Influence of age, gender characteristics, chronotype on the expression of core clock genes Per1 , Clock , Bmal1 and Cry1 in buccal epithelium

The purpose of the study is to determine the expression of the core clock genes in buccal epithelial cells of healthy people with different chronotypes. Materials and methods . Fourteen healthy volunteers with a healthy periodontium and oral mucosa (7 women and 7 men) were selected for participation in the trial. The buccal epithelium sampling was performed at 07:00 am and 07:00 pm in one day by cytological brush. The surveyed patients were examined chronotypically using the Horn-Ostberg test. The determination of the mRNA expression of the Per1, Clock, Bmal1, Cry1 genes was performed by quantitative real-time PCR. Statistical analysis was performed using two-way analysis of variance followed by Bonferroni post hoc tests. Results . Per1 expression was higher in the morning, regardless of chro-notype, age, and gender. The expression of the Clock demonstrated the prevalence of the evening in both chronotypes, in both men and women. Bmal1 was better expressed in the evening, regardless of age, gender, and chronotype. The expression of Cry1 did not show statistically significant differences between the indicators. Con-clusions . The evening expression of Clock was higher in people with the evening chronotype than in people with the morning chronotype. The chronotype did not show any effect on the expression of Per1, Bmal1, and Cry1 . Age and sex did not show any effect on the expression of the core clock genes.


INTRODUCTION
Daily endogenous rhythms are triggered by mechanisms called circadian clock.A circadian clock, or circadian oscillator, is a biochemical oscillator that cycles with a stable waves and is synchronized with 24 hours (the earth's current day).In most living things, internally synchronized circadian clocks allow the organism to anticipate daily environmental changes that correspond to the day-night cycle and adjust its biology and behavior accordingly.
Robust circadian rhythms in physiological functions and behaviors are conserved across all organisms, from cyanobacteria to humans.Cyanobacteria were the first prokaryotes reported to have the circadian clock regulated by a cluster of three genes: kaiA, kaiB, and kaiC, wich are responsible for fundamental physiological processes such as the regulation of nitrogen fixation, cell division, and photosynthesis.More than 20 circadian clock-related genes have been identified in Arabidopsis, with homologs of these present in other plants, including crops.Such circadian clock genes as CCA1 (Circadian Clock-Associated1), LHY (Late Elongated Hypocotyl), GI (Gigantea), and others are involved in internal metabolic and hormonal signals, ranging from the control of metabolism, photosynthesis, growth, and development.The discovery of genes such as Per (Period), Tim (Timeless), and DBT (Doubletine) in Drosophila and later in mice contributed to the functioning of the circadian clock in humans (Dodd et al., 2015;Panter et al., 2019).
The human biological clock plays a fundamental role in regulating the rhythmic course of all physiological processes occurring in the body, including organismal, organ, and cellular ones.Body temperature, pulse rate, respiration, blood pressure, brain activity, hormone production, cell regeneration, and other processes are subordinated to circadian rhythm (Jagannath et al., 2017;Douma et al., 2018;Nirvani et al., 2018;Farshadi et al., 2020).
The dynamics of daily biorhythms parameters is called chronotype.It referrs to behavioral patterns or manifestations based on biological processes controlled by circadian rhythms.Based on the intrinsic circadian rhythm of the individual, individuals differ in his preferred time of sleep and activity.The tradition of dividing people by the type of their peak of activity on owls (evening peak) and larks (morning peak) arose in 1939 (Potter et al., 2016;Chi-Castañeda et al., 2018;Montaruli et al., 2021).
Clock genes, as key regulators of physiological function and circadian clock dysfunction have been linked to various diseases and multiple morbidities such as diabetes mellitus, obesity, thrombosis, neurodegenerative diseases, cardiovascular disease, cancer, psychiatric disorders, autoimmune and inflammatory diseases, and sleep disorders.Perturbation of the internal clock system has been found to be comorbid with major oral, head and neck pathologies, such as oral cancer and Sjögren syndrome (Papagerakis et al., 2014;Adeola et al., 2019).
Circadian regulation of normal physiological and metabolic processes comes from fluctuations in the expression of core genes of the clock and the proteins they encode and differ in each individual organ or tissue.Key genes include Per (Per), Cry (Cryptochrome), Bmal1 (Brain Muscle Arnt-Like Protein-1) and Clock (Circle Output Kaput) (Andreani et al., 2015;Hurley et al., 2016;Panda, 2016;Anna et al., 2021).
Analysis of scientific data has revealed that the circadian clock is the main determinant of cellular homeostasis of the maxillofacial region, which regulates the proliferation and differentiation of salivary glands, odontoblastst cells and oral cavity epithelium; influences on the differentiation of ameloblasts and mineralization of dental tissue, influences on pulp sensibility.Some publications also suggest a correlation between circadian periodicity, cross-striations, and incremental lines in histological tooth sections.The circadian rhythm of gene expression was detected in basal cells of the oral epithelium, including the palatal and connective epithelium, remnants of the Malassez epithelium; in ameloblasts and odontoblast cells, tooth pulp cells, periodontal dental ligament cells, osteoblasts, and alveolar bone osteoblasts (Lech et al., 2016;Panda, 2016;Adeola et al., 2019;Janjic et al., 2019).
For example, Bmal1, Cry2, and Per2 influence bone mass and bone volume trough regulation of osteoclast parameters and differentiation, playing an important role during enamel formation (Xu et al., 2016).However, the specific functions of the respective peripheral clocks in oral tissues and the mechanisms that are implied on the way to the fulfillment of such a function or behavior are still widely unknown.
Recently, there are limited data on the correlation between chronotype and mRNA expression of the core genes of the clock Per1, Clock, Bmal and Cry1 in human buccal epithelial cells at different times of the day.
The purpose of the study is to determine the expression of core clock genes in buccal epithelial cells of healthy people with different chronotypes.

