Targeting the Gut-Brain Axis With Ganoderma Lucidum Spores Ameliorates Depression in Thyroid Cancer Patients: a Randomized, Double-blind, Placebo-controlled Trial
Phase: NA
Conditions: Depression, Thyroid Cancer
Sponsor: Ling Zhiqiang

Targeting the gut–brain axis with Ganoderma lucidum
spores ameliorates depression in thyroid cancer patients: a
randomized, double-blind, placebo-controlled trial
Study Site ：Zhejiang Cancer Hospital
Principal Investigator：
Protocol Amendment Date: March 12, 2026
Version：3.0

Protocol Signature Page
I have read this protocol. I understand and agree with its contents and
consent to conduct the clinical study in accordance with this protocol. I
agree to maintain confidentiality regarding this protocol and related
information.
Study Site ：Zhejiang Cancer Hospital
Principal Investigator：
Date：March 12, 2026

Research Protocol Summary
Title
Targeting the gut–brain axis with Ganoderma lucidum spores ameliorates
depression
in
thyroid
cancer
patients:
a
randomized,
double-blind,
placebo-controlled trial
Intervention
Group
Sporoderm-removed Ganoderma lucidum Spore powder (RGLS)
Control Group
placebo control
Trial Center
Zhejiang Cancer Hospital
Trial Objectives
To clarify the clinical efficacy of RGLS intervention on depressive symptoms
accompanying post-operative papillary thyroid carcinoma.To elucidate the
antidepressant mechanism of RGLS.
Trial Design
Randomized, Double-Blind, Placebo Parallel-Controlled Trial
Study Population
Patients with post-operative papillary thyroid carcinoma accompanied by
depressive symptoms
Sample Size
300 cases; RGLS group: 200 cases, placebo group: 100 cases
Treatment
Regimen
From the first day after enrollment, subjects take the trial drug (RGLS or
placebo) orally at 4g/day for 90 days.
Efficacy
Endpoints
1.Primary Endpoint:Hamilton Depression Rating Scale-24 (HAMD-24) score.
2.Secondary Endpoints:
(1) Pittsburgh Sleep Quality Index (PSQI) score.
(2) Changes in gut microbial diversity and metabolites.
3.Exploratory Endpoint: Thyroid Function.
Safety Endpoints
Incidence of Adverse Events
Statistical
Analysis
Efficacy analyses will be based on the Full Analysis Set (FAS) and the
Per-Protocol Set (PPS), while safety analyses will be based on the Safety
Analysis Set (SAS). Appropriate statistical methods will be applied to compare
both primary and secondary endpoints between groups. All tests will be
two-sided, with a P < 0.05 considered statistically significant.

Flowchart
Process
baseline
Visit 1
Final Visit
baseline
Month 1
Month 3
Informed Consent
√
Collection
of
Demographic
Data
√
Collection of Medical History
(Current, Past, Medication)
√
Psychiatric Examination
√
HAMD-24 Assessment
√
√
√
Inclusion/Exclusion
Criteria
Check
√
Randomization
√
Stool Sample Collection
√
√
PSQI Assessment
√
√
√
Residual Blood Collection
√
√
Concomitant
Medication
Recording
√
√
√
Adverse Event Recording
√
√
Trial Drug Dispensing
√
√
Trial Drug Retrieval
√
√
√

Contents
1. Trial Background.....................................................................................................................................1
2. Research Objectives................................................................................................................................3
3. Research Significance............................................................................................................................ 3
4. Trial Design..............................................................................................................................................4
5. Technical Roadmap................................................................................................................................ 4
6. Technical Route.......................................................................................................................................4
7. Subject Selection.....................................................................................................................................5
7.1 Inclusion Criteria..............................................................................................................................5
7.2 Exclusion Criteria.............................................................................................................................5
7.3 Exclusion Criteria (Post-enrollment)........................................................................................... 6
7.4 Criteria for Subject Withdrawal from the Trial.........................................................................6
8. Trial Protocol........................................................................................................................................... 6
8.1 Sample Size and Allocation...........................................................................................................6
8.2 Study Drug Information..................................................................................................................6
8.3 Intervention Protocol Design.........................................................................................................7
8.4 Drug Management and Retrieval..................................................................................................8
9. Concomitant Medication Restrictions................................................................................................ 8
10. Study Procedures...................................................................................................................................9
10.1 Screening/Baseline Period (Baseline)....................................................................................... 9
10.2 Visit 1 (Month 1)............................................................................................................................9
10.3 Visit 2 / Final Visit (Month 3).....................................................................................................9
10.4 Principles of Indicator Collection............................................................................................ 10
11. Efficacy Evaluation Criteria.............................................................................................................11
11.1 Primary Efficacy Endpoint........................................................................................................11
11.2 Secondary Efficacy Endpoints..................................................................................................11
11.3 Exploratory Endpoint..................................................................................................................13
12. Adverse Event Evaluation................................................................................................................ 13
12.1 Definition of Adverse Event (AE)...........................................................................................13
12.2 Causality Assessment................................................................................................................. 13
12.3 Monitoring Process..................................................................................................................... 13
13. Data Management...............................................................................................................................14

