Effects of propofol on mammalian central and peripheral circadian clocks CURRENT STATUS: POSTED

Background Circadian rhythm has a significant correlation with the occurrence and development of many diseases. Studies have shown that the anesthetic agent propofol can alter the rhythms of body temperature and activity in rats. Methods U2OS cells and ex vivo liver tissue were treated with different concentration of propofol, followed by recording the oscillation of the circadian clock. And two month-old mice were exposed to propofol (10mg/kg and 20mg/kg) or vehicle, detecting the expression of the clock genes. Results The results showed that propofol reduced the amplitude and lengthened the period of Per2 oscillation. Treatment with 10 mg/kg propofol significantly increased the expression of D-box binding PAR BZIP transcription factor (Dbp) and clock circadian regulator (Clock) in the liver. Treatment with 20 mg/kg propofol significantly decreased expression of cryptochrome circadian regulator 1 (Cry1), Dbp, nuclear receptor subfamily 1 group D member 1 (Nr1d1) and Clock and significantly increased the hypoxia signaling pathway genes hypoxia inducible factor 1 subunit alpha (Hif-1α), Egl-9 family hypoxia inducible factors Egln1, Egln2 and Egln3 in the hypothalamus. Conclusion The above results indicate that the general anesthetic propofol can change the circadian clock of ex vivo and in vivo mammalian liver tissues.

(Clock) in the liver. Treatment with 20 mg/kg propofol significantly decreased expression of cryptochrome circadian regulator 1 (Cry1), Dbp, nuclear receptor subfamily 1 group D member 1 (Nr1d1) and Clock and significantly increased the hypoxia signaling pathway genes hypoxia inducible factor 1 subunit alpha (Hif-1α), Egl-9 family hypoxia inducible factors Egln1, Egln2 and Egln3 in the hypothalamus. Conclusion The above results indicate that the general anesthetic propofol can change the circadian clock of ex vivo and in vivo mammalian liver tissues.

Background
The clinical data showed that in patients with surgical aortic valve replacement, the incidence of postoperative complications in patients undergoing surgery in the morning was 2 times higher than that for patients undergoing surgery in the afternoon [1]. Therefore, it is proposed that the difference in postoperative complications of surgical aortic valve replacement is related to the circadian clock.
Almost all living organisms on the earth have a circadian clock of approximately 24 h, which is evolved to adapt to the earth rotation period [2,3]. Except for regulated by the environment factors like light and temperature, the circadian clock is controlled by clock genes to maintain its stability.
The expression of clock genes is represented by a sinusoidal oscillation; therefore, period, amplitude and phase are used to describe circadian clock [4]. The core clock is composed of a transcriptiontranslation feedback loops engaged with positive (CLOCK and BMAL1) and negative ( CRYPTOCHROME and PERIOD) factors [5]. In mammals, the central pacemaker in the hypothalamic suprachiasmatic 4 nuclei (SCN) coordinates peripheral tissue clocks to perform physiological functions [6].
While the central clock in SCN is stable, which can be regulated by light and some metabolites [7], the peripheral clock is relatively independent of SCN and can be regulated by more factors, like temperature, chemicals and feeding. A research has shown that dexamethasone does not affect the expression of clock genes in the SCN but that it can change the phase of the clock genes in liver, kidney, and heart [8] .Clinical study mentioned above indicated that the cause of difference in postoperative complications may be related to the fluctuant expression of the intrinsic clock gene Rev-Erbα(Nr1d1) in the morning and afternoon [1]. However, it did not consider the effects of various drugs and external environmental factors on circadian clock during the surgery, additionally, the difference between central and peripheral clock was not taken into consideration. During the surgery, general anesthesia is an important and essential factor, which may influence the circadian clock.
As one of the most commonly used anesthetics in clinical treatment, propofol is used not only in painless gastrointestinal endoscopy as a low dose but in continuous intraoperative perfusion as well.
And propofol can act on both the central nervous system and peripheral tissues. The circadian clock of peripheral tissues is regulated by the SCN and self-regulated as well [9]. For peripheral tissues, selfregulated circadian clock is most important for the maintenance of the function [10]. Furthermore, after mice were anesthetized with propofol, their respiratory rate slowed, which may affect the hypoxia signaling pathway. Previous studies have confirmed that the reciprocal regulation between the circadian clock and hypoxia signaling at the genome level in mammals [11].
Summarizing the current research, it was found that propofol can alter the body temperature rhythm and locomotor activity rhythm in rats [12,13]. It was also reported that 6 h after being anesthetized with propofol, the expression of D-box binding PAR BZIP transcription factor (Dbp) in the whole brain of rats was increased and the expression of period 2 (Per2) was reduced [14]. It's reported the changes in the expression of clock genes in whole brain, but it did not clarify the change in the central and peripheral circadian clock, nor on the hypoxia signaling pathway, nor did it study the effect of different propofol concentrations on the circadian clock. In peripheral tissues, liver is an important metabolic organ, and many basic metabolic pathways in liver (such as glycolysis, fatty acid 5 metabolism, and cholesterol synthesis) are also rhythmic [10,15].

Anestheic exposure
Eight-week old male C57BL/6J mice were randomly allocated to 10 mg/kg propofol (n=4), 20 mg/kg propofol (n=4) or vehicle (saline, n=8) treatment groups. Tail vein injection was performed at ZT10, and the tail vein injection with the same volume of normal saline was given to the mice in the control group. The anesthetic level of loss of righting reflex (LORR) was determined in mice administered propofol. All animals were sacrificed by cervical dislocation.