MATERIALS AND METHODS
Fourteen healthy volunteers with a healthy periodontium and oral mucosa (7 women and 7 men) were selected for participation in the trial.All participants were fully informed about the nature, potential risks and benefits of their participation in the study and signed an inform consent form.The study protocol was reviewed and approved by the ethical committee of Poltava State Medical University (№ 188, 25.11.2020).Research was carried out in full accordance with the Helsinki Declaration of 1975, as revised in 2013.

Inclusion and Exclusion Criteria
Inclusion criteria were as follows: 1) the age 36-45 years (middle adult age group) (Tsyigankov et al., 2009); 2) good general health; 3) healthy periodontium and oral mucosa; 4) written informed consent form.Exclusion criteria were as follows: 1) antibiotics or anti-inflammatory medications were in the preceding 3 months; 2) periodontal therapy within the previous 6 months; 3) pregnancy and breast feeding; 4) the presence of sever uncontrolled (decompensated) internal organ disease, or neuropsychiatric disorders; 5) the presence of other conditions that determined the participant's inability to understand the nature and possible consequences of the study.

The chronotype
The chronotype was identified by the Horn-Ostberg test that assessed belonging to a certain type of biorhythm by the sum of scored points.It is a self-assessment questionnaire, whose main purpose is to measure whether a person's circadian rhythm produces peak alertness in the morning, in the evening, or between.The Horn-Ostberg assessment consists of 23 multiple-choice questions, each having four or five response options.The responses to the questions are combined to form a composite score that indicates the degree to which the respondent favors morning versus evening (Reiter et al., 2021).The study included only people of the morning chronotype and the evening chronotype.

Sampling of the buccal epithelium
The buccal epithelium sampling was performed at 07:00 am and 07:00 pm in one day in the autumn-winter period.Sampling was performed early in the morning and late in the evening, i.e., at the two extreme points of the 24-hour cycle, in order to record the peak expression of morning and evening genes and to obtain the statistical significance of the differences between the expression levels.The study was carried out by the minimally invasive method using a cytological brush with a narrow plastic bristle and a blunt end.For a tighter contact of the cytological brush with the buccal mucosa, rotational movements were performed in place for 10 s in one direction with pressure on the buccal mucosa.The brush was then removed and immediately immersed in RNA stabilizing solution (RNAlater™ Stabilization Solution Invitrogen™ (ThermoFisher, USA) at room temperature and frozen at -80°C for further usage.Before RNA isolation, the samples were kept at -20°C overnight (Gu et al., 2021).