13.1 Case Report Form (CRF) Management..................................................................................14
13.2 Data Entry Standards.................................................................................................................. 14
13.3 Database Locking Criteria.........................................................................................................14
14. Clinical Trial Management and Quality Control.........................................................................14
14.1 Ethical Compliance Management............................................................................................14
14.2 Standardized Operating Procedures (SOPs)..........................................................................15
14.3 Document Management Standards..........................................................................................15
14.4 Quality Monitoring Mechanism...............................................................................................15
15. Statistical Analysis of Trial Data.................................................................................................... 15
15.1 Full Analysis Set (FAS)............................................................................................................. 15
15.2 Per-Protocol Set (PPS)............................................................................................................... 16
15.3 Safety Analysis Set (SAS).........................................................................................................16
16. Statistical Analysis Plan....................................................................................................................16
17. Blinding, Unblinding, and Emergency Unblinding.................................................................... 17
17.1 Blinding Design........................................................................................................................... 17
17.2 Formal Unblinding Process.......................................................................................................17
17.3 Emergency Unblinding Criteria............................................................................................... 17
18. Trial Schedule......................................................................................................................................18
References...................................................................................................................................................18
Appendix 1: Hamilton Depression Rating Scale (HAMD)..............................................................20
Appendix 2: Pittsburgh Sleep Quality Index (PSQI).........................................................................21

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1. Trial Background
Depression, as a major global public health issue, has become a significant component of the
global disease burden due to its high rates of disability and mortality. With socioeconomic
development and epidemiological transition, non-communicable diseases have replaced infectious
diseases as the primary health threat, among which the incidence and mortality of cancer show a
sustained upward trend. The immense psychological stress associated with the cancer diagnosis
and treatment process leads to comorbid depression in approximately 50% of patients,
significantly increasing suicide risk, severely impacting quality of life, and creating a heavy
socioeconomic burden. The pathological mechanism of cancer comorbid with depression involves
the interaction of multiple factors, including cancer-related fatigue, nutritional and metabolic
disorders, chronic stress, systemic inflammation, immune dysfunction, and neurometabolic
imbalance, collectively leading to impaired neuroprotective mechanisms and accumulation of
neurotoxic substances. Existing evidence indicates that poor prognosis and decreased quality of
life are significantly correlated in cancer patients with comorbid depression.1 Papillary thyroid
carcinoma, as the most common pathological type of thyroid cancer, although associated with
good postoperative prognosis and long-term survival rates, is marked by significantly reduced
quality of life, often accompanied by depressive symptoms and sleep disturbances. Studies show
that thyroid hormones are crucial for brain development and functional maintenance, and
dysfunction of the hypothalamic-pituitary-thyroid (HPT) axis can lead to neuropsychiatric
abnormalities.2 In clinical management, to prevent recurrence and metastasis, patients require
long-term levothyroxine sodium administration to maintain a subclinical hyperthyroid state. This
iatrogenic dysregulation of the thyroid axis further increases the risk of psychiatric disorders such
as depression, anxiety, and insomnia. Notably, existing evidence indicates a significant association
between thyroid dysfunction and gut microbiota dysbiosis.3
Notably, epidemiological data clearly indicate that women represent a high-risk population
for both thyroid cancer and cancer-related depression.4 This susceptibility stems from a complex
interplay of multiple factors: the unique biological foundation involving interactions between sex
hormones (e.g., estrogen) and the thyroid axis; the distinct threat and anxiety posed by cancer
diagnosis and treatments (such as radioactive iodine therapy and TSH suppression therapy) to
women's reproductive function and pregnancy planning; and the specific psychosocial pressures

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arising from the multiple roles women often undertake within society and family.5 These
intertwined factors collectively render female post-operative thyroid cancer patients a high-risk
group for depressive symptoms. To focus on this vulnerable and mechanistically distinct
population, this study will restrict participants to women, aiming to obtain more homogeneous and
in-depth scientific insights and to provide more targeted interventional evidence for this group.
The gut microbiota plays a key role in neuropsychiatric disorders through the bidirectional
regulatory mechanisms of the gut-brain axis, influencing central nervous system function via the
following pathways: 1) Microbial metabolites (e.g., SCFAs) directly act on vagal nerve endings,
transmitting signals via the nucleus tractus solitarius-paraventricular thalamic pathway; 2)
Regulating neurotransmitter balance and neurotrophic factor expression; 3) Modulating systemic
inflammatory responses and oxidative stress levels; 4) Affecting blood-brain barrier integrity.
When gut microbiota is disrupted, this regulatory network becomes imbalanced, leading to the
development of neuropsychiatric diseases such as depression through multiple interactions within
the neuro-immune-endocrine system.6 There are 56 ethnic subgroups in China, and their intestinal
flora differs.7 Therefore, in this study, we will only select the Han Chinese population.
Current societal awareness and acceptance of mental illnesses like depression remain
inadequate. Influenced by stigma and social prejudice, coupled with a lack of public mental health
knowledge, a large number of patients fail to seek professional diagnosis and treatment in a timely
manner. This delay in seeking medical care causes most patients with depression to miss the
golden window for early intervention, severely affecting disease prognosis and outcomes.
The pathological basis of depression involves dysfunction of central neural networks, and its
pathogenesis exhibits characteristics of multisystem interaction. Major hypotheses include:
neurotransmitter
imbalance
(monoamine
hypothesis),
neurotrophic
dysfunction,
hypothalamic-pituitary-adrenal
(HPA)
axis
dysregulation,
neuroinflammation,
immune
dysregulation, and the microbiota-gut-brain axis disruption hypothesis. Clinical treatment adopts a
comprehensive intervention strategy combining pharmacotherapy, psychotherapy, and physical
therapy. Among these, first-line antidepressants such as SSRIs (Selective Serotonin Reuptake
Inhibitors) and SNRIs (Serotonin-Norepinephrine Reuptake Inhibitors), although widely used,
have significant limitations: delayed onset of action (2-4 weeks), unstable maintenance of efficacy,
non-response in some patients, and drug side effects such as sexual dysfunction, central nervous

