Based on the Theory of Gut-brain Axis, the Intervention Effect and Related Mechanism of Ganoderma Lucidum Spore Powder on Depressive Symptoms in Patients With Thyroid Cancer Were Investigated
Phase: NA
Conditions: Depression, Thyroid Cancer
Sponsor: Ling Zhiqiang

Based on the ' gut-brain axis ' theory, the intervention
effect
and
related
mechanism
of
wall-removed
Ganoderma lucidum spore powder on depressive
symptoms
in
patients
with
thyroid
cancer
were
investigated
SETTING : Zhejiang Cancer Hospital
Ethical approval number：IRB-2023-307
Scheme revision time : 2023.3.28
Version number : 3.0

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Scheme signature page
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content of this program, agree to follow this program for
clinical research. I agree to keep this scheme and related
content confidential.
Research Center : Zhejiang Cancer Hospital
Date : 2023.3.28

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Ⅰ.Test background
Depression is an important cause of global disability and mortality, and its
prevalence continues to rise. Compared with the general population, the incidence of
depressive symptoms in patients with thyroid cancer is high. The psychological
survey of thyroid cancer patients after thyroid cancer surgery found that the number of
positive symptom self-rating scale, depression, anxiety and terror factors, and
pessimism factors in the thyroid cancer postoperative group were higher than those in
the control group. In modern society, the acceptance of mental diseases such as
depression is generally low. Patients with depression often do not choose to seek
medical treatment because of their stigma and lack of understanding of mental illness.
As a result, many patients miss the best treatment opportunity because they do not get
timely and accurate diagnosis.
Due to the characteristics of multi-component, multi-target, dynamic and holistic
in the research of traditional Chinese medicine, it is necessary to continuously
develop new strategies to study the therapeutic mechanism of active components of
traditional Chinese medicine. Metabonomics combined with intestinal metagenomics,
metagenomics and other multi-omics platforms can jointly identify new functional
metabolites and functional bacteria, laying a good foundation for the study of the
mechanism of active ingredients related to traditional Chinese medicine. It has been
found that the composition and function of intestinal microorganisms in patients with
depression are imbalanced. Imbalanced microbiota may induce depression and
anxiety through the microbial-gut-brain axis ( biosynthesis and metabolic pathways of
tryptophan ). It may also be involved in the occurrence of depression through other
flora metabolites ( amino acids, fatty acids, short-chain fatty acids, etc. ). Therefore,
this study used the wall-removed Ganoderma lucidum spore powder to intervene in
patients with thyroid papillary carcinoma accompanied by depressive symptoms, and
explored the antidepressant mechanism of the wall-removed Ganoderma lucidum
spore powder through microbial 16 s diversity analysis and metabolomics methods.
Ganoderma lucidum spores are rich in polysaccharide peptides, adenine

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nucleosides, proteins, enzymes, trace elements and other special components. The key
effective components known now are Ganoderma lucidum polysaccharides and
Ganoderma lucidum triterpenes. Ganoderma lucidum is widely used as a medicinal
fungus to prevent and treat many diseases. Studies have shown that Ganoderma
lucidum spore powder has antidepressant effect.
This study was carried out only in this research institution. A total of 300 patients
with papillary thyroid carcinoma accompanied by depressive symptoms were
recruited to participate in this clinical study. This study was divided into study group
and control group, ratio ( 2 : 1 ). From the first day, the experimental group was given
oral administration of 4g of Ganoderma lucidum spore powder ( 1 bag at a time, 2
times a day, 2g / bag ) for 90 days, and the control group was given oral
administration of 4g ' placebo ' ( 1 bag at a time, 2 times a day, 2g / bag ) for 90 days.
Ganoderma powder product instructions : daily care dose is 2g / d, 4g / d can achieve
the therapeutic effect. In the previous study, ' wall-removing Ganoderma lucidum
spore powder ' was used in clinical practice, prevention and reduction of
cancer-related fatigue symptoms in cancer patients, and improvement of the quality of
life of cancer patients in the Second Zhejiang Hospital.Ganoderma lucidum
medication method 2g / time, once in the morning and once in the evening, a total of
4g. The same dose was used.
Ⅱ.test purpose
1.In this study, the antidepressant mechanism of ganoderma lucidum spore powder
was studied by microbial 16 s diversity analysis and metabolomics method, and the
clinical efficacy of ganoderma lucidum spore powder intervention on postoperative
depressive symptoms of papillary thyroid carcinoma was determined.
2.To investigate the effect of Ganoderma lucidum spore powder on the depression of
patients through the communication pathway of microbial-intestinal-brain axis
( biosynthesis and metabolic pathway of tryptophan ), or through other microbial
metabolites ( amino acids, fatty acids, short-chain fatty acids, etc. ) to inhibit the
occurrence of depression in patients. To clarify the antidepressant mechanism of