Quantification of the expression of the Per1, Clock, Bmal1, Cry1 genes in the buccal epithelium
General RNA was isolated from biological samples using a set of reagents for isolation and purification of RNA with a magnetic sorbent (UkrGenTech, Kyiv, Ukraine).A set of reagents for the reverse transcription reaction (UkrGenTech, Kyiv, Ukraine) was used to obtain cDNA.For each reaction, we used: 5×PCR mixture containing 5 mm deoxynucleotide triphosphate, 2.5 mm MgCl2 in appropriate buffer solution, random hexameric primer at a final concentration of 20pM, reverse transcriptase ML-RT at a final concentration of 100U, deionized water free of RNases.Reverse transcription was
PCR was carried out under the following conditions: the first cycle -95°C -300 s and the next 45 cycles: 55-60°C -40 sec; 95°C -15 sec.The GAPDH gene was used as a reference gene.The relative 2 -ΔCT method was used for data analysis (Biassoni et al., 2014;Fraga et al., 2014).

Statistical analysis
Statistical analysis «SPSS for Windows» 13.0 was used by means of two-way ANOVA followed by Bonferroni post-hoc tests.P values of <0.05 were considered statistically significant in all analyses (MacFarland, 2012).
The null hypothesis tested was that chronotype, age, and gender had no influence on expression of core genes of the clock in buccal epithelium cells of healthy volunteers.