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system effects (headache, drowsiness), and gastrointestinal adverse reactions, leading to reduced
long-term treatment adherence.8
Ganoderma lucidum,9 a rare and precious variety among traditional Chinese medicinal herbs,
has long been revered as the "elixir herb," with its medicinal value recorded in ancient texts such
as Shennong's Classic of Materia Medica. Modern research indicates that Ganoderma spores, the
reproductive cells of the fungus, are rich in bioactive components, primarily including: 1)
Ganoderma polysaccharides (crude polysaccharide content ≥8.0g/100g) with immunomodulatory
effects; and 2) Triterpenoids (total triterpenoids ≥4.0g/100g) exhibiting antitumor activity.
Sporoderm-removed
Ganoderma
lucidum
Spore
powder
(RGLS)
significantly
improves
bioavailability and is now widely used in the field of adjuvant cancer therapy. Preliminary clinical
observations suggest that RGLS can significantly improve depressive symptoms in patients with
colorectal
adenoma
(HAMD-24
score
reduction
≥30%)
and
enhance
immune
function
(CD4+/CD8+ ratio increased by 1.5-fold).
2. Research Objectives
This study employs microbial metagenomics combined with metabolomics technology to
systematically investigate the interventional effects and mechanisms of RGLS on depressive
symptoms in patients after papillary thyroid carcinoma surgery. The focus is to elucidate: 1) Its
clinical efficacy (improvement in HAMD-2410/PSQI11 scores); 2) The molecular mechanisms
through which it regulates depressive states via the microbiota-gut-brain axis; and 3) The
regulatory role of key microbial metabolites in the development of depression. The research
findings will provide evidence-based medical support for the clinical application of Ganoderma
spore powder.
3. Research Significance
Based on the multi-component, multi-target characteristics of traditional Chinese medicine,
this study innovatively integrates multi-omics technologies such as microbial metagenomics and
metabolomics to systematically analyze the molecular mechanisms underlying the antidepressant
effects of RGLS. The research is grounded on two major scientific findings: 1) Characteristic gut
microbiota dysbiosis exists in patients with depression; and 2) Ganoderma spore powder exhibits
potential antidepressant activity. By constructing a multidimensional regulatory network of
"microbiota-metabolites-gut-brain axis," the study aims to elucidate: ①The clinical improvement

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effect of RGLS on depression after thyroid cancer surgery; ②The regulatory role of key
functional microbiota and their metabolites; and ③The mediating mechanism of the
microbiota-gut-brain axis in the development of depression. This research strategy provides an
innovative methodological paradigm for studying the mechanisms of complex traditional Chinese
medicine systems.
4. Trial Design
This study adopts a randomized, double-blind, placebo parallel-controlled clinical trial
design.
5. Technical Roadmap
(1) Integrating microbiomics and metabolomics technologies to construct a multidimensional
analysis system of "Microbiota-Metabolites-Gut-Brain Axis":
(2) Pre-intervention: Baseline clinical assessment (HAMD-24/PSQI) and biological sample
collection.
(3) Intervention process: Strictly quality-controlled standardized drug administration
protocol.
(4) Post-intervention: Multi-omics data collection and analysis.
(5) Integrated Analysis: Correlation modeling of microbiota, metabolites, and clinical
indicators.
6. Technical Route

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7. Subject Selection
7.1 Inclusion Criteria
(1) Outpatients at Zhejiang Cancer Hospital with histologically confirmed papillary thyroid
carcinoma (post-surgery) and depressive symptoms (HAMD-24 score ≥8, confirmed by a clinical
psychologist).
(2) Han Chinese ethnicity.
(3) No history of depression or other psychiatric disorders.
(4) Age 18-80 years.
(5) Female.
7.2 Exclusion Criteria
(1) Suffering from other gastrointestinal system diseases.
(2) History of gastrointestinal surgery prior to intervention.
(3) Including those with other concurrent malignancies requiring chemotherapy, radiotherapy,
biological therapy, or traditional Chinese medicine treatment.
(4) Received antibiotic treatment or microecological modulators within 3 months prior to
intervention.
Allocated to RGLS group (n=200)
Received allocated RGLS (n=)
Did not receive allocated RGLS (give reasons) (n=)
Allocated to placebo group (n=100)
Received allocated placebo (n=)
Did not receive allocated placebo (give reasons)
(n=)
Discontinued RGLS (give reasons) (n=)
Lost to follow-up for primary outcome (give reasons)
(n=):
Completed (n=)
Discontinued placebo (give reasons) (n=)
Lost to follow-up for primary outcome (give reasons)
(n=):
Completed (n=)
Analysed for primary outcome (n=)
Excluded from analysis (give reasons) (n=)
Analysed for primary outcome (n=)
Excluded from analysis (give reasons) (n=)
Allocation
Follow-Up
Analysis
Randomised ( n=300)

