Study on the Correlation Between Metabolomics and Anxiety and Depression in Bronchiectasis

Non-cystic fibrosis bronchiectasis is a common pulmonary disorder. Recurrent acute exacerbations of bronchiectasis can severely impair lung function, accelerate the decline of pulmonary capacity, diminish quality of life and shorten survival duration. While previous studies have identified factors such as deteriorating lung function and Pseudomonas aeruginosa infection as contributing to acute exacerbations, eliminating these factors does not entirely prevent such episodes in patients with bronchiectasis. Current research suggests that 20-40% of patients with bronchiectasis experience anxiety and/or depression. These conditions are associated with an increased risk of disease exacerbation, with depression specifically linked to a shorter time to first exacerbation. Therefore, investigating the intrinsic role of anxiety and depression in disease progression is crucial.

According to the inclusion and exclusion criteria, this study will enroll participants diagnosed with bronchiectasis at Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Guizhou Provincial People's Hospital, and Yichang Central Hospital between December 20, 2025, and December 31, 2030. The study protocol was approved by the research ethics boards at each hospital. At enrolment, the following demographic information will be collected: gender, age, height (in metres) and weight (in kilograms). Medical history, previous exacerbations and blood test results (including complete blood count and biochemical parameters) will also be obtained during the initial consultation. Pathogenic examination results will also be recorded. Additionally, pulmonary function tests (e.g. FEV1% predicted) and high-resolution computed tomography (HRCT) scans of the lungs will be performed. Patients' breathlessness will be assessed using the modified Medical Research Council (mMRC) dyspnoea scale, while anxiety and depression status will be evaluated using the Hospital Anxiety and Depression Scale (HADS). With informed consent, biological samples, including blood, sputum and stool, were collected. Participants underwent follow-up every three months post-enrolment, with records maintained of exacerbation frequency, hospitalisation episodes and survival status. Upon completion of data collection, metabolomic analysis will be performed on samples including blood, sputum, and stool to investigate whether patients' anxiety and depression states are associated with the aforementioned metabolomic findings, identify corresponding biomarkers, and analyze the role of anxiety and depression in the progression of bronchiectasis.

Materials and Methods

1. Data Collection Inclusion and exclusion criteria

Inclusion Criteria: * Age ≥18 years * Participants' pulmonary imaging findings and clinical presentation met the diagnostic criteria for bronchiectasis * Clinically stable (no antibiotics or oral corticosteroids within 4 weeks prior to enrolment); * Patients who are willing to sign the consent form and participate in the study.

Exclusion Criteria: * Age \<18 years * Does not meet the diagnostic criteria for bronchiectasis * Participants with cystic fibrosis or previous lung transplantation * Participants who are unable to cooperate with the study due to dysfunction of vital systems such as heart, brain, liver, and kidneys, or who are unable to participate in the study due to comorbid serious diseases * Participants with active disorders, including active tuberculosis, active allergic bronchopulmonary aspergillosis, active nontuberculous mycobacterial infection and malignancy or secondary traction bronchiectasis associated with pulmonary fibrosis * Pregnant or lactating females * Who are not able to provide informed consent or who refuse to participate in the clinical study

Collection of patient demographic indicators and laboratory test results. Demographic indicators (age, height, weight), mMRC score, laboratory test results, pulmonary function (FEV1% predicted), radiological scores (modified Reiff, Bhalla score) were collected upon participant enrolment.

Assessment of Anxiety and Depression Status：All patients were requested to complete the Hospital Anxiety and Depression Scale (HADS) at baseline. The score for each subscale (HADS depression and anxiety) ranges from 0 to 21 points, with a score ⩾8 indicating probable depression or anxiety .

Assessment of Disease Severity：Disease severity was evaluated using the Bronchiectasis Severity Index (BSI). 2. Metabolomics Experimental MethodsMetabolomics Experimental Methods 2.1 Sample Preparation 50 μL of collected sample was thawed on ice and mixed with 200 μL of cold methanol:acetonitrile (1:1, v/v) containing the IS (1 μg/mL). The mixture was vortexed vigorously, incubated at -20°C for 2 h, and then centrifuged at 14,000 g for 15 min at 4°C. The supernatant was transferred to a new vial for LC-MS analysis. A pooled quality control (QC) sample was prepared by combining equal aliquots from all individual samples.

