Assessment of circadian rhythm protein levels in the pathogenesis of infantile colic

Introduction

The circadian rhythm (CR) is a repetitive cycle with periods of approximately 24 h that regulates various biological processes. In mammalian cells, these rhythms are generated and regulated by four main genes/proteins, including Cryptochrome (Cry1 and Cry2) and Period (Per1 and Per2), Circadian Locomotor Output Cycle Kaput (Clock), Brain-Muscle-Arnt-Like protein-1 (Bmal1) [1]. Numerous studies highlight a bidirectional relationship between the desynchronization of circadian clock genes/proteins and disease severity, where disruptions in CR can exacerbate various conditions. This may predispose individuals to many diseases, such as neurological, psychiatric, cardiometabolic, and immune disorders [2], obesity [3], insomnia, gastrointestinal system (GIS) disorders, and cancer [4], 5]. Since the link between CR disruption and diseases has been recognized, the treatment of these diseases has been reorganized [3].

The functions of the GIS are regulated by the circadian system and exhibit rhythmic changes. Colon motor activity increases in the morning; however, the secretion of gastric acid is heightened during the night. The gut microbiota in both humans and mice has been reported to display circadian oscillations [6]. Disruption of CR due to shift work or jet lag has been associated with GIS symptoms such as abdominal bloating, abdominal pain, diarrhea, and constipation [7]. Rao and Lin (2022) observed that Per1/2 double-knockout mice exhibited a loss of rhythmic muscle contractions in the intestine and rhythmic fluctuations in colon pressure [6]. CR genes/proteins could be potential cancer biomarkers [8]. Colon adenocarcinoma disease (COAD) is one of the cancer types closely associated with circadian disruption [6]. Bmal1 is reported as a tumor suppressor gene in COAD [9]. Mostafaie et al. (2009) revealed that individuals diagnosed with COAD exhibited elevated levels of the Clock gene [10]. Furthermore, several reports suggest that Cry2 and Per2 could be evaluated as potential prognostic biomarkers in COAD. Per2 has also been demonstrated to serve as a prognostic predictor in several human malignancies, including gastric, colorectal, and lung cancer [11], [12], [13]. These findings indicate that CR genes/proteins responsible for biological rhythms are directly linked to GIS physiology [14].

Disruptions in the circadian system may contribute to the development of infantile colic, a condition of unknown etiology that is likely multifactorial [15]. Besides, hormonal, neurodevelopmental, psychological, and gastrointestinal variables are among the hypothesized causes [16]. Infantile colic is a clinical syndrome observed in infants aged 0–3 months, characterized by episodes of restlessness, agitation, and excessive crying, particularly during the late afternoon and evening hours. These episodes occur for at least 3 h a day, three days a week, and persist for a minimum of 3 weeks, without any identifiable underlying cause [14].

Studies on hormones, particularly components of the sleep-wake cycle such as serotonin and melatonin, associated with intestinal motility in the pathophysiology of infantile colic, are remarkable [17], 18]. However, while the levels of these hormones have been examined in infants with colic, the role of CR genes/proteins involved in their regulation remains poorly understood. This highlights a critical gap in understanding the mechanisms underlying infantile colic. Our study aims to investigate the levels of CR core proteins in infantile colic and gain a novel perspective on the pathophysiology of the disease. In addition, we analyzed the effects of vitamin D supplementation in infants and the use of propofol and curareen during caesarean deliveries on biological rhythm disruptions. Our sample consisted of infants diagnosed with infantile colic and healthy controls. Plasma samples of the participants were collected and the levels of CR proteins Cry1, Cry2, Per1, Per2, Clock, and Bmal1 were measured. We aim for the findings to enhance understanding of the mechanisms behind infantile colic and guide the development of novel therapeutic approaches by targeting the regulation of the CR gene and protein.

Materials and methods

Results

A total of 61 participants, including the control group (n=30) and the patient group (n=31), completed the study. Information was collected from the participants’ families under the main headings of participant characteristics, maternal information, and household details before blood samples were taken (Table 1).