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(5) Acute intestinal obstruction.
(6) Patients who are currently taking antidepressant medication, or those whom the
investigators judge require immediate initiation of antidepressant medication treatment.
(7) Organic brain diseases, brain trauma.
(8) History of psychiatric disorders, use of psychoactive substances (e.g., drugs).
(9) Severe liver or kidney dysfunction.
(10) Pregnancy or lactation.
7.3 Exclusion Criteria (Post-enrollment)
(1) Subjects found not to meet inclusion criteria after enrollment.
(2) Subjects who did not take RGLS according to the study protocol or did not complete the
intervention course.
(3) Poor compliance, voluntary withdrawal mid-study, or inability to assess efficacy or safety
due to missing primary endpoint data or incomplete records.
7.4 Criteria for Subject Withdrawal from the Trial
(1) The investigator deems it medically or ethically necessary to stop the trial.
(2) Occurrence of adverse events, complications, or special physiological changes making
continued participation inadvisable.
(3) Pregnancy during the treatment period.
8. Trial Protocol
8.1 Sample Size and Allocation
Sample size calculation was performed using G*Power 3.1.9.7 software. Parameters were set
as follows: independent samples t-test, two-tailed, significance level α=0.05, power (1-β)=0.90,
experimental to control group ratio of 2:1. Based on previous study,12 the effect size (Cohen's d)
was set to 0.5 (mean difference between groups equals 0.5 times the standard deviation), assuming
equal variances. The calculated required sample size was 128 for the RGLS group and 64 for the
control group. The final sample of 300 (200 RGLS / 100 control) was determined considering:
(1) Statistical power assurance.
(2) Ethical optimization: Using a 2:1 allocation reduces placebo use.
(3) Quality control: Presetting a 15% dropout rate buffer.
8.2 Study Drug Information

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(1) Experimental Drug
Generic Name: Sporoderm-removed Ganoderma lucidum Spore powder (RGLS)
Specification: 2g/sachet
Quality Control: Complies with relevant standards of the Chinese Pharmacopoeia 2025
Edition.
(2) Control Drug
Composition: placebo control.
Specification: 2g/sachet
Simulant Characteristics: Appearance/packaging identical to the experimental drug.
Quality Control Standard: Same as the experimental drug.
(3) Drug Source
Manufactured
on
the
GMP-certified
production
line
of
Jinhua
Shouxian
Valley
Pharmaceutical Co., Ltd., with full batch quality inspection reports provided.
8.3 Intervention Protocol Design
(1) Grouping Design
This is a single-center, randomized, double-blind, placebo parallel-controlled trial. A total of
300 patients with post-operative papillary thyroid carcinoma accompanied by depressive
symptoms (HAMD-24 score ≥8) will be enrolled. Subjects will be randomized in a 2:1 ratio
(RGLS group to control group). To maintain strict double-blinding, a three-code (A, B, C)
operational grouping system will be implemented:
Two of the code groups receive the same active intervention (RGLS), while the third code
group
receives
a
placebo identical
in
appearance,
packaging,
and administration.
The
correspondence between codes A, B, C and the assigned intervention is sealed and concealed from
both investigators and participants. All statistical analyses will be performed after unblinding,
comparing the combined RGLS group against the placebo group.
(2) Administration Regimen
Dosage and Frequency: Twice daily, 2 g (one sachet) each time.
Intervention Duration: Continuous intervention for 90 days.
Administration Method: Dissolve in 50 ml of warm water to form a homogeneous suspension
for oral intake.

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(3) Ethical Oversight:
Approved by the Ethics Committee of Zhejiang Cancer Hospital (IRB-2026-288, IIT). All
subjects will provide written informed consent. The study will be conducted in accordance with
GCP guidelines and the Declaration of Helsinki. Case data will be de-identified, and biological
samples will be anonymized. Pre-intervention samples will be labeled with codes 001-300, and
post-intervention samples with codes 001(2)-300(2).
(4) Drug Packaging Specifications
A three-level packaging system will be used:
Primary Packaging Unit: Aluminum foil sachet (2g/sachet, containing RGLS or placebo).
Secondary Packaging: 60 sachets/box (equivalent to a 30-day supply).
Total Supply: 3 boxes/subject (covering the 90-day course).
Quality Control:
All packaging materials comply with the YBB00132002 standard for pharmaceutical
aluminum foil. Full batch traceability is implemented.
8.4 Drug Management and Retrieval
(1) Storage Conditions:
Environmental requirements: Cool, ventilated area (temperature ≤25°C, relative humidity
≤60%).
Security measures: Dedicated drug cabinet with dual locks.
Special precautions: Stored in an area inaccessible to children.
(2) Dispensing and Supervision:
Authorized research personnel dispense drugs according to the visit schedule.
Dual signature for medication records (investigator-subject).
Real-time registration of drug batch number/expiry date.
(3) Retrieval Process:
Used sachets are collected and counted at each follow-up visit.
Final verification of total drug amount at trial end.
Discrepancy recording: Missing quantities and reasons annotated in the CRF.
9. Concomitant Medication Restrictions
This study strictly prohibits the following concomitant medications that may affect efficacy