3.
Monitoring the circadian rhythm of U2OS cells 3.5. Tecan microplate reader analysis: U2OS cells was added to a 96-well plate. The medium in each well was aspirated, and 100 μL of XM was added to each well. After sealing with a membrane, the plate was placed in a Tecan.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Mice were sacrificed by cervical dislocation. The hypothalamus (including the SCN) and the liver were collected. RNA was extracted from the obtained tissues using TRIzol. The extracted RNA was reverse transcribed using 5× PrimeScript TM RT Master Mix. PCR was conducted using a CFX96 real-time PCR system (95℃ for 3 min, 95℃ for 3 s, and 60℃ for 30 s (40 cycles)) to detect the expression of clock genes (the primer sequences are shown in supplementary data).

Monitoring the circadian rhythm in ex vivo liver tissue of mice
A total of 1,200 μL of XM medium was added to a 3.5 cm culture dish, and a tissue culture membrane (pore size, 0.45 μm) was placed in the dish (avoiding bubbles). Mice were sacrificed by cervical dislocation. A small piece of liver tissue was from the edge of the liver and transferred onto the membrane. The dish was sealed with a piece of glass and placed in a LumiCycle. 6.

Data analysis
The recorded data were analyzed using LumiCycle Analysis software. The results from the Tecan 7 microplate reader were exported using Excel. Statistical significance was determined using two-sided Student's tests, and differences with P<0.05 were considered significantly different (*p<0.05, **p<0.01). All data were plotted with GraphPad Prism 7.

Propofol changed the amplitude of Per2 in U2OS cells
To verify whether propofol has an effect on circadian clock and to determine the effect of different concentrations of propofol on circadian clock, human myeloma U2OS cells, which are the most widely used in circadian clock studies, were used. U2OS cells after the stable transfection of Per2:dLuc were used as a reporter system [16], and the dynamic change in the fluorescence signal was used to analyze the changes in the oscillation of Per2 after propofol was applied to the U2OS cells. The U2OS cells were seeded into a 96-well plate. Propofol was added to XM medium to prepare a mother liquor in the U2OS cells in the control group and the experimental group have no significant changes ( Figure   1D).

2.
Propofol at a high concentration prolonged the period of Per2 in ex vivo liver tissue After determining that propofol affects the oscillation of Per2 in cells, to investigate whether propofol 8 directly affects the circadian clock of ex vivo liver tissue without the SCN, liver tissue from PER2::LUC transgenic mice were placed in a LumiCycle to record the oscillation of Per2. 10 μM propofol was added to the medium at the peak and trough of Per2. The treated and untreated ex vivo liver tissues was continuously recorded in the LumiCycle . The medium was replaced with untreated (no propofol) medium after 3-4 days, and the recording was continued. The most significant effect occurred when propofol was added at the peak of Per2; 10 μM propofol lengthened the period of Per2 in the ex vivo liver tissue by 5 h (Figure 2A). Treatment when the trough of Per2 lengthened the period of Per2 in the ex vivo liver tissue by 2 h (Figure 2B). Statistical analysis of all data showed that propofol treatment at the peak and trough of Per2 could lengthen the period of Per2 in ex vivo liver tissue ( Figure 2C); no significant effect on amplitude was observed ( Figure 2D).

3.Propofol changed the expression of clock genes in in vivo liver tissue and hypothalamic tissue
To investigate the effect of propofol on the circadian clock of in vivo central tissue and peripheral liver tissue, the expression of clock genes were detected in samples collected from mice after they were anesthetized with propofol. The circadian clock of hypothalamic tissue in mice is usually used as an indicator of central circadian clock. According to Figure 2, the effect of propofol on the circadian clock of ex vivo liver tissue was the most significant at the peak of Per2. Because the effect of propofol on mice was the most significant 2 h after the treatment was applied, the expression of Per2 in mouse liver reached the peak point at zeitgeber time (ZT)12; thus, tail vein injection of propofol at doses of 10 mg/kg (n=4) or 20 mg/kg (n=4) was performed at ZT10. The anesthetic level of loss of righting reflex (LORR) was determined in mice administered propofol [17], and the tail vein injection with the same volume of normal saline was given to the mice (n=4) in the control group. At ZT12, liver and hypothalamus samples were collected to detect the expression of clock genes by qRT-PCR (including Per1, Per2, Dbp, nuclear receptor subfamily 1 group D member 1 (Nr1d1), brain and muscle ARNT-like 1 (Bmal1), clock circadian regulator (Clock), and cryptochrome circadian regulator 1 (Cry1)) and hypoxia inducible factor 1 subunit alpha (Hif-1α) and its response genes (including vascular endothelial growth factor A (Vegfa), Egl-9 family hypoxia inducible factor 1 (Egln1), Egln2, and Egln3).

9
The results showed that the expression of Dbp and Clock in liver tissue was significantly increased with 10 mg/kg propofol ( Figure 3A), while the expression of clock genes in the hypothalamus did not significantly change ( Figure 3B). There was no significant change in the expression of clock genes in liver tissue with 20 mg/kg propofol ( Figure 3C). The expression levels of the clock genes Cry1, Dbp, Nr1d1 and Clock in the hypothalamus were significantly decreased, while the expression levels of the hypoxia-related genes Hif-1α, Egln1, Egln2, and Egln3 were significantly increased ( Figure 3D).

Discussion
In this study, cells harboring the Per2 promoter-driven luciferase reporter were first used to reveal Egln2 and Egln3 in the hypothalamus. Studies have confirmed that Hif-1α binds directly to the promoter region of Per2 to affect its expression [11]; therefore, it is hypothesized that the treatment with propofol at a high concentration for 2 h may affect hypothalamic clock genes of mice by activating the hypoxia signaling pathway, which can affect the expression of Per2 and affect the entire feedback loop, thereby significantly reducing the expression of a large number of clock genes.
The reduction in hypothalamic clock genes will affect clock-controlled genes, which will, in turn, affect the metabolism and periodic activities of the body [19].