The mean ages of the infants in the control and patient groups were 42.23±11.81 days and 39.58±12.21 days, respectively. Of the total participants, 34.40 % were female (n=21) and 65.60 % were male (n=40). 30 percent of the control group was female (n=9) and 70 percent was male (n=21). The patient group consisted of 38.70 % females (n=12) and 61.30 % males (n=19). No significant differences were observed between the groups for variables other than nutrition, vitamin D supplementation, and nocturnal sleep, as shown in Table 1. While 73.33 % of the subjects in the control group were breastfed, this rate was 38.71 % in the patient group (p=0.01). In addition, most 0subjects with colic (45.16 %) were fed infant formula in addition to breast milk (p=0.01). While 67.7 % of the subjects in the colic group received vitamin D supplementation, this rate was 36.7 % in the control group (p=0.02). Night sleep duration was significantly longer in the control group than in the patient group (p=0.04).

Circadian rhythm protein levels

The changes in CR protein levels in plasma samples between the groups are shown in Table 2 and Figure 1. Analysis of the test statistics revealed statistically significant differences between the groups for Cry1 (U=265.5, p=0.058), Cry2 (t-test=2.188, p=0.033), Per2 (U=261, p=0.049), Clock (U=260.5, p=0.048), and Bmal1 (U=529, p=0.050) protein levels. Conversely, no statistically significant difference was observed for Per1 (U=458, p=0.920) between the comparison groups. Cry1 and Per2 levels were significantly increased in the patient group compared to the control group (p=0.058 and p=0.049, respectively). Cry2 level was decreased in the patient group compared to the control group (p=0.033). The patient group exhibited a significant increase in Clock protein levels, accompanied by a notable decrease in Bmal1 protein levels (p=0.048 and p=0.050, respectively).

Table 2:

CR protein levels in plasma samples.

Protein	Control group, ng/L Median (Q1–Q3)	Patient group, ng/L Median (Q1-Q3)	p-Value
Cry1	200 (150–313.34)	253.33 (193.33–638.89)	0.058 b
Cry2	71.25 (45.94–88.13)	54.38 (40.31–69.37)	0.033 a
Per1	140.45 (107.32–202.53)	135.38 (111.72–157.58)	0.920
Per2	329.94 (260.68–471.15)	407.54 (314.66–598.85)	0.049 a
Clock	390 (124.58–520)	495 (233.33–624.19)	0.048 a
Bmal1	791.84 (518.56–1061.79)	635.12 (468.77–1649.85)	0.050 a

1. Results are shown as median (Q1-Q3) (Q1: 25th percentile; Q3: 75th percentile). CR protein levels are expressed in ng/L., CR, circadian rhythm. Normality tests (Kolmogorov-Smirnov p=0.200, Shapiro-Wilk p=0.315) indicated that the Cry2 variable was normally distributed. Therefore, the p-value reported for Cry2 was obtained from an independent samples t-test. For all other variables, which did not exhibit normal distribution, the Mann-Whitney U test was applied. Bold values indicate statistically significant results (^ap<0.05; ^bp<0.10).

Figure 1:

Comparison of circadian rhythm proteins in control and patient groups. (A) Cry1 level in control and patient groups; (B) Cry2 level in control and patient groups; (C) Per1 level in control and patient groups; (D) Per2 level in control and patient groups; (E) clock level in control and patient groups; (F) Bmal1 level in control and patient groups. Results are shown as median (Q1-Q3) (Q1: 25th percentile; Q3: 75th percentile). Circadian rhythm protein levels are expressed in ng/L. Mann–whitney U test, *p<0.05.