2.2 LC-MS Analysis Chromatographic separation was performed on a Waters ACQUITY UPLC system using a HSS T3 column (2.1 × 100 mm, 1.8 μm) maintained at 40°C. The mobile phase consisted of (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. A linear gradient was applied as follows: 0-2 min, 2% B; 2-10 min, 2% to 98% B; 10-12 min, 98% B; 12-12.1 min, 98% to 2% B; 12.1-15 min, 2% B. The flow rate was 0.4 mL/min and the injection volume was 2 μL.

\> Mass spectrometry was conducted on a SCIEX TripleTOF 6600+ system operated in both positive and negative ESI modes. The parameters were set as follows: Ion Source Gas 1: 60 psi, Ion Source Gas 2: 60 psi, Curtain Gas: 35 psi, Source Temperature: 550°C, Ion Spray Voltage: ±5500 V. Data were acquired in information-dependent acquisition (IDA) mode.

2.3 Data Processing and Multivariate Statistical Analysis Raw data files were processed using MS-DIAL software for peak picking, alignment, and normalization against the IS. The resulting data matrix was imported into SIMCA-P software (v16.0, Umetrics, Sweden) for multivariate analysis. Unit variance scaling and mean centering were applied prior to PCA and OPLS-DA. The OPLS-DA model was validated by a 200-time permutation test.

Metabolites with Variable Importance in Projection (VIP) \> 1.0 from the OPLS-DA model and p-value \< 0.05 from univariate Student's t-test were considered statistically significant.

2.4 Metabolite Identification Significant metabolites were identified by comparing their accurate mass (mass error \< 10 ppm) and MS/MS spectra with entries in the HMDB database. Where possible, identification was confirmed by comparison with authentic standards (Level 1 confidence). 3. Clinical Data Statistical Analysis and Methods:

The data obtained during the study were pre-collated. For continuous data, normality tests were first performed. If all groups met normality, the Student's t-test was used for comparison between groups. Otherwise, the non-parametric Wilcoxon rank sum test was considered. For categorical variables, the χ2 test was used. Statistically significant data were subjected to multivariate logistic regression analysis. P \< 0.05 was deemed statistically significant.

Statistical analysis of all data was performed through SPSS (IBM SPSS Statistics 26.0, SPSS Inc., Chicago, IL) and R language (version 4.1.3, www.R-project.org/). All statistical tests were two-sided, and statistical significance was set at 0.05.

• Hospital Anxiety and Depression Scale (HADS) [Срок оценки: Lasts for 5 years]
• Lung Function FVC measured in liters (L) [Срок оценки: recorded at baseline and re-tested every year, up to 5 years.]
• Lung Function FEV1% [Срок оценки: recorded at baseline and re-tested every year, up to 5 years.]
• Lung Function - The FEV1/FVC ratio [Срок оценки: recorded at baseline and re-tested every year, up to 5 years.]
• modified Medical Research Council (mMRC) dyspnea score [Срок оценки: At baseline and updated once a year for 5 years]
• Bhalla scores on CT of participants' lungs [Срок оценки: Assess once a year from the start of enrollment, for a total duration of five years.]
• Frequency of hospitalisation [Срок оценки: Starting from inclusion in the study, data will be summarized once per year for a total duration of five years.]
• Mortality rate [Срок оценки: Five-year mortality rate]
• Bronchiectasis severity Score (BSI) [Срок оценки: At baseline and updated once a year for 5 years]
• E-FACED score [Срок оценки: At baseline and updated once a year for 5 years]
• Quality of Life Questionnaire-Bronchiectasis [Срок оценки: At baseline and updated once a year for 5 years]
• Microbiology checklist [Срок оценки: At least 1 year before enrollment and 5 years during the follow-up]