2.1 Study Subjects
MDD Patient Group Source:
All subjects were adolescent MDD patients hospitalized in the first psychiatric ward of the First Affiliated Hospital of Zhengzhou University. The study group included 99 adolescent depression patients, comprising 33 males and 66 females, with an age range of [15.0 (14.0, 17.0)] years.
Inclusion Criteria:
(1)Meets the DSM-5 diagnostic criteria for Major Depressive Disorder (MDD).
(2)A total score of ≥ 20 on the 24-item Hamilton Depression Rating Scale (HAMD-24).
(3)Age between 12 and 18 years, with an education level of middle school or high school.
(4)No history of immunosuppressant or immunoenhancer use in the past six months.
(5)Han ethnicity.
Subgroup Classification:
(1)MDD with Evening Chronotype (MDD-ET+): This subgroup included 61 cases with an MEQ-5 total score of < 12, aged [16.0 (15.0, 17.0)] years, including 22 males and 39 females.
(2)MDD without Evening Chronotype (MDD-ET–): This subgroup included 38 cases with an MEQ-5 total score of ≥ 12, aged [15.0 (14.0, 17.0)] years, including 11 males and 27 females.
Control Group Source:
The control group consisted of individuals recruited from the community, selected based on age matching with the study group. A total of 57 individuals were included, comprising 41 males and 16 females, with an age range of [15.0 (15.0, 16.0)] years.
Inclusion Criteria:
(1)Does not meet the diagnostic criteria for Major Depressive Disorder (MDD) according to DSM-5.
(2)Total score on the 24-item Hamilton Depression Rating Scale (HAMD-24) ≤ 8.
Age between 12 and 18 years, with an education level of middle school or high school.
(3)No history of immunosuppressant or immunoenhancer use in the past six months.
Control Group Subgroup Classification:
(1)Control Group with Evening Chronotype (HC-ET+): This subgroup included 30 cases with an MEQ-5 total score of < 12, comprising 20 males and 10 females, with an age range of [15.0 (15.0, 16.0)] years.
(2)Control Group without Evening Chronotype (HC-ET–): This subgroup included 27 cases with an MEQ-5 total score of ≥ 12, comprising 21 males and 6 females, with an age range of [15.0 (15.0, 16.0)] years.
Exclusion Criteria for Both Groups:
(1)Diagnosis of schizophrenia, bipolar disorder, or anxiety disorders.
(2)Diagnosis of hypertension, diabetes, or significant organ (including heart, brain, lung, kidney, etc.) diseases.
(3)History of substance dependence or abuse.
(4)History of infection within the two weeks prior to enrollment.
(5)Pregnant or lactating women.
Ethical approval
for this study was obtained from the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Ethical Review Number: SB201901012, 2023-KY-0220-001). Prior to the survey, informed consent was obtained from all participants, and consent for the control group was also obtained from relevant school authorities.
2.2 Data Collection
2.2.1General Information Collection Form
A self-designed general demographic data collection form was used, including fields for:Name,Age,Gender,Ethnicity,Years of education.
2.2.2 Assessment of Mental Health Level and Circadian Rhythm
(1)Morning and Evening Questionnaire 5 (MEQ-5)[35]
The MEQ-5 scale was used to assess the circadian rhythm type of adolescents. It consists of 5 items, with a total score of 25 points. Based on the score, < 12 points indicate an evening chronotype, 12–17 points indicate an intermediate chronotype, and ≥ 18 points indicate a morning chronotype. In this study, a score of < 12 points was considered indicative of an evening chronotype (ET+), while a score of ≥ 12 points was considered indicative of a non-evening chronotype (ET-).
(2) Hamilton Depression Rating Scale-24 (HAMD-24)[36]
The HAMD-24 scale is designed to measure the intensity of depressive symptoms and comprises 24 items. Scores range from 0 to 74 points, where ≤ 8 points indicate no depressive symptoms, 9 to 19 points indicate possible depressive symptoms, ≥ 20 points indicate mild to moderate depression, and ≥ 35 points indicate severe depression symptoms. In this study, a total score of ≥ 20 points on the HAMD-24 was used as the criterion for depressive symptoms.
