Safety and Efficacy of Jiaotai Wan in Patients with Depression: Study Protocol for a Randomized Controlled Trial

Backgroud: Depression is a common affective disorder characterised by marked and lasting melancholia, with corresponding thought and behaviour changes. Due to an accelerated pace of life and increased work pressure, the incidence of depression has risen sharply, causing great harm to family and social life. Jiaotai Wan (JTW) is a Chinese herbal formula that is commonly prescribed for depression and insomnia in clinical treatment, and exhibits antidepressant effects as shown in animal experimental research. However, there are no standard clinical trials to confirm its efficacy in treating depression and insomnia. Therefore, this study aims to assess the efficacy and safety of JTW in the treatment of depression. Methods: A randomized, multicentre clinical trial with parallel groups was designed in this study. A total of 40 patients with depression were included and randomly divided to either the treatment or the control group with a ratio of 1:1. The patients received JTW plus fluoxetine or fluoxetine alone once per day for eight weeks. The primary outcome was analysed using the Hamilton Depression Rating Scalescore for patients in the treatment group compared with that in the control group. The secondary outcomes included Traditional Chinese medicine syndrome integral scale scores, Wisconsin Card Sorting Test, blood metabonomics, urine metabonomics, and brain structure and function by functional magnetic resonance imaging. Discussion: The results of this trial will find changes in brain structure, brain function, and metabolism in patients with depression, and provide critical evidence for JTW in the treatment of depression.

Depression is a common mood disorder with high incidence rates of recurrence, disability and suicide [1]. The World Psychiatric Association survey has shown that current incidences of depression are 4.2% globally and 6.9% in China, respectively.
Depression has now become a serious worldwide public health and social issue to affect patients' physical and mental health and reduce the quality of life. Moreover, depression can even lead to aggressive behaviours, and cause harm to individuals and families [2][3][4][5].
Traditional Chinese medicine (TCM), with outstanding treatment effect on the depression-related symptoms, has been extensively applied in China to treat the depression [6][7]. Traditional antidepressant western medicine including tricyclic compounds, monoamine oxidase inhibitors and selective 5-hydroxytryptamine reuptake inhibitors, etc. can effectively relieve depressive symptoms. However, many deficiencies exist such as long medication cycle, serious toxic and side effects, poor medication compliance, repeated disease delay, etc., which can greatly affect the prognosis and clinical application of the disease [8]. TCM also has therapeutic effects on depression-related symptoms For example, Xiaoyaosan [9] is used to soothe liver, relieve depression and invigorate spleen; Kai-xin-san [10] is applied to invigorate qi, nourish heart and tranquilize mind; Chaihu Shugan San [11], etc. A meta-analysis result has shown that [12] Chaihu-longgu-muli decoction or combination of antidepressants is superior to antidepressants alone in the treatment of depression, which can significantly reduce the incidence of adverse reactions during medication. JTW comprises two TCM herbs, Coptis chinensis Franch and Cinnamomum cassia Presl, which are mainly used to treat depression and insomnia. Several experimental animal studies have demonstrated the effect of JTW on depression mouse models, but no standard clinical evidences can support those claims [13][14][15][16]. Therefore, in this study, a clinical study was designed to evaluate the efficacy and safety of JTW on patients diagnosed with depression.

Study design
This is a multicentre, randomized and controlled trial to evaluate the efficacy and safety of JTW in patients with depression. A flowchart for the study is shown in Qualified patients were randomly divided into the treatment and control groups with a ratio of 1:1, respectively. Recruited patients received JTW plus fluoxetine or fluoxetine alone once per day for eight weeks. After the 8-week treatment period, the patients were then followed up for four weeks. The outcome measures included the Hamilton Depression (HAMD) Rating Scale scores, TCM syndrome integral scale scores, Wisconsin Card Sorting Test (WCST), blood metabonomics, urine metabonomics and brain structure and function on functional magnetic resonance imaging (fMRI).

