using SPSS Version 15.0 or GraphPad Prism 5. P value less than 0.05 was considered statistically significant.

Bavachalcone Induces ROR 1 Expression and Regulates Bmal1
Circadian Rhythm. In the screening of natural ingredient activity, bavachalcone exhibited characteristics that can activate ROR 1 expression (Table 1). Bavachalcone's dose effect relationship showed that it dose-dependently activated ROR 1 luciferase reporter gene activity, causing a more than 3-fold increase at a dose of 20 mol/L ( = 3, < 0.01, Figure 1). Moreover, bavachalcone dose-dependently induced ROR 1 expression in mRNA and protein levels, causing a more than 2-fold increase in mRNA expression ( = 3,     Figure 3: Bavachalcone-enlarged expression amplitude of the circadian gene Bmal1. After HUVECs were treated with 50% serum shock for 1 h, the medium was replaced with 10% serum DMEM with or without 20 mol/L bavachalcone. The mRNA expression levels at the indicated time were measured using semiquantitative (a) and real-time quantitative PCR (b) ( = 3 each). Data are expressed as the mean ± SD. * * < 0.01 versus vehicle control. < 0.01, Figure 2(a)) and a 6-fold increase in protein expression ( = 3, < 0.01, Figure 2(b)) at a dose of 20 mol/L. Furthermore, bavachalcone dynamically regulated circadian gene Bmal1 mRNA expression, a ROR 1 downstream signal. As shown in Figures 3(a) and 3(b), from 24 to 48 h after 50% serum shock, treatment with bavachalcone enhanced the circadian amplitude of Bmal1 mRNA expression on 28, 36, and 40 h, which was far wider than other time points compared with the control, with the differences being significant ( = 3, < 0.01) at each time point.

Discussion
ROR is a member of the ROR family, and its ROR function has been implicated in several pathological processes, such as cancer, autoimmune diseases, inflammation, osteoporosis, and metabolic syndrome [16]. Our results show that bavachalcone activates ROR 1 luciferase reporter activity and mRNA and protein expression. Our previous findings show that bavachalcone activates AMPK activity and induces MnSOD expression [14]. In addition, ROR 1 activates AMPK activity [17,18], and reduces oxidative stress through the induction of MnSOD and GPx1 expression [9]; our results are consistent with these reports. Numerous studies have indicated that the transcriptional activating function of ROR 1 is regulated not only by its natural ligands, such as cholesterol and cholesterol derivatives, but also by synthetic ligands [16].
Our results show that bavachalcone delays cell senescence and inhibits mRNA expression of p16 ink4a (a maker of replicative senescence [19]) and IL-1 (a proinflammatory cytokine of the senescence-associated secretory phenotype [20]). These inhibitory effects could be partially reversed by the ROR inhibitor VPR-66. The Bmal1 promoter region contains ROR response elements where the ROR families can bind [21], supporting our results that the dynamic expression of Bmal1 gene is regulated by bavachalcone-enhanced ROR 1 activity. Studies have reported that deficiency of Bmal1 increases vascular superoxide production; endothelial nitric oxide synthase uncoupling; and COX-2, Nox4, MMP2, and MMP9 expressions and underlies vascular stiffness [22][23][24]. In addition, studies have shown that deficiency of the circadian clock transcriptional factor BMAL1 impairs glucose homeostasis, exhibits premature age-associated disorders, reduces lifespan [25][26][27], and increases sensitivity to genotoxic stress [28]. A recent study reported that reactive oxygen species exhibits a wave in the circadian rhythm in the brains of wild-type mice, but not in Bmal1 deficient mice [29]. In addition, deficiency of clock gene, a Bmal1 partner, reduces lifespan and increases age-related characteristics [30].
In conclusion, through a luciferase assay, we showed that bavachalcone induced ROR 1 expressions at luciferase reporter, mRNA, and protein levels in human endothelial cells and dynamically regulated Bmal1 mRNA expression. In addition, bavachalcone suppressed replicative senescence in human endothelial cells partially through the ROR -Bmal1 pathway. Our results demonstrate that bavachalcone, a natural medicine ingredient, has a pharmacological function in regulating ROR .