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evaluation:
(1)
Immunomodulators:
Including
biological
agents,
traditional
Chinese
medicine
compounds, and functional health products.
(2) Psychotropic drugs: Antidepressants, sedative-hypnotics, and other central nervous
system drugs.
(3) Microecological agents: Antibiotics, probiotic/prebiotic preparations.
(4) Others: Dietary supplements with neuroregulatory or immunomodulatory activity.
10. Study Procedures
10.1 Screening/Baseline Period (Baseline)
(1) Signing of informed consent.
(2) Verification of inclusion/exclusion criteria.
(3) Collection of demographic and medical history data.
(4) Psychiatric examination and HAMD-24 assessment.
(5) PSQI assessment.
(6) Stool sample collection.
(7) Residual blood collection for thyroid function tests.
(8) Urine pregnancy test for women of childbearing age.
(9) Recording of concomitant medications.
(10) Drug dispensing.
10.2 Visit 1 (Month 1)
(1) HAMD-24 assessment.
(2) PSQI assessment.
(3) Recording of concomitant medications and adverse events.
(4) Drug retrieval and dispensing.
10.3 Visit 2 / Final Visit (Month 3)
(1) HAMD-24 assessment.
(2) PSQI assessment.
(3) Stool sample collection.
(4) Residual blood collection for thyroid function tests.
(5) Recording of concomitant medications and adverse events.

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(6) Drug retrieval.
10.4 Principles of Indicator Collection
(1) Psychological Scale Assessment
HAMD-24 Scoring: Independently completed by two senior psychiatrists (Kappa value
≥0.75).
PSQI Scoring: Self-administered by the subject under the supervision of a psychologist.
Assessment Time Points: Baseline, Mid-intervention (Month 1), End of intervention (Month
3).
(2) Stool Sample Collection Method:
Place a stool collector in the toilet bowl. Defecate into the collector. Use a long-handled
scoop to thoroughly mix the fresh stool sample (avoid urine contamination).
Aliquot the mixed stool sample into two pre-labeled 5-10 mL sterile tubes or pre-sterilized
collection tubes, filling each to approximately 2-3 mL.
Use a wooden stick to transfer about 1 mL of the sample from the 5-10 mL collection tube to
a 1.5 mL centrifuge tube; or directly transfer about 1 mL from the mixed stool sample in step ①to
a 1.5 mL centrifuge tube.
Cap the centrifuge tube tightly, check the seal, place in a sample box in order of code, and
store in a -80°C freezer.
If collecting stool at home, place the collection tube in a ziplock bag and deliver to the
hospital sample reception point as soon as possible (within 2 hours).
Sample Collection and Storage Notes:
Samples should not be exposed to room temperature for more than 2 hours.
Collection should be performed at the same time of day to avoid circadian rhythm effects on
metabolism.
Samples should be mixed immediately after collection, aliquoted, and frozen.
Prepare duplicate samples to avoid repeated freeze-thaw cycles.
(3) Blood Sample Collection:
Residual serum from routine clinical thyroid function tests was used. Approximately 500 μL
to 1 mL of supernatant was transferred into 1.5 mL centrifuge tubes (multiple aliquots
recommended), placed in a cryobox, and stored at –80°C. Sample labeling followed the same

11 / 27
numbering
system
as
the
corresponding
fecal
samples:Pre-intervention:
001–300.
Post-intervention: 001(2)–300(2).
(4) Stool and Blood Sample Collection Timing, Preservation, and Transport:
Collection Time Points: Baseline, End of intervention (Month 3).
Preservation: Flash-freeze in liquid nitrogen for 15 minutes before transferring to a -80°C
freezer.
Ensure continuous freezer operation without power interruption, as power failure will cause
sample thawing.
11. Efficacy Evaluation Criteria
11.1 Primary Efficacy Endpoint
Change in Hamilton Depression Rating Scale-24 (HAMD-24) score:
(1) Scale Characteristics: A 24-item standardized tool for assessing depressive symptoms,
covering emotional, cognitive, and somatic dimensions.
(2) Clinical Severity Grading:
No depression (0-7 points)
Subclinical depression (8-20 points)
Mild to moderate depression (21-35 points)
Severe depression (≥36 points)
(3) Application Value: Widely used as a gold standard for evaluating antidepressant treatment
efficacy.
(4) Definition of Clinical Remission: After 90 days of intervention (Month 3), meeting both
criteria:
HAMD-24 score decreases to the non-depressed range (<8 points) or improves by ≥1 severity
level.
Inter-group comparison shows statistical significance (P < 0.05, two-tailed test).
11.2 Secondary Efficacy Endpoints
(1) Sleep Quality Assessment (PSQI)
Scale Structure: 7-dimension self-assessment scale (Cronbach's α=0.83).
Cut-off Value:
Normal sleep (<7 points)

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Sleep disturbance (≥7 points)
Effective Criteria:
Individual level: PSQI score decreases to the non-sleep disturbance range (<7 points).
Inter-group comparison: Difference is statistically significant (P < 0.05).
(2) Gut microbiota Indicators
After 90 days of intervention (Month 3):
①Alpha Diversity Analysis
Indices: Shannon, Simpson, and Richness indices.
Method: The differences in alpha diversity indices between the two groups post-intervention
will be compared using an independent samples t-test (if normality and homogeneity of variance
assumptions are met) or the Mann-Whitney U test (nonparametric).
②Beta Diversity Analysis
Metric: Based on Bray–Curtis dissimilarity.
Method: Dimensionality reduction and visualization will be performed using Principal
Coordinate Analysis (PCoA) and Non-metric Multidimensional Scaling (NMDS).
③Differential Species Analysis
Taxonomic Level: Analysis will be conducted at the genus and species levels.
Methods:
LEfSe (Linear Discriminant Analysis Effect Size) will be applied to identify biomarker
species, with a threshold of LDA score > 2.5 and P < 0.05.
DESeq2 will be used for differential abundance testing. Significant differential species will
be defined as those with an FDR-adjusted P < 0.05 and |log₂ fold change| > 0.
(3) Serum metabolite Indicators
Method: An Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) model will
be constructed based on untargeted metabolomic data.
Screening Criteria: Metabolites satisfying both Variable Importance in Projection (VIP) > 1.0
and Student’s t-test P < 0.05 (or the corresponding nonparametric test) will be defined as
differential metabolites between groups. KEGG pathway enrichment analysis will be performed
on the identified differential metabolites. Post-intervention differences in these indicators between
the RGLS and placebo groups will be compared. Statistical significance is defined as P < 0.05 (or