Discussion

Although infantile colic is classified as a functional disorder with an unidentified etiology and multifactorial mechanisms, regulating CR may have benefits in managing these complex processes [15]. Recent studies focusing on hormones such as melatonin and serotonin suggest that infantile colic is associated with circadian misalignment; biological clock desynchronization may be linked to colic symptoms. Serotonin causes smooth muscle contraction, while melatonin induces relaxation. According to Engler et al. (2014), although the effect of serotonin in infants begins during the first weeks after birth, the melatonin cycle is regulated after the third month. As a result, melatonin cannot counteract serotonin’s contraction-inducing effects during the first three months, leading to severe intestinal contractions, particularly in the late afternoon and evening [17]. Similarly, İnce et al. (2018) observed reduced melatonin levels in colic infants, whereas Brett et al. (2024) reported elevated cortisol concentrations [15], 18]. Furthermore, Cuesta et al. (2008) demonstrated that the administration of serotonergic receptor agonists, which induce intestinal motility and smooth muscle contraction, led to a decrease in Per1 and Per2 expression [21]. In our study, Cry2 and Bmal1 levels were significantly decreased (p=0.033 and p=0.050) in the colic group, whereas Cry1, Per2, and Clock levels were significantly elevated (respectively, p=0.058, p=0.049, and p=0.049). In healthy individuals, CR genes/proteins exhibit synchronized oscillations, although variations may occur due to environmental and metabolic factors. Several studies have reported fluctuations in these genes/proteins, particularly among patients with COAD [10], 13], 22]. He et al. (2022) reported that the expression of Clock and Cry1 was elevated, whereas Bmal1, Cry2, and Per1 were diminished in patients diagnosed with COAD. COAD patients with higher Cry2 and Per2 expression levels exhibited a worse prognosis. This suggests that the Cry2 and Per2 genes/proteins may contribute to tumor formation or accelerate cancer progression in the colon. Therefore, the elevation of Cry2 and Per2 gene/protein levels can be considered potential prognostic biomarkers [13]. Recent findings suggest that suppressing the Bmal1 gene in COAD promotes cell invasion, indicating that Bmal1 acts as a tumor suppressor. Additionally, high Bmal1 expression levels have been associated with longer lifespans in COAD patients compared to lower levels [9]. Another study supporting our findings revealed that individuals with COAD exhibited elevated Clock gene expression and decreased Per1 levels [10]. Yoon et al. (2025) suggested that the increase in Clock and Cry1 expression contributes to cancer formation, while the increase in Bmal1, Per1, Per2, and Cry2 expression demonstrates a tumor-suppressing effect. Additionally, the same study reported that the loss of Bmal1 was associated with the downregulation of the Per1, Per2, and Per3 genes [8]. Based on this information, we hypothesized that the fluctuations in Per2 observed in our patient group may result from the suppression of Bmal1. In certain instances, the expression of specific genes may demonstrate opposite alterations. If one CR gene is downregulated, another functionally synchronized gene may show a compensatory increase. Failure of the compensatory mechanism can elevate the risk of developing pathological processes. Based on the common findings of our study and other studies, we suggest that the decrease in Cry2 and Bmal1 protein levels, and the increase in Cry1, Per2, and Clock protein levels, may have a potential relationship with GIS diseases.

The majority of colicky infants were fed with infant formula in addition to breast milk, whereas most infants in the healthy group were breastfed (p<0.05). Breastfeeding has been shown to improve nocturnal sleep due to its melatonin content, thereby reducing colic symptoms [23]. Infants aged 0–3 months receive melatonin through breast milk due to their physiological deficiency. An absence of melatonin from breast milk has been associated with various pathologies, particularly colic [24].

In this study, moderate positive correlations were observed between Cry1 levels and nocturnal sleep, sibling count, and vitamin D supplementation (p<0.05). Per1 and Per2 levels showed moderate positive correlations with gestational diabetes, while Per2 levels exhibited a negative correlation with age. Additionally, Clock levels demonstrated moderate correlations with sibling count and vitamin D supplementation (positive), as well as with age (negative). The Per1, Per2, and Clock genes have significant effects on sleep regulation and metabolic disease (obesity risk) [23], [24], [25], [26]. While their associations with sleep disorders and certain metabolic risk factors are well established, their direct relationships with sibling count, vitamin D supplementation, and gestational diabetes have not been clearly demonstrated in current research. To the best of our knowledge, our study provides a novel clinical contribution by investigating these variables and demonstrating moderate associations, thereby offering preliminary evidence for their potential relevance in regulating circadian protein expression. As a routine practice, vitamin D supplementation is provided to infants when the nutrient content of breast milk is insufficient [27]. In this study, we found that a higher proportion of infants in the colic group received vitamin D supplementation compared to the control group (p=0.021). Our findings indicated a positive relationship between vitamin D supplementation and increased levels of Cry1 and Clock proteins, emphasizing caution with high doses in pharmaceutical toxicology. Further research using larger data sets is required to elucidate the relationship between vitamin D, colic, and CR.