Inclusion criteria
To participate in the study, patients should meet the following criteria: (2) Conform to the diagnostic criteria of TCM syndrome of disharmony between the heart and kidney; (3) HAMD 24-item score of ≥20 points and <35 points; (4) Age, 18-65 years; (5) Total HAMA score of ≤21 points, depressed mood (item 6) score ≥2 points and anxious mood (item 1) score <3 points; (6) Those who have not taken antidepressants or have taken them but have stopped taking them for two weeks; (7) Sign the informed consent form.

Exclusion criteria
Patients with the following criteria will be excluded: (1) Secondary to other mental or physical illness and depression with severe psychotic symptoms; (2) Severe anxiety (HAMA total score >21 points); (3) Allergic constitution or previous research on drug allergy; (4) Suicidal thoughts; (5) Bipolar disorder, refractory depression; (6) Intracranial cerebrovascular disease, neurodegenerative diseases, intracranial tumour, high blood pressure, or diabetes, leading to brain vascular distribution or abnormal blood flow of systemic disorders; (7) Serious diseases of other systems or severe heart, liver and renal insufficiency; (13) Poor compliance or inability to be interviewed regularly.

Withdrawal and discontinuation
During the trial, if they had any adverse reactions, the participants could stop the trial at any time. No strategy was adopted to improve adherence to the study drug.
During the trial, the drugs related to depression were banned, while those not related to depression could be used. Patients with severe illnesses could withdraw the trial and take the appropriate treatment.

Ethics and recruitment
All patients signed the informed consent forms prior to inclusion, which included the research protocols, the benefits and risks, confidentiality, costs, and voluntary principles. The study has been approved by the Ethics Committee of Tianjin University of Traditional Chinese Medicine. If the protocol requires modification, ethical approval will be reapplied. Any revisions in the study protocol will be

Sample size
In reference to the relevant literature, the specific process to estimate the sample content of each group was as follows [17][18][19]. The sample size estimation formula for clinical trials is written as where n is the number of cases required for each group, μ 1 and μ 2 are the expected averages of the treatment and control groups, respectively, and σ 2 is the standard deviation of the control group, α = 0.05, β = 0.1, the output ƒ(α, β) of the look-up table is 10.5. Taking the score of HAMD scale after treatment as the main evaluation index, through consulting previous literatures [20][21][22] and taking the average, μ 1 = 9.39, μ 2 = 12.49, and σ 2 of the control group is 2.97, the above research data were substituted into the above formula to calculate that n is 19.2, and the sample size of both the treatment and control groups was 20 cases with total 40 cases.

Randomization
Treatment allocation was done when participants met the inclusion criteria and signed the informed consent form. Patients were randomly divided into two groups, the treatment and control groups, with a 1:1 distribution ratio according to the random numbers generated by IBM SPSS Statistics software version 19.0. The recruiter obtained a sequence number from the distributor when a patient was eligible for the trial. The subjects' allocation was concealed inside the sealed, opaque and serialized envelopes to avoid the influence from the biases of the researchers, who took responsibility of recruitment and treatment assessment. To ensure strict confidentiality, the envelopes were not transparent, even under intense light. The envelopes would be opened, and the assigned intervention type would be recognized only after each subject finished all baseline evaluations.

Blinding methods
This study was a single-blind trial. The evaluators knew the group allocations and the treatment protocol for better operation, while the subjects and statisticians were unaware of the group allocations in order not to be disturbed by subjective factors. The treatment assignments were also kept secret for the principal investigator, statistician and outcome evaluator throughout the trial before locking the database.

Interventions
Patients randomized to the treatment group were administered one bag of JTW

4.
Brain structure and function on fMRI: MRI can reveal brain tissue abnormalities in patients with depression in the aspects of brain microstructure, brain function changes and brain metabolite changes, and fMRI combines the three aspects of function, anatomy and imaging [23][24][25]. Diffusion tensor imaging (DTI) technique was used to detect differences and changes in fractional anisotropy values in the whole brain. The changes in cerebral white matter integrity in patients with depression before and after treatment were compared in order to study the effect of JTW on brain metabolites and the changes in brain metabolites by magnetic resonance spectroscopy (MRS). The comparison of the brain structure and function on fMRI discovered that the mean scores of 8 weeks were different from 0 weeks.