(3)Hamilton Anxiety Scale (HAMA)[37]
The HAMA scale is used to assess the severity of anxiety symptoms and consists of 14 items. The total score ranges from 0 to 56 points, where ≤ 7 points indicate no anxiety symptoms, 8 to 13 points indicate possible mild anxiety, ≥ 14 points indicate mild anxiety, ≥ 21 points indicate moderate anxiety, and ≥ 29 points indicate severe anxiety.
2.2.3 IL-6, CRP, ACTH, COR, and Copeptin Detection Methods
ACTH and COR:
To assess the overall level and rhythmic changes of COR, blood samples were collected from patients after a 12-hour fast at 8:00 AM, 4:00 PM, and 24:00 AM the next day. Approximately 5 mL of venous blood was collected in EDTA anticoagulant tubes and sent to our hospital's laboratory center for ACTH and COR detection. We utilized the Siemens IMMULITE2000 automatic immunoassay analyzer from Germany along with corresponding reagent kits for the analysis.
Detection Procedure:
Plasma COR (COR8, COR16, and COR24) and ACTH (ACTH8, ACTH16, and ACTH24) levels were measured at 8:00 AM, 4:00 PM, and 24:00 AM.
Calculation of Parameters:
We further calculated the area under the curve (AUC) for each patient to assess the total cortisol output over the specified time period. AUC reflects the total secretion of COR over a certain period, equivalent to the area under the trapezoidal curve.
Additionally, two slopes (COR8-16 and COR16-24) were calculated to determine the rate of COR concentration decline from morning to evening.
These measurements and calculations provide insights into the secretion pattern and rhythmic changes of COR and ACTH, allowing for a comprehensive evaluation of the adrenal axis function in the study participants[38].
![](https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1719319156.png)
CRP and IL-6 Detection Methods
All participants underwent blood collection of approximately 5 mL from the cubital vein between 7:00 AM and 9:00 AM, with EDTA used as the anticoagulant. After blood collection, samples were sent to our hospital's laboratory center for the detection of CRP and IL-6 levels.
(1)CRP levels were measured using the immunoturbidimetric assay with the C-reactive protein test kit (Roche, Switzerland) on the cobas e 801 fully automated biochemical analyzer (Roche, Switzerland).
(2)IL-6 levels were measured using the electrochemiluminescence immunoassay with the interleukin 6 test kit (Roche, Switzerland) on the cobas 8000 series fully automated biochemical analyzer (Roche, Switzerland).
Copeptin: Blood samples of 5 mL were collected from the cubital vein on an empty stomach at 8:00 AM and transferred to sterile centrifuge tubes. Within 2 hours of collection, the whole blood was centrifuged for 10 minutes at a speed of 3000 revolutions per minute and a radius of 10 centimeters to prepare serum. During serum preparation, the supernatant was extracted to avoid hemolysis, and the serum was stored in a refrigerator at -80°C. The determination of serum copeptin levels was performed using the RayBio Human Copeptin EIA Kit (RayBiotech, USA, EIA-COP-1) with a competitive colorimetric method. The intra- and inter-assay coefficients of variation for this kit were both less than 10% and 15%, respectively, with a detection limit of 0.5 ng/mL.
2.3 Data Analysis
The data analysis was conducted using SPSS 25.0 software. Frequency (n) and percentage (%) were used to describe count data, while mean ± standard deviation was used for normally distributed continuous variables, and median (interquartile range) was used for skewed distributed continuous variables. Differences between the MDD-ET + group and the MDD-ET- group were analyzed using independent samples t-test for continuous variables, and non-parametric tests (Mann-Whitney U test) were used for continuous variables that did not follow a normal distribution. Chi-square test was used for analyzing categorical variables. Repeated measures analysis of variance was used to compare COR and ACTH values at different time points between the MDD-ET + and MDD-ET- groups. Binary logistic regression analysis was employed to identify independent risk factors for evening chronotype depression, with odds ratio (OR) and 95% confidence interval (95% CI) used for effect estimation.
For comparisons among the MDD-ET+, MDD-ET-, HC-ET+, and HC-ET- groups, Kruskal-Wallis test was used to analyze continuous variables. Before conducting covariance analysis, ln transformation was performed to approximate normal distribution and meet the assumption of homogeneity of variance. Subsequently, 2×2 covariance analysis was conducted with HAMA total score as a covariate to examine the main effects and interactions of copeptin levels.