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wall-removed Ganoderma lucidum spore powder and provide objective basis for its
application.
3.To construct a clinical database of patients with depressive symptoms after papillary
thyroid carcinoma surgery, and to explore the optimal mode of intervention of
exfoliated ganoderma lucidum spore powder in patients with depressive symptoms.
The multi-omics platforms such as metabolomics and intestinal flora were used to
directly find the functional components related to the intervention effect of
wall-removed Ganoderma lucidum spore powder ; combined with clinical data,
mining potential clinical indicators or more sensitive biomarkers ; on this basis, the in
vivo function and mechanism of the target flora ( potential metabolic beneficial
bacteria ) or metabolites ( potential epigenetic or parabiotic ) alone or in combination
will be verified.
Ⅲ.experimental design
1.design description: A randomized, double-blind, parallel, placebo-controlled study.
2.technical route
Patients with depressive symptoms after
papillary thyroid carcinoma surgery
Baseline data
test team
control
Oral administration of ganoderma lucidum spore powder / placebo, 4g / day * 90 days
Data collection :
1.Depression and sleep assessment ;
2.Fecal specimen collection : analysis of microbial 16s diversity ; analysis of
intestinal flora metabolites ;
3.Blood sample collection : analysis of immune indicators
statistical analysis
randomization

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Ⅳ.case selection
4.1 inclusion criteria
( 1 ) Patients with depressive symptoms after papillary thyroid carcinoma
surgery in the outpatient department of Zhejiang Cancer Hospital ( patients with
depressive symptoms by clinical psychiatrist and Hamilton Depression Scale score of
8-20 points ) ;
( 2 ) Han nationality ;
( 3 ) no previous depression and other mental diseases ;
( 5 ) 18-80 years old ;
( 6 ) Women ;
( 8 ) BMI : 19 ~ 24. ( BMI calculation method : BMI = weight ( kg ) / height 2
( m ) )
4.2 excluded criteria
( 1 ) suffering from other diseases of the intestinal system ;
( 2 ) Have received gastrointestinal surgery before intervention ;
( 3 ) Including patients with other malignant tumors, who need chemotherapy,
radiotherapy, biological therapy or traditional Chinese medicine treatment ;
( 4 ) received antibiotics or microecological modulators within 3 months before
the intervention ;
( 5 ) Acute intestinal obstruction ;
( 6 ) Patients with severe depressive symptoms who must receive antidepressant
treatment ;
( 7 ) organic diseases such as heart and brain diseases, brain trauma ;
( 8 ) history of mental illness, use of psychoactive drugs such as drugs ;
( 9 ) Severe liver and kidney dysfunction ;
( 10 ) Pregnancy, lactation
4.3rejecting standard
( 1 ) After enrollment, it was found that the included objects did not meet the
inclusion criteria ;

