2.1.1 Ethical approval and study registration.

This study protocol was approved by the Medical Ethics Committee of the First Teaching Hospital of Tianjin University of Traditional Chinese Medicine (item number: TYLL2023[Z]004) and was registered with the China Clinical Trial Registry (item number: ChiCTR2300071386)

2.1.2 Informed consent.

The study will be carried out following the Declaration of Helsinki formulated by the World Medical Association and relevant clinical research regulations in China. Before patients are enrolled in this study, the researcher is responsible for fully and comprehensively introducing the purpose, procedure, and possible risks of this study to him or his designated representative in written form. Patients should be made aware of their right to withdraw at any time. All participants will obtain written informed consent before enrollment. The researcher will ensure that each patient signs an informed consent form before participating in the study, which will be kept in the study file. Patients who may be pregnant must be informed that if they get pregnant during the study, the study may endanger the fetus. Before taking part in this study, the patient should agree to use contraception during the study.

2.2.1 Inclusion criteria.

Participants who meet the following criteria will be included.

1. Diagnosed with stroke and meet the diagnostic criteria for “Mood disorder characterized by depression or accompanied by severe depressive episode caused by a stroke can also be characterized by mania or mixed characteristics (DSM- Ⅳ)” and “Depressive disorder due to another medical condition” in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-V) [22].
2. Experience a first episode of stroke onset within 6 months.
3. Age between 35 and 80 (inclusive).
4. The HAMD score of 8 to 17 (inclusive).
5. Clear consciousness, stable vital signs, cooperative physical examination.

2.2.2 Exclusion criteria.

1. Patients who have received antidepressant treatment within 2 weeks or are participating in other clinical trials for antidepressants.
2. Patients who have consciousness disorder or obvious cognitive impairment function (Mini-mental State Examination score <17).
3. Patients who have severe aphasia and cannot communicate.
4. History of depression prior to this stroke event.
5. Patients with severe liver and kidney dysfunction (alanine aminotransferase more than 3 times the upper limit of normal, and serum creatinine is more than 180 μmol/L).
6. Pregnant and lactating women.
7. Intolerance or refusal to receive acupuncture or electroacupuncture.

2.2.3 Shedding criteria.

1. Patients who have severe adverse effects will be removed from the experiment at the discretion of the researcher.
2. Patients whose condition progresses during the experiment, influencing efficacy and safety decisions.
3. Patients with low compliance (80% treatment compliance) or patients who received treatments not part of this trial.

2.2.4 Termination criteria.

1. Severe safety issues occurred during the trial.
2. The situation where the study does not have any clinical value, i.e., The effect of acupuncture or electroacupuncture is much lower than that of modern medicine on the market.

2.5.1 Control group.

Patients in the control group will receive conventional rehabilitation treatment with medication and acupuncture for 4 weeks.

1. Acupuncture treatment based on XNKQ Acupuncture for stroke dysfunction, acupoint locations are as described in Table 2. Acupoints include Shuigou (DU26), bilateral Neiguan (PC6), affected side Sanyinjiao (SP6), affected side Jiquan (HT1), affected side Chize (Lu5), affected side Weizhong (BL40), affected side Hegu (LI4), affected side Zusanli (ST36). The patient is placed in a supine position. After skin disinfection, the acupuncturist holds a disposable acupuncture needle (0.25mm×40mm Hwato brand, Suzhou Medical Supplies Factory Co., Ltd., Suzhou, China) for acupuncture operation. It should be noted that XNKQ Acupuncture uses a specific operation method, which is explained in Table 2. The acupuncture method is used to “De Qi” before the acupuncture is performed. Then all needles would be removed after being retained for 30 minutes. All patients will receive treatment six times each week for four weeks.
2. Oral escitalopram tablets 10mg Qd for 4 weeks.

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Table 2. Specific acupuncture methods and locations of acupoints.

https://doi.org/10.1371/journal.pone.0303162.t002

2.5.2 Antidepressant acupuncture group.

The patients will be placed in a semi-supine position, i.e., the bed rocker is elevated to 45°, and after skin disinfection, the treatment proceeds as follows.

1. The exact XNKQ acupuncture as in the control group is given, and the same acupuncture method is used to “De Qi” before the antidepressant acupuncture is performed [23]. Lifting-thrusting and twirling-rotating manipulations are acupuncture procedures used to make patients feel "De Qi," such as soreness, numbness, and swelling.
2. Antidepressant acupuncture is given. The acupoint selections and ear-treating areas are Baihui (DU20), Yintang (EX-HN3), Fengfu (DU16), and auricular concha (corresponding to the auricular point Xin (CO15) and auricular point Shen (CO10)). The specific acupoint locations and operation diagram are shown in Table 2 and Fig 2. The specific operations are as follows. The Baihui (DU20) point is inserted to a depth of 3 to 5 mm at an angle of 15°. The Yintang (EX-HN3) point is inserted to a depth of 10 to 15 mm at an angle of 45° (Needle tip towards the root of the nose). The Fengfu (DU16) point is inserted to a depth of 15 to 20 mm at an angle of 45° (Needle tip towards the Laryngeal node area). The auricular concha is equivalent to the position of the auricular point Xin (CO15) and auricular point Shen (CO10) of the auricular acupoint, with a depth of 2 to 3 mm and an angle of 90°. The two electrode clamps of the electroacupuncture instrument are then clamped to the shanks of the two acupuncture needles. Baihui (DU20) and Yintang (EX-HN3), connected to a group of electro-acupuncture, auricular acupoint Xin (CO15) and acupoint Shen (CO10) connected to another group of electro-acupuncture (Hwato SDZ-IIB type electro-acupuncture instrument, Suzhou, China). The researcher set the frequency at 2/15Hz, the intensity at 2mA, and the time at 30min, and alternately acupuncture/electro-acupuncture at bilateral auricular acupoints. All patients will be treated once a day, 6 sessions per week for 4 weeks.