RESULTS
The study population consisted of 14 healthy volunteers.These participants were divided into two chronotypes -morning (n=8) and evening (n=6) due to the results of the Horn-Ostberg test.
The results of the expression level determination of these genes depending on the chronotype are represented in Table 2.
Per1 was determined to be better expressed early in the morning than late in the evening, regardless of chronotype (P 1 <0.05).The difference between the indicators of morning and evening expression of Per1 in the two chronotype groups was not statistically significant.The level Clock expression of was higher in the evening in both "larks" and "owls" (P 1 <0.05).The difference between the indicators of the morning expression of Clock in the two groups was not statistically significant.The difference between the indicators of the Clock expression at 07:00 pm in the two chronotype groups was statistically significant (P 2 <0.05).The expression of Bmal1 in the two groups was found to be higher in the evening than in the morning (P 1 <0.001).There was no statistically significant difference between indicators of morning and evening expression of Bmal1 in the two groups.Cry1 demonstrated the same night prevalence in both chronotypes, but was not statistically significant.Additionally, there were no statistically significant differences between indicators of morning and evening Cry1 expression in two chronotype groups.
Thus, the results obtained indicated the statistically significant morning prevalence of Per1 expression and the evening prevalence of Clock and Bmal1 expressions in both chronotype groups of the study population.In addition, the difference between the indicators of evening expression of Clock in the two chronotype groups was statistically significant.The expression of Per1, Bmal1, and Cry1 was not demonstrated statistically significant difference in the indicators depending on chronotype.
The results of the determination the gene expression level according toage are presented in Table 3.
The expression of Per1 was found to be higher in the morning than in the evening in both age groups (P 1 <0.001).There was no statistically significant difference between indicators of morning and evening Per1 expression in the two groups.The level of Clock expression was higher at 07:00 pm among participants of Thus, the results obtained indicated the statistically significant morning prevalence of Per1 expression and the evening prevalence of Bmal1 expression in both age groups of the study population.The expression of Per1, Clock, Bmal1 and Cry1 did not show statistically significant differences in the indicators depending on age.
The gender dependencies of the expression level of these genes are illustrated in Table 4.
The level of Per1 expression was higher early in the morning in both women and men (P 1 <0.05).There was no statistically significant difference between the indicators of morning and evening expression of Per1 in the two gender groups.The Clock was expressed better in the evening than in the morning in both men and women (P 1 <0.05).There was no statistically significant difference between the indicators of morning and evening expression of the Clock in the two gender groups.Bmal1 demonstrated the prevalence of expression in the evening, regardless of gender (P 1 <0.05).The difference between the indicators of morning and evening expression of Bmal1 in two gender groups was not statistically significant.The level of Cry1 expression was higher in the evening in women and almost equal at 07:00 am and 07:00 pm in men, but was not statistically significant.There was no statistically significant difference between the indicators of morning and evening expression of Cry1 in two age groups.
Thus, the results obtained indicated the statistically significant morning prevalence of Per1 expression and the evening prevalence of Clock and Bmal1 expressions in both gender groups of the study population.The expression of Per1, Clock, Bmal1, and Cry1 did not demonstrated statistically significant differences between the indicators depending on gender.
Furthermore, the analysis of scientific literature showed the presence of a large number of articles that have studied the expression of core clocl genes in human oral squamous cell carcinoma cells (Li et al., 2016;Zhao et al., 2016;Qin et al., 2017;Yang et al., 2020;Gong et al., 2021).In contrast, there are a relatively few scientific papers that have studied the expression of core clock genes in the healthy oral mucosa depending on chronotype.We revealed the expression of the Per1, Сlock, Bmal1 and Cry1 genes in the healthy oral mucosa in patients without any somatic or dental disease and traced the level of expression of each individual gene depending on chronotype, age, and gender.
As in other similar scientific works, we had a small sample size, which limited the assessment of the correlation between gene expression and population characteristics (Bjarnason et al., 2001;Bjarnason et al., 2007;Zieker et al., 2010;Kurbatova et al., 2014;Cho et al., 2016;Gu et al., 2021;Sato et al., 2021).Bjarnason G.A. and others (Bjarnason et al., 2001;Bjarnason et al., 2007), Zieker and others (Zieker et al., 2010), Cho and others (Cho et al., 2016) and Gu and others (Gu et al., 2021), who investigated the expression of core genes of the clock in the healthy oral mucosa, the sampling were carried out every 4 hours within 24 hours.In our study, we performed the sampling at 07:00 am and 07:00 pm, in order to record the peak expression of morning and evening genes and to obtain the statistical significance of the differences between the expression level.
Analysis of the study results revealed statistically significant time differences in the expression of the core clock genes Per1, Clock, Bmal1 and Cry1.
Previously published data demonstrated that Per1expression in the oral mucosa and in human skin is maximal in the morning and gradually decreases during the day (Bjarnason et al., 2001;Gu et al., 2021).Gu and others (Gu et al., 2021) in their study determined that eight participants had peak times of Per1 expression between 10:30 am and 02:10 pm and three participants had peak times at 6:30 am, 7:00 am, and 9:00 am.The average peak times of of Per1' RNA expression were approximately 10:45 am.It was also found that a later Per1 peak time was found in the elderly, but the small number of samples analyzed did not reach statistical significance.Regarding the chronotype, they did not find correlations between gene expression peak times and chronotype (Gu et al., 2021).
In our study, it was found that Per1 exhibits the same rhythmic expression that peaks early in the morning (at 07:00 am) and decreases in the evening, regardless of the chronotype, age, and gender.The chronotype, age, and gender did not have influence on Per1 expression, which correlates with previous published data.
According to the study of Bjarnason and others the Clock expression was not rhythmic (Bjarnason et al., 2001).On the contrary, Zhanfeng and others in their study determined that the expression peak of Clock was at 12:00 pm (Zhanfeng et al., 2019).But in our study Clock expression demonstrated the evening prevalence in both morning and evening chronotypes, in both men and women.The evening expression of the Clock was higher among the population with evening chronotype than with morning chronotype.Sex and age did not influence Clock expression.
In a previously published study of Bjarnason and others it was found that Bmal1 shows a rhythmic expression, peaking at night (late activity) (Bjarnason et al., 2001).On the contrary, Zieker and others in their study indicated that maximum expression of Bmal1 was observed at 06:00 am (Zieker et al., 2010).But according to the study Zhanfeng and others, the expression peak of Bmal1 was at 12:00 pm (Zhanfeng et al., 2019).
In our study, Bmal1 expression showed a maximal level in the evening (at 07:00 pm) and gradual decrease in early in the morning (at 07:00 am), regardless of chronotype, gender, and age.Chronological, gender, and age did not influence Bmal1 expression.
Previous analyses of Cry1 detected peak gene expression in oral mucosa during the afternoon (Bjarnason et al., 2001;Zieker et al., 2010).In the study of Bjarnason and others, it was found that Cry1 demonstrates peak of expression at late afternoon (Bjarnason et al., 2001).In the study of Zieker and others, it was observed that Cry1 shows peak time of expression at 06:00 pm (Zieker et al., 2010).
In our study, the results obtained about Cry1 expression in different chronotype groups, different age groups, and among men and women were not statistically significant.Chronotype, sex, and age did not influence Cry1 expression.
We can hypothesize that the buccal epithelium might be regulated not only by the dark-light cycle but by the direct influence of meal and fluid on the oral mucosa (Fig. 1).
Our study has several limitations.The small sample size of this study limited the statistical power to find genes with circadian rhythmic expression and to assess correlations between genes expression and population characteristics.Future studies with a larger sample size are needed to confirm our findings.The sampling was carried out exclusively at 07:00 am and 07:00 pm on one day in the autumn-winter period.Therefore, we were limited in time and were unable to determine the expression level of core genes of the clock at other times of the day or in the other season of the year.Additionally, we did not take into account the influence of such factors as lifestyle, diet, intensity of physical activity, and sleep quality.
The prognosis for further investigation is to study the correlation between the expression of core clock genes and mealtime, as well as with the concentrations of rhythmically expressed hormones such as melatonin.
The results of the study on the expression of Per1, Cry1, Bmal1, Clock in the healthy oral mucosa can also be used in patients with periodontitis.In the future, we plan to study the expression of core genes of the clock Per1, Cry1, Clock and Bmal1 in buccal epithelial cells of patients with periodontitis.