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( 2 ) Subjects who did not take oral ganoderma lucidum spore powder or
intervene in the course of dissatisfaction after enrollment according to the study
protocol ;
( 3 ) poor compliance, midway active withdrawal or lack of main indicators,
incomplete data, etc., can not determine the efficacy or safety.
4.4 The subjects withdrew from the test standard
( 1 ) It is necessary for researchers to stop the trial from the perspective of
medical ethics ;
( 2 ) patients with severe adverse reactions ( SAE ), complications or special
physiological changes should not continue to accept the test ;
( 3 ) Pregnancy during treatment.
Ⅴ.test scheme
5.1 Number of cases and distribution of cases
According to the ' Drug Registration Management Method ', the study sample
should be no less than 100 pairs. It is assumed that the case shedding rate is 15 %, and
the sample size is not less than 117 pairs. Finally, the sample size was determined to
be 300 cases. In order to reduce the use of placebo in the control group, so that more
patients benefit from treatment, the experimental group : the control group was
assigned to 2 : 1, that is, 200 cases in the experimental group and 100 cases in the
control group.
5.2 Research Drug Information
（1）testing medicine
The main component is the wall-removed Ganoderma lucidum spore powder,
specification : 2g / bag, batch number : the same batch number ;
（2）control drug
Placebo : the main component is the wall shell and accessories of the
wall-removed Ganoderma lucidum spore powder, specification : 2g / bag, batch
number : same batch number
（3）Drug source

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The above drugs were produced and provided by Jinhua Shouxiangu
Pharmaceutical Co., Ltd., and all research drug tests were qualified. If the drug
expiration test is not completed, the drug needs to be re-prepared and blinded.
5.3 medication regimen
（1）From the first day after enrollment, the subjects were given oral research
drugs ( wall-removing spore powder / placebo ), once in the morning and once in the
evening, 1 packet ( 2g ) each time, for 90 days. Taking method : 50ml warm boiled
water into suspension.
（2）dosage adjustment：If this scheme has not been revised in writing, the dose
of the test drug shall not be adjusted.
5.4 drug packaging
One patient in the trial was treated with 6 medium packs of medicine, each
containing 30 small bags. The drugs in the pouch are ganoderma lucidum spore
powder ( 2g ) or placebo ( 2g ).
5.5 Drug storage and recovery
The storage conditions of the drugs studied were : closed, placed in a clean,
ventilated, shady place, away from children 's contact.
All test drugs must be stored safely and sealed, kept and distributed by a special
person, and supervised the subjects to take them on time as required. The researcher
should register and sign the medication record form after each administration to the
patient, and be responsible for recycling the used test drug packaging bags.
Researchers and sponsor supervisors must count all drugs at the end of the trial and
check with the number of drugs issued, the number of missing drugs and the cause of
CRF. At the end of the trial, all unused drugs should be returned to the sponsor, and
the relevant handover procedures and records should be completed according to GCP
requirements.
Ⅵ.drug combination
It is forbidden to use any interfering drugs, such as biological therapy, traditional
Chinese medicine treatment, antibiotic treatment, microecological modulators,
antidepressants, other health care products with immune regulation function,

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psychotropic drugs and so on.
Ⅶ.research process
7.1 Screening / Baseline ( Week 0 )
( 1 ) Sign informed consent ;
( 2 ) Complete the verification of inclusion and exclusion criteria ;
( 3 ) to obtain demographic data and medical history data ;
( 4 ) Psychiatric examination and HAMD-24 scale evaluation ;
( 5 ) PSQI scale evaluation ;
( 6 ) Stool specimen collection ;
( 7 ) Thyroid function test residual blood collection ;
( 8 ) Women of childbearing age to do urine pregnancy test ;
( 9 ) Record the combined medication ;
( 10 ) Drug distribution.
7.2 Visit 1（D28±3d）
( 1 ) Evaluation of HAMD-24 scale ;
( 2 ) PSQI scale assessment ;
( 3 ) Record drug combination and adverse events ;
( 4 ) Drug recovery and distribution.
7.3 Visit 2（D90±3d）
( 1 ) Evaluation of HAMD-24 scale ;
( 2 ) PSQI scale assessment ;
( 3 ) Fecal specimen collection ;
( 4 ) Thyroid function test residual blood collection ;
( 5 ) Record the combined medication and adverse events ;
( 6 ) Drug recovery.
7.4 Principles of index collection
( 1 ) Hamilton Depression Scale ( HAMD-24 ) score and Pittsburgh Sleep
Quality Index score :
The HAMD-24 was evaluated by two senior clinical psychologists, who were
responsible for the assessment of HAMD-24 by Dr. Qiu Yaju. PSQI was evaluated by