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Fig 2. Auricular concha acupoints Shen(CO10) Xin(CO15) acupoints Bai-Hui(Du20) Yin-Tang(EX-HN3) Feng-Fu(DU16).

https://doi.org/10.1371/journal.pone.0303162.g002

2.5.3 Sample size.

In this study, HAMD-17 is used as the leading indicator for sample size calculation. Based on the results in the preliminary clinical study, i.e., observation of the Hamilton Depression Scale score reduction (ΔHAMD) results of 6.13 ± 1.96 Mean ± SD reduction in the test group and 4.57 ± 1.87 reduction in the control group, according to the sample size calculation formula.

α = 0.05, 1-β = 0.9, σ = 1.96, δ = (6.13–4.57) = 1.56

α is set to 0.05, the statistical power is set to 0.9, and a two-sided test is conducted. Based on the calculated dropout rate of 20%, each group will eventually require 42 patients; that is, a total of 84 participants will be randomly divided into two groups in a 1:1 ratio.

2.6.1 The primary outcome.

2.6.1.1 Hamilton Depression Scale. The Hamilton Depression Rating Scale (HAMD-17) is used to measure the severity of depressive symptoms in people with depressive disorders. The scoring content includes 17 symptom entries of depression, guilt, suicide, difficulty sleeping, lack of deep sleep, early awakening, work, interest, retardation, irritability, psychogenic anxiety, and physical anxiety, which is currently the most widely used.

In addition, the clinical effective rate is calculated by the reduced value of the HAMD-17 score (ΔHAMD), and the curative effect is judged. According to the Nimodipine method, the curative effect index was calculated according to the reduced score of the Hamilton Depression scale (HAMD-17). The formula was as follows,

A = Total score after treatment. B = Total score before treatment

The curative effect is judged as follows:

Recovery: symptom score reduction ≥ 75%.

Significantly effective: 50%≥ symptom score reduction < 75%.

Effective: 25%≥ symptom score reduction < 50%.

Invalid: symptom score reduction < 25%.

Total effective rate according to the formula

R = Recovery. S = Significantly effective. E = Effective. T = Total cases

2.6.2 The second outcome.

2.6.2.1 Self-rating Depression Scale. Self-rating depression scale (SDS) is used to assess patients with post-stroke depression. When evaluating the curative effect, the self-evaluator should evaluate once at the baseline and once at the end of the study at two weeks and the fourth week of treatment. The SDS has 20 items, ten of which are positive and ten others negative. Before the self-evaluator evaluates, the researcher must make the subject understand the filling method of the full scale and the meaning of each question, and then make an independent self-assessment.

2.6.2.2 National Institute of Health Stroke Scale. The National Institute of Health stroke scale (NIHSS) [24] is used to evaluate the severity of neurological deficit symptoms in stroke patients. Researchers need to check and record the results quickly. Researchers cannot train or tell the patient hints unless necessary (such as repeatedly asking the patient to make a particular effort). If some items are not evaluated, they should be explained in detail in the table. Unassessed items can be reviewed and studied by monitoring video and other technical means and discussed with inspectors.

2.6.2.3 Modified Barthel Index. The modified Barthel Index (MBI) is used to evaluate the self-care ability of stroke patients; the higher the score, the stronger the patient’s self-care ability, including defecation, grooming (personal hygiene), toileting, eating, transferring, walking on level ground, dressing, going up and down stairs, and bathing. Each item has five levels and associated points. The lower the score or level of an item, the more the patient’s reliance on it.

2.6.2.4 Serum laboratory index and Serum metabolomics analysis. Serum laboratory index: determination of IL-1β, IL-10, 5-HT, and BDNF. Serum metabolomics analysis: After thawing the serum samples at room temperature, 100μL was absorbed and transferred to a fresh test tube, followed by 300μL methanol and 10μL internal standard (2- chlorophenyl alanine 2.5g/L). After the samples were mixed and centrifuged, 200μL of supernatant was sucked into the sample tube for detection by liquid chromatography-tandem mass spectrometry. The detection equipment was the AcquityTM PLC-Q-TOF-MS platform (Alfa Chemistry, USA). The advanced data features of the obtained data are extracted, processed by XCMS in R, normalized, and formed into a two-dimensional data matrix.

2.7.1 Adverse event record.

In the case report form, an "adverse event record form" is set up, requiring researchers to truthfully fill in the occurrence time, severity, duration, measures taken, and outcome of adverse events.

2.7.2 Adverse event report.

Severe adverse events are reported, and in that case, the researcher must take immediate measures to protect the safety of the subjects and report it to the unit in charge of the research and the ethics committee in time. The researcher should sign and date the report. The applicant will ensure that all reporting procedures required by laws and regulations are met.

2.9.1 Quality control.

Qualified acupuncturists, doctors, researchers, and statisticians in the First Teaching Hospital of Tianjin University of Traditional Chinese Medicine monitored and revised this experimental scheme. They also execute process quality control and data quality control, including compliance control implemented by the researcher, subject compliance control, and follow-up information integrity control, as well as creating a quality control team. Data quality control includes the composition of data supervisors, responsibilities and workflow, input process, interim analysis plan, research termination standard, data verification, data cleaning, etc. In addition, there will be regular supervision by clinical experts. Upon completion of the study, we will provide repository information for our data, all data underlying the findings will be made available as part of the manuscript or supporting information, or deposited in a public repository where the data will be recorded.