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FDR < 0.05).
11.3 Exploratory Endpoint
To compare changes in thyroid function parameters (TSH,T3,T4,FT3,FT4,TPOAb,Tg) from
baseline to Month 3 between the RGLS and placebo groups.For between-group comparisons,
continuous variables will be analyzed using a t-test if normally distributed, or the Mann-Whitney
U test if not normally distributed.
12. Adverse Event Evaluation
12.1 Definition of Adverse Event (AE)
Any unfavorable or unintended occurrence during the clinical trial period, including:
(1) New clinical symptoms/signs.
(2) Laboratory abnormalities (worsening by ≥1 grade compared to baseline).
(3) Exacerbation of pre-existing conditions (according to NCI-CTCAE v5.0 standards).
Regardless of its relationship to the trial drug.
12.2 Causality Assessment
Using the WHO-UMC Causality Assessment System.
Required Tests: Blood routine, high-sensitivity C-reactive protein (hs-CRP), liver and kidney
function (ALT/AST/Scr).
Supportive Tests: Based on clinical presentation, may include ECG, thyroid function tests,
etc.
12.3 Monitoring Process
(1) Recording Standards:
Complete documentation on a dedicated CRF page: Time of onset, severity (Grade 1-5),
management measures, outcome.
Investigator signs the "Adverse Event Assessment Form."
(2) Reporting Mechanism:
Serious Adverse Events (SAE): Investigators must report to the principal investigator and the
institutional ethics committee within 24 hours of becoming aware of the event, and subsequently
submit a detailed follow-up report within 7 days. For fatal or life-threatening SAEs, an additional
report must be submitted to the Center for Drug Evaluation of the National Medical Products
Administration within 24 hours.

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(3) Safety Dataset: Based on the Full Analysis Set (FAS) principle, including all exposed
subjects.
13. Data Management
13.1 Case Report Form (CRF) Management
(1) Use standardized CRF forms.
(2) Implement three-level quality control: Investigator completion →Monitor verification →
Data manager review.
13.2 Data Entry Standards
(1) Software System: EpiData 3.1 Professional.
(2) Entry Process:
Double data entry by independent personnel (Kappa consistency ≥0.9).
Logical checks (pre-set value ranges/skip logic).
Query resolution mechanism.
13.3 Database Locking Criteria
(1) Prerequisites: Completion of blind data review.
(2) Locking Procedure:
Joint signature by statistician, investigator, and sponsor.
Generation of a read-only database (with audit trail enabled).
Backup of the original database (encrypted with SHA-256).
14. Clinical Trial Management and Quality Control
14.1 Ethical Compliance Management
(1) Any protocol amendments must receive written approval from the Ethics Committee
(Approval document archive number:IRB-2026-288, IIT).
(2) Implement a dual-signature system for informed consent (investigator - subject/guardian).
(3) Informed Consent Process: Prior to the screening visit, a trained investigator will provide
a detailed explanation to potential subjects regarding the study purpose, procedures, potential risks
and benefits, alternative treatments, confidentiality provisions, and the principle of voluntary
participation. Subjects will be given adequate time to ask questions and will sign the written
informed consent form in a pressure-free environment. A copy of the signed consent form will be
provided to the subject for their retention.

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14.2 Standardized Operating Procedures (SOPs)
(1) Personnel Training:
Unified training for GCP-certified investigators (training records archived).
Consistency assessment for scale assessors (ICC ≥0.8).
(2) Data Collection:
Source Data Verification (SDV) ratio ≥20%.
Outlier determination: Refer to standard reference ranges of the laboratory LIS system.
14.3 Document Management Standards
(1) Recording Requirements:
CRF completion follows ALCOA principles (Attributable, Legible, Contemporaneous,
Original, Accurate).
Modification standards: Preserve original entries; modifications signed and dated by the
person making the change.
(2) Retention Period:
Original documents (including HAMD-24/PSQI scales) retained for 5 years after trial
completion.
Electronic data backed up to the National Clinical Research Data Platform.
14.4 Quality Monitoring Mechanism
(1) Implement three-level quality control: Investigator self-check →Institutional QC →
Sponsor audit.
(2) Key document list management (Essential Document List).
15. Statistical Analysis of Trial Data
Statistical work will be undertaken by Professor Yi-Min Zhu, an expert from the Division of
Biostatistics, Department of Public Health at Zhejiang University. He will participate in trial
design and protocol implementation, be responsible for data management and statistical analysis,
and complete the statistical summary report.
15.1 Full Analysis Set (FAS)
(1) Inclusion Criteria:
Randomized
patients
with
post-operative
papillary
thyroid
carcinoma
(≥30
days
post-operation).