Safety reporting
Adverse events (AEs) are defined as any unfavourable and unintended sign,

Data collection, management and quality control
The hard copies of CRFs were used to collect relevant data for each participant, and participants were assessed every four weeks during the study. Data on HAMD, WCST and TCM syndrome integral scale were collected at each visit (week 0 ± 3 days, week 4 ± 3 days, week 8 ± 3 days and week 12 ± 3 days). Blood metabolomics, urine metabolomics and fMRI were carried out only at the first and third visits (week 0 ± 3 days and week 8 ± 3 days, respectively). The SPIRIT flowchart of the trial is shown in Table 1.
After being reviewed by clinical inspectors, completed CRFs were sent to a specified statistics centre, and data input and processing were performed by two individual data administrators to ensure data accuracy. All documents gathered in this study were stored securely. Participants were referred to by a specific randomization number rather than by their names in all other documents in this study, except for the informed consent form. Statistical analysis was conducted as an intention-to-treat analysis by using SPSS Statistics version17.0 software. A two-sided P-value of <0.05 was considered to be statistical significant. Baseline differences among the groups were evaluated using 1-factor ANOVA for measurement data and the χ 2 test for enumeration data, respectively. In addition, χ 2 test was used for categorical variables. The changes in scores from baseline to endpoint of treatment were assessed using a paired t test for measurement data and a signed rank test for enumeration data, respectively.
Comparisons between groups were performed using ANOVA and a rank test.
To limit the extent of missing data, when the assigned treatment was discontinued, efforts were made to obtain the participant's consent to collect the data on treatments and outcomes.
The researchers conducting the data analysis were not involved in experimental or clinical decision-making processes to avoid any bias caused by subjective factors from the researchers.

Discussion
Depression is one of the most common mental disorders and social issues worldwide. In the modern society, the incidence of depression has risen dramatically due to an accelerated pace of life and increased work pressure [26]. Treatment of depression by Western medicine has more side effects, more adverse reactions and shorter duration of action than that by TCM; therefore, Western medicine should not be used for a long term. In addition, many patients do not respond adequately to the treatment [27][28]. Prevention and treatment of depression by TCM can date back thousands of years ago. TCM is more stable and effective, with fewer side effects and better patient compliance than Western medicine; that is one of the reasons why China has become a hot spot for medical tourism in the recent years [29].
JTW is a commonly used formula for clinical treatment of insomnia and depression.
The research team used mouse suspension experiments and mouse forced swimming experiments to study the antidepressant effect of JTW in different proportions, the results showed that JTW had antidepressant effect, and the antidepressant effect was dose-dependent. The research group also conducted a mouse reserpine antagonism experiment, and found that JTW can exert antidepressant effects by regulating the levels of NE and 5-HT in hippocampal tissue and cortex [13][14][15][16]. Clinically, the addition and subtraction of JTW as the main prescription can obviously relieve the symptoms of palpitations, insomnia, anxiety, and irritability in patients with depression, but no standard clinical evidences can support those claims. Therefore, we conducted a multi-center clinical randomized controlled trial, using the HAMD scale and the TCM syndrome integral scale as the evaluation criteria for the efficacy, combined with the application of multimodal fMRI technology and combining metabolomics, to analyze the changes in brain structure and brain function, blood, urine metabolic fingerprints of patients with depression, and the intervention effects of JTW, to study the efficacy and mechanism of anti-depression of JTW, and provide scientific basis for clinical treatment of depression in JTW.
Our study has several limitations. First, since this study is undertaken in China, it is uncertain whether the effects of JTW would be similar in other ethnic groups.
Second, because of the small sample size, the extrapolation of the results of this test is poor. Moreover, at present, the diagnosis of depression only depends on the subjective judgment of clinicians on the type and severity of symptoms, and lacks objective laboratory diagnostic methods, which also brought uncertainty for us to evaluate whether the clinical symptoms of depression were alleviated. Important changes to the protocol will be submitted to the TJUTCM for review.
Informed consent will be obtained from all participants in the trial.

Consent for publication
The results will be offered for publication in peer-reviewed journals.
and YT were in charge of the recruitment of patients, data collection and management. All authors read and approved the final manuscript.