Circadian Clock Proteins in Gingival Crevicular Fluid of Individuals With and Without Circadian Rhythm Disruption

Investigation of Circadian Clock Gene/Proteins in Individuals With and Without Disrupted Circadian Rhythm: A Prospective Cross-Sectional Clinical Study

This prospective cross-sectional clinical study aims to investigate the relationship between circadian rhythm disruption and periodontal inflammation by evaluating circadian clock protein levels, inflammatory (IL-1beta) and anti-inflammatory (IL-10) cytokine levels in gingival crevicular fluid (GCF) of individuals with and without circadian rhythm disruption.

Participants aged 20-50 years will be classified into four groups based on their circadian rhythm status (disrupted/normal) and gingival health status (gingivitis/healthy). Clinical periodontal parameters including plaque index, gingival index, bleeding on probing, and probing depth will be assessed. Circadian rhythm status will be determined using validated questionnaires (Morningness-Eveningness Questionnaire and Munich Chronotype Questionnaire). Night-shift workers will represent the circadian rhythm disruption group.

GCF samples will be analyzed for circadian clock proteins (BMAL-1, CLOCK, PER-1, PER-2, PER-3, CRY-1, CRY-2, REV-ERB-beta, MTNR1B) and cytokines (IL-1beta, IL-10) using ELISA. Serum cortisol and melatonin levels will be measured for biochemical verification of circadian rhythm status.

Gingivitis groups will receive standard periodontal treatment and be re-evaluated at 2 weeks post-treatment. A total of 116 participants (29 per group) are planned for enrollment.

Gingival tissue plays a critical role in maintaining oral health as a fundamental component of periodontal tissues. Recent evidence suggests that circadian rhythm mechanisms, beyond microbial factors, may significantly influence periodontal disease pathogenesis through modulation of host immune responses.

The circadian rhythm is an endogenous mechanism regulating biological processes in approximately 24-hour physiological and behavioral cycles, controlled centrally by the suprachiasmatic nucleus in the hypothalamus. At the molecular level, core clock genes (CLOCK, BMAL1, PER1-3, CRY1-2) operate through the Transcription-Translation Feedback Loop (TTFL). CLOCK and BMAL1 proteins form heterodimers in the cell nucleus, bind to E-box regions on DNA, and initiate transcription of PER and CRY genes. Subsequently, PER and CRY proteins synthesized in the cytoplasm return to the nucleus and inhibit the CLOCK-BMAL1 complex, creating approximately 24-hour rhythmic gene expression cycles. Regulatory nuclear receptors such as REV-ERBa and ROR also contribute to the fine-tuning of this mechanism.

Recent studies have demonstrated the presence of functional peripheral clock mechanisms in periodontal tissues. CLOCK, BMAL1, PER1-3, and CRY1-2 gene expression has been identified in gingival fibroblasts and periodontal ligament fibroblasts. Experimental studies have shown that circadian rhythm disruption can increase inflammatory processes, enhance macrophage activation, elevate inflammatory cytokine production, and accelerate alveolar bone loss in periodontitis models.

Despite these findings, human studies evaluating the relationship between inflammatory cytokines and circadian rhythm proteins in gingival crevicular fluid (GCF) remain limited. This study addresses this gap by evaluating gingival health status using clinical periodontal parameters in individuals with and without circadian rhythm disruption.

Study Design:

Participants will be classified into four groups based on circadian rhythm status and periodontal health:

• Group 1: Normal circadian rhythm / Periodontally healthy (control)
• Group 2: Normal circadian rhythm / Gingivitis
• Group 3: Circadian rhythm disruption / Periodontally healthy
• Group 4: Circadian rhythm disruption / Gingivitis
• Group 5: Group 2 at 2 weeks post-periodontal treatment
• Group 6: Group 4 at 2 weeks post-periodontal treatment

Time Points:

T0 (Baseline): Clinical periodontal examination, GCF, saliva, and serum sampling in all groups.

T1 (Day 14): Re-evaluation of clinical parameters and biological sampling in gingivitis groups after standard periodontal treatment.

Clinical Periodontal Assessment:

All periodontal measurements will be performed by a single examiner using a standard periodontal probe at six sites per tooth (mesiobuccal, buccal, distobuccal, mesiolingual, lingual, distolingual). Parameters include plaque index (PI), gingival index (GI), bleeding on probing (BOP), probing depth (PD), and clinical attachment level (CAL).

GCF Sampling:

GCF samples will be collected using sterile Periopaper strips placed in the gingival sulcus for 30 seconds after supragingival plaque removal and isolation. GCF volumes will be quantified using a Periotron device. Samples contaminated with blood will be excluded.

Saliva Sampling:

Unstimulated saliva samples will be collected as an alternative biological material if circadian rhythm proteins cannot be detected at sufficient levels in GCF.

Serum Sampling:

Venous blood samples will be collected from the antecubital vein for biochemical evaluation of circadian rhythm status through cortisol and melatonin level analysis.

All biological samples will be collected during standardized morning hours (09:00-11:00) to minimize diurnal variation in biomarker levels.

Laboratory Analysis:

GCF samples will be analyzed using ELISA for circadian rhythm-related proteins (MTNR1B, BMAL-1, CLOCK, PER-1, PER-2, PER-3, CRY-1, CRY-2, REV-ERB-beta) and inflammatory biomarkers (IL-1beta and IL-10).

Statistical Analysis:

Data will be analyzed using SPSS. Normality will be assessed by Shapiro-Wilk test. Comparisons among independent groups will use one-way ANOVA or Kruskal-Wallis test, with Tukey or Dunn post-hoc tests. Pre- and post-treatment comparisons will use paired t-test or Wilcoxon signed-rank test. Correlations will be assessed by Pearson or Spearman analysis. Multivariate regression models will control for age, sex, BMI, and waist circumference as covariates. Significance level: p < 0.05.

This study was approved by the Inonu University Health Sciences Scientific Research Ethics Committee and will be conducted in accordance with the Declaration of Helsinki. Written informed consent will be obtained from all participants.

Inclusion Criteria:

• Aged between 20 and 50 years
• Systemically healthy
• Diagnosed as periodontally healthy or with gingivitis based on clinical periodontal examination
• Having at least 20 natural teeth in the oral cavity
• Willing to provide written informed consent
• Agreeing to participate in circadian rhythm assessment and completing the Morningness-Eveningness Questionnaire (MEQ) and Munich Chronotype Questionnaire (MCTQ)
• Sleep duration of 6-9 hours per day
• For periodontally healthy: PD <=3 mm, BOP <10%, CAL=0, no radiographic bone loss
• For gingivitis: PD <=3 mm, 10%<=BOP<=30%, CAL=0, no radiographic bone loss
• For circadian rhythm disruption group: being a night-shift worker

Exclusion Criteria:

• Use of antioxidant or melatonin supplements
• Presence of any systemic disease
• Use of antibiotics, anti-inflammatory drugs, or immunosuppressants within the past 3 months
• Having received periodontal treatment within the past 6 months
• Pregnancy or lactation
• Use of tobacco or tobacco products
• Presence of acute oral infection, abscess, or advanced periodontal disease
• Currently undergoing orthodontic treatment or having fixed orthodontic appliances
• Inability to comply with study procedures or refusal to provide informed consent
• Self-reported sleep duration less than 6 hours or greater than 9 hours per day