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Received at least one dose of the trial drug.
(2) Data Handling: Missing values addressed using Multiple Imputation.
(3) Analysis Purpose: Intention-to-Treat (ITT) analysis for primary efficacy endpoints.
15.2 Per-Protocol Set (PPS)
(1) Criteria:
Fully adhered to the trial protocol.
Medication compliance ≥80%.
No major protocol deviations.
(2) Analysis Purpose: Sensitivity analysis.
15.3 Safety Analysis Set (SAS)
(1) Composition Criteria:
Received at least one dose of the trial drug.
Has post-baseline safety assessment data.
(2) Analysis Purpose:
Calculation of adverse event incidence rates.
Analysis of laboratory abnormalities.
16. Statistical Analysis Plan
Statistical analysis will be performed using SPSS 25.0, GraphPad Prism 9 and R 4.3.1.
(1) Subject Distribution: Comparison of dropout and exclusion rates between groups using χ²
test or Fisher's exact test.
(2) Comparability Analysis: Comparison of demographic data and other baseline indicators to
assess group comparability. Use χ² test, Fisher's exact test, CMH test, t-test, ANOVA, or
non-parametric tests based on data type and distribution.
(3) Compliance Analysis: Comparison of whether subjects in both groups used the trial drug
on time and in the correct dosage and avoided prohibited medications. Primarily use χ² test or
Fisher's exact test.
(4) Efficacy Analysis: Statistical methods for two independent samples to compare
differences between the experimental and control groups. If normality and homogeneity of
variance assumptions are met, use t-test; otherwise, use Mann-Whitney U test (Wilcoxon
rank-sum
test).
Furthermore,
perform
repeated
measures
mixed
linear
model
analysis

17 / 27
incorporating group (experimental/control), time point (baseline, end), and group-by-time
interaction as fixed effects.
(5) Safety Analysis: Descriptive statistics for adverse reactions. Comparison of adverse
reaction rates between groups using χ² test or Fisher's exact test.
(6) All statistical tests should report the test statistic and corresponding p-value. For Fisher's
exact test, report the p-value directly. A p-value ≤0.05 indicates statistical significance.
17. Blinding, Unblinding, and Emergency Unblinding
17.1 Blinding Design
(1) Randomization Coding System:
Use block randomization method (block size=6).
Generate random allocation sequence using SAS 9.4.
Establish three treatment groups: A/B/C.
(2) Drug Blinding Specifications:
Double-blind design: Both subjects and research team remain unaware of group assignment.
Emergency envelopes: Sealed and stored in the institutional pharmacy (temperature
monitored).
Blind code custody: Three copies (statistics team/sponsor/institutional office).
17.2 Formal Unblinding Process
(1) Prerequisites for Unblinding:
Database locked (confirmed by three parties).
Final version of the Statistical Analysis Plan (SAP) completed.
(2) Unblinding Procedure:
First-level unblinding: Reveal the attribute of groups A/B/C (e.g., which is active/placebo).
Second-level unblinding: Disclose the specific intervention scheme for each group.
Opening of the blind code requires witnessing by the statistician, investigator, and monitor.
17.3 Emergency Unblinding Criteria
(1) Indications:
Serious Adverse Event (SAE, ≥CTCAE Grade 4).
Life-threatening complications.
(2) Execution Process:

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Three-level approval: Investigator →Institutional Office →Independent Data Monitoring
Committee (IDMC).
Submit the "Unblinding Record Form" within 24 hours after unblinding.
(3) Handling Principles:
The unblinded case is immediately withdrawn from the study.
Initiate the alternate randomization number mechanism.
18. Trial Schedule
Clinical trial period: March 2026 - July 2026.
Completion of final report: August 2026.
References
1.Polityńska, B.et al. Is depression the missing link between inflammatory mediators and cancer?
Pharmacology & therapeutics.240,108293 (2022).
2.Duval,
F.et
al.
Thyroid
axis
activity
and
dopamine
function
in
depression.
Psychoneuroendocrinology.128,105219 (2021).
3.Liu, X.et al. Complex relationship between gut microbiota and thyroid dysfunction: a
bidirectional two-sample Mendelian randomization study. Frontiers in endocrinology.14,1267383
(2023).
4.Pizzato, M.et al. The epidemiological landscape of thyroid cancer worldwide: GLOBOCAN
estimates
for
incidence
and
mortality
rates
in
2020.
The
lancet
Diabetes
&
endocrinology.10,264-72 (2022).
5.Chen, W.et al. Association of Total Thyroidectomy or Thyroid Lobectomy With the Quality of
Life in Patients With Differentiated Thyroid Cancer With Low to Intermediate Risk of Recurrence.
JAMA surgery.157,200-9 (2022).
6.Mayneris-Perxachs, J.et al. Microbiota alterations in proline metabolism impact depression. Cell
metabolism.34,681-701.e10 (2022).
7.Chen, R.et al. Progress in research on gut microbiota in ethnic minorities in China and
consideration of intervention strategies based on ethnic medicine: A review. Frontiers in cellular
and infection microbiology.12,1027541 (2022).
8.Goodwin, G. M.et al. Single-Dose Psilocybin for a Treatment-Resistant Episode of Major
Depression. The New England journal of medicine.387,1637-48 (2022).
9.Guo, C.et al. Ganoderma lucidum polysaccharide modulates gut microbiota and immune cell
function to inhibit inflammation and tumorigenesis in colon. Carbohydrate polymers.267,118231
(2021).
10.Zhang, Y.et al. Early Enhancement of Neuroplasticity Index, the Ratio of Serum Brain-Derived
Neurotrophic Factor Level to HAMD-24 Score, in Predicting the Long-Term Antidepressant
Efficacy. Frontiers in behavioral neuroscience.15,712445 (2021).
11.Yin, X.et al. Effect of Electroacupuncture on Insomnia in Patients With Depression: A
Randomized Clinical Trial. JAMA network open.5,e2220563 (2022).