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the subjects themselves and guided by psychologists.
（2）Fecal sample collection method :
①The stool collector was placed in the toilet, the feces were discharged into the
collector, and the fresh fecal samples were fully mixed with a slender handle spoon
( Note : to avoid urine contaminated feces ).
②The mixed fecal samples were divided into two 5 ~ 10 mL sterile tubes
marked with good names or pre-sterilized collection tubes, and about 2 ~ 3 mL of
each tube was collected.
③About 1 ml fecal samples were sub-packed into 1.5 ml centrifuge tubes from
5-10 ml sample collection tubes with long-stem wooden rods. Or take about 1 ml to
1.5 ml centrifuge tube directly from the mixed fecal samples in 1.
④The centrifuge tube was covered well, and the fit was checked. According to
the numbering order, it was placed in the sample box and placed in a refrigerator at −
80 °C for cryopreservation.
If the feces are collected at home, the collection tube is packed with a
self-sealing bag and sent to the hospital sample receiving place as soon as possible
( within two hours ).
Notes for sample collection and preservation :
Samples should not be exposed to room temperature for more than 2 hours.
Sample collection should be carried out at the same time of the day to avoid the
influence of rhythm on metabolism.
The sample should be mixed immediately after collection, and frozen after
separation.
Prepare two samples at the same time to avoid repeated freezing and thawing.
（3）Blood sample collection：
①The remaining venous blood after thyroid function test in the laboratory was
injected into the serum / plasma separation tube, and mixed upside down for 5-8
times.
②The whole blood was placed at room temperature ( 20 °C ~ 25 °C ),
standing for about 30min ( no more than 2h ), and the blood was completely solidified,

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and the anticoagulant was added as plasma.
③After the serum / plasma was fully separated, centrifuged at 1000 g for 10 min.
About 500μl-1mL of the supernatant was taken to a 1.5mL centrifuge tube
( recommended multi-tube subpackage ) and placed in a cryopreservation box.
④. The cryopreservation box was placed in a refrigerator at −80 °C and sent
by dry ice.
（4）Stool and blood samples preservation and transportation：①Liquid nitrogen
rapid cryopreservation for 15 min and then transferred to -80 °C refrigerator
cryopreservation.②Ensure that the refrigerator continues to work constantly power,
power will cause the sample freeze-thaw③Shunfeng and other dry ice transport ( in
transit 5kg / day ) or cold chain transport.
VIII.response evaluation criteria
8.1 leading indicator
( 1 ) Hamilton Depression Scale ( HAMD-24 ) score classification
No obvious depressive symptoms : total score 0-7 points ; may have depression :
total score 8-20 points ; mild or moderate depression : total score 21-35 ; severe
depression : total score 36-76 points.
( 2 ) Validity standard
Depression relief : After 90 days of treatment, HAMD score decreased.
8.2 secondary index
( 1 ) Changes of intestinal microbial diversity and metabolites in patients
( 2 ) Remission of sleep status : After 90 days of treatment, the Pittsburgh Sleep
Quality Index score decreased.
( 3 ) Immune enhancement
Ⅸ.Evaluation of adverse events
1.Definition of adverse events : Any disease that occurs during a drug clinical
trial, new symptoms, signs or abnormal laboratory tests, or deterioration of the
original symptoms and signs, whether or not related to the test drug, is an adverse
event.