CONCLUSIONS
The data obtained indicate that the chronotype has its influence on the evening expression of Clock, which was higher in people with the evening chronotype than in people with the morning chronotype.The addition to the morning or evening chronotype did not show any effect on the expression of the core clock genes Per1, Bmall and Cry1.The chronotype does not have significant effect on the circadian regulation of physiological and metabolic processes in the buccal epithelium.Age and sex did not show any effect on the expression of core clock genes.

Figure 1. Schematic illustration of the main mechanisms of action in both the central nervous system and peripheral clocks
The central pacemaker of the circadian system is located in the hypothalamic suprachiasmatic nucleus (SCN).Oscillating neurons and astrocytes are integrated as a single circadian unit and produce a coordinated circadian signal.The SCN forwards the rhythmic signal trough the hypothalamus-pituitary-adrenal (HPA) axis and the autonomic nervous system (Wang et al., 2021).The mammalian circadian clock network at the molecular level consists of the transcription of genes that contain E-box elements in their promoters, such as Per1-3, Cry1/2, Rorα, Rev-erbα, and Dbp.The Per and Cry proteins translocate back into the nucleus and repress the activity of the Bmal1/Clock complex, which subsequently inhibits their own expression.Rorα and Rev-erbα fine-tune the expression of Bmal1 and Nfil3, trough activation and repression, respectively.The stability and nuclear translocation of circadian clock proteins are modulated by post-transcriptional modifications (PTMs).Cricadian clocks also control the rhythmic expression of numerous clock-contolled genes (CCGs) and biological processes (Wang et al., 2021).Due to the complex interaction between external Zeitgeber and internal circadian rhythms, the greatest beneficial effects of entrainment on circadian function are seen when food intake coincides with the activity phase (Salgado-Delgado et al., 2013;Schilperoort et al., 2019), while an inhibitory effect occurs when food is taken during the rest phase (Yasumoto et al., 2016).