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12.Yan, N.&Hu, S. The safety and efficacy of escitalopram and sertraline in post-stroke depression:
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Appendix 1: Hamilton Depression Rating Scale (HAMD)
Hamilton Depression Rating Scale (HAMD)
Name_______________ Age_______Gender_______________ID _________
Outpatient/Hospital No._______________Diagnosis_______________
Date_________
Home Address______________________Contact Phone_____________Rater_________
Circle the score that best fits the patient's condition.
1．Depressed Mood
0
1
2
3
4
2．Feelings of Guilt
0
1
2
3
3．Suicide
0
1
2
3
4
4．Insomnia (Early)
0
1
2
5．Insomnia (Middle)
0
1
2
6．Insomnia (Late)
0
1
2
7．Work and Activities
0
1
2
3
4
8．Retardation
0
1
2
3
4
9．Agitation
0
1
2
3
4
10．Anxiety (Psychic)
0
1
2
3
4
11．Anxiety (Somatic)
0
1
2
3
4
12．Somatic Symptoms (Gastrointestinal)
0
1
2
13．Somatic Symptoms (General)
0
1
2
14．Genital Symptoms
0
1
2
15．Hypochondriasis
0
1
2
3
4
16．Loss of Weight
0
1
2
17．Insight
0
2
18．Diurnal Variation: A. Morning
0
1
2
B. Evening
0
1
2
19．Depersonalization & Derealization
0
1
2
3
4
20．Paranoid Symptoms
0
1
2
3
4
21．Obsessional Symptoms
0
1
2
22．Helplessness
0
1
2
3
4
23．Hopelessness
0
1
2
3
4
24．Worthlessness
0
1
2
3
4
Note：
Total Score：

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Appendix 2: Pittsburgh Sleep Quality Index (PSQI)
Pittsburgh Sleep Quality Index (PSQI)
Name _________ Gender ____ Age ____ Education Level _________
Instructions: The following questions relate to your usual sleep habits during the past month only.
Your answers should indicate the most accurate reply for the majority of days and nights in the
past month. Please answer all questions.
1.During the past month, what time have you usually gone to bed at night? (Please use 24-hour format)
Bedtime: ____ Hour ____ Minute
2.During the past month, how long (in minutes) has it usually taken you to fall asleep each night?
①Less than 15 minutes ②16-30 minutes ③31-60 minutes ④More than 60 minutes
3.During the past month, what time have you usually gotten up in the morning? (Please use 24-hour
format)
Wake-up time: ____ Hour ____ Minute
4.During the past month, how many hours of actual sleep did you get at night? (This may be different
than the number of hours you spent in bed.)
Actual sleep time: ____ Hours ____ Minutes
For each of the remaining questions, check the one best response. Please answer all questions.
During the past month, how often have you had trouble sleeping because you.
5.Cannot get to sleep within 30 minutes
①Not during the past month ②Less than once a week
③Once or twice a week ④Three or more times a week
6.Wake up in the middle of the night or early morning
①Not during the past month ②Less than once a week
③Once or twice a week ④Three or more times a week
7.Have to get up to use the bathroom
①Not during the past month ②Less than once a week
③Once or twice a week ④Three or more times a week
8.Cannot breathe comfortably
①Not during the past month ②Less than once a week
③Once or twice a week ④Three or more times a week
9.Cough or snore loudly
①Not during the past month ②Less than once a week
③Once or twice a week ④Three or more times a week
10. Feeling too cold

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①Not during the past month ②Less than once a week
③Once or twice a week ④Three or more times a week
11. Feeling too hot
①Not during the past month ②Less than once a week
③Once or twice a week ④Three or more times a week
12. Having bad dreams
①Not during the past month ②Less than once a week
③Once or twice a week ④Three or more times a week
13. Experiencing pain
①Not during the past month ②Less than once a week
③Once or twice a week ④Three or more times a week
14. Other reasons (please describe)
During the past month, how often have you had trouble sleeping because of the reasons listed above?
①Not during the past month ②Less than once a week
③Once or twice a week ④Three or more times a week
15. How would you rate your overall sleep quality during the past month?
①Very good ②Fairly good ③Fairly bad ④Very bad
16. During the past month, how often have you taken medication (prescribed by a doctor or "over the
counter") to help you sleep?
①Not during the past month ②Less than once a week
③Once or twice a week ④Three or more times a week
17. During the past month, how often have you had trouble staying awake while driving, eating meals,
or engaging in social activities?
①Not during the past month ②Less than once a week
③Once or twice a week ④Three or more times a week
18. During the past month, how much of a problem has it been for you to keep up enthusiasm to get
things done?
①No problem at all ②Only a very slight problem
③Somewhat of a problem ④A very big problem
19. Do you have a bed partner or roommate?
①No bed partner or roommate ②Partner/roommate in other room
③Partner in same room, but not same bed ④Partner in same bed