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2.Relationship between adverse reactions and experimental drugs : Searching
through the CNKI database, no side effects and related adverse reactions of
Ganoderma lucidum were found in the current study. If complained of discomfort,
through laboratory tests such as blood routine, liver and kidney function, platelets,
hemoglobin, determine or determine whether it is related to the test drug.
3.Adverse event recording and reporting channels :
In the event of adverse events, the occurrence time, clinical manifestations,
treatment process and duration, outcome and the relationship between the test drugs
should be recorded in detail on the case report form page. Serious adverse events
should fill in the serious adverse events form and report to SFDA Registry Drug
Research and Supervision Office within 24 hours, notify the team leader unit and the
bidder, and report to the center ethics committee, and submit details within 7 days
thereafter.
Safety evaluation at the end of the trial should include all patients who have used
the test drug, regardless of whether they meet the evaluable criteria.
X.Statistical analysis of test data
1. Data Selection for Statistical Analysis
( 1 ) Full Analysis Set ( FAS ) : Constituted of all cases with depressive
symptoms after papillary thyroid carcinoma surgery that were randomized into groups
and at least 1 month after surgery, that is, Intention-to-treat. The FAS set was used for
efficacy analysis, and the missing data was filled with the last observed data filling
( LOCF ) criterion.
( 2 ) Per-Protocol Set ( PPS ) : PPS is composed of all cases that meet the test
plan, have good compliance, do not use prohibited drugs during the test, and complete
the content of CRF requirements. The PPS set is used for efficacy analysis.
( 3 ) Safety Analysis Set ( SAS ) : It is composed of all cases that have been
randomly grouped, used at least one drug and have safety evaluation. The SAS set is
used for safety analysis.
2. statistical analysis plan

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Statistical analysis was performed using SPSS 9.1.3 statistical software.
( 1 ) Distribution of two groups of cases : the comparison of the shedding and
elimination rates of the two groups was performed by χ2 test or exact probability
method.
( 2 ) Comparability analysis : Demographic data and other basic indicators were
compared to measure the comparability of the two groups. According to the type and
distribution of data, χ2 test or exact probability method, CMH test, t test, variance
analysis or non-parametric statistical method were used.
( 3 ) Compliance analysis : To compare whether the two groups of patients use
the test drugs on time and in quantity. The drugs that were not banned in the regimen
were mainly tested by χ2 test or exact probability method.
( 4 ) Validity analysis : Repeated measures were used to compare the differences
in measurement data such as depression scores and sleep scores between the
experimental group and the control group, or linear regression models were used to
control baseline depression scores or sleep scores. Differences in depression scores
and sleep scores between the two groups. Compare the score after the end of the
experiment with the baseline score, calculate the difference before and after the
change, and use the t test to compare the difference between the score changes of the
experimental group and the control group.
( 5 ) Safety analysis : adverse reactions were statistically described, and the
incidence of adverse reactions in the two groups was compared by χ2 test or exact
probability method.
( 6 ) All statistical tests should give test statistics and corresponding p values.
When using the exact probability method, the p value is given directly. P ≤0.05
indicated that the difference was statistically significant.
XI.Setting up Blindness, Uncovering Blindness and Emergency Uncovering
Blindness
1.Blinding
( 1 ) Subjects were randomly numbered and grouped : The cases were numbered

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according to the time sequence of enrollment, and divided into three groups : A, B,
and C. The patients were divided into three groups according to the time sequence of
enrollment.
( 2 ) Randomly determined grouping : Randomly determined by drawing lots,
group A, group B and group C correspond to ' experimental group ' or ' control group ',
respectively.
2.Provisions on Blinding
Uncover the blind bottom envelope : The three groups of A, B, and C are
provided in the letter as test drugs or reference drugs. The groups corresponding to
each number are assigned to statistical analysts for statistical analysis and statistical
conclusions are made. According to the statistical conclusions, combined with
professional knowledge, make professional conclusions.
3. Emergency Blinding
When the patient has serious adverse reactions or emergency events need to
determine the group for treatment, when the patient has serious complications or
symptoms of deterioration must take emergency measures, emergency cases do need
to break the blind, by the researchers in the center of the person in charge, the person
in charge after consultation with the bidding unit can read the emergency letter to
break the blind. Once the corresponding numbered emergency letter is opened, the
case is treated as a shedding case.
XII.Test schedule arrangement
2023.3-2024.5 Time for clinical trials
2024.9 Complete the summary repor

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