Regression analyses were conducted to assess the associations between plasma CR protein levels, including Cry1, Cry2, Per1, Per2, Clock, and Bmal1, and various natal and sociodemographic variables. None of the variables in the models had a significant effect on plasma Cry1 and Cry2 levels (p>0.05), suggesting that the analyzed variables may not play a substantial role in determining these protein levels. An increase in night-time sleep duration was found to significantly decrease Per1 and Per2 levels, whereas Per1 and Per2 levels were significantly higher in women with gestational diabetes and higher sibling counts. Clock levels were found to decrease significantly with increasing age and were significantly lower in individuals receiving vitamin D supplementation. However, although the Per1 and Clock models met statistical criteria such as model fit and coefficient significance, they failed to satisfy key regression assumptions and are therefore not considered reliable. A larger sample size, the inclusion of different variables, or alternative analytical methods may provide a more comprehensive understanding of these relationships. Only the Per2 model was both statistically significant and met all necessary assumptions. This model demonstrated that gestational diabetes, sibling count, and nocturnal sleep exert strong effects on Per2 levels. Similarly, Hoang et al. (2021) reported that alterations in Per2 contribute to variable sleep phenotypes [28]. We claim that managing gestational diabetes and increasing night-time sleep duration may reduce colic symptoms. Our study found that having more siblings may be associated with the development of infantile colic. In larger family structures, environmental factors such as more frequent disruptions in the infant’s sleep–wake cycle or irregular caregiving routines may contribute to this association. Additionally, factors such as genetic predispositions, parental stress levels, and variations in parenting styles should also be considered.

Our study has some limitations, including that the sample consisted of infants and only one blood sample could be collected per participant during the day. Although this situation limits the examination of changes in CR during the day, this limitation can be ignored since the samples were collected from both groups at similar times. The study was conducted with infants aged 0–3 months, and obstacles such as parental leave limited the sample size. Our sample size is consistent with the research on the subject in the literature [18]. On the other hand, expanding the sample size could lead to more comprehensive results.

Despite its high prevalence, the pathogenesis of infantile colic remains incompletely understood [18]. Due to the uncertainties surrounding its etiology, no single effective treatment method has been developed for colic. The development of synthetic CR proteins as part of drug formulations may offer a novel pharmacotherapeutic approach to managing this condition. Innovative methodologies for modulating the mammalian circadian clock with synthetic molecules are being developed. Synthetic derivatives of CR proteins have been produced, such as KL001 and KL044 for Cry1 and Cry2, KL101 and KL201 for Cry1, TH301 for Cry2, and KS15 for Clock-Bmal1. CR modulators are expected to have significant market potential in the pharmaceutical industry, and reports suggest that these molecules could offer drug candidates for various conditions, including jet lag, shift work, metabolic diseases, cancer, Alzheimer’s disease, and Parkinson’s disease [29]. The findings of our study may contribute to developing a treatment strategy for infantile colic by activating Bmal1 and Cry2 or suppressing Clock and Per2, using their synthetic derivatives.

This study is the first to explain that infantile colic may be linked to biorhythm disruption through the primary rhythm-regulating proteins, thereby addressing a critical gap in the field and offering an innovative perspective on treatment. This study, which contributes to the understanding of the pathophysiology of the disease, aims to prevent unnecessary examinations and applications, while improving sleep patterns and quality of life for both infants with colic and their parents.

Conclusions

In this study, differences were found in the levels of CR proteins Cry1, Cry2, Per2, Clock, and Bmal1 in infants with colic. Our results indicate that CR proteins are influenced by clinical and sociodemographic factors, with gestational diabetes, nocturnal sleep duration, number of siblings, and vitamin D supplementation emerging as the most significant variables. Our study will form a basis for further studies elucidating the disease mechanism and developing new treatment strategies for infantile colic. This research may also pave the way for new studies exploring how changes in the levels of CR-related genes/proteins could play a role not only in infantile colic but also in the etiology of various other diseases. Our findings suggest that regulation of circadian oscillators could be an effective approach for the prevention and management of diseases with limited treatment options, such as colic.