Efficacy of verum and sham acupoint catgut embedding in treatment of simple adult obesity: study protocol for a randomized, placebo-controlled trial

Obesity has became a global health issue. The previous studies suggested that acupoint catgut embedding (ACE) might be a potential treatment option for obesity. In this study, we will conduct a rigorous and normative trial to determine the efficacy of ACE for obesity. A total of 99 eligble patients diagnosed with obesity will be recruited. They will be randomly allocated to either the verum ACE group, sham ACE group or waiting-list group with 33 patients in each group. Each patient in two ACE groups will recieve 8 sessions of treatment over 8 weeks. The primary outcomes will be the reduction of body mass index after treatments. Secondary outcomes will include waist circumference, hip circumference, waist hip rate, body fat percentage, blood lipid level, as well as the subcutaneous fat area, visceral fat area and the scale assessments of World Health Organization Quality of Life. All outcomes will be evaluated at baseline, end of 8 weeks treatments and 3-month follow up. The evaluators and data analysts will be blinded to group allocation. The finding of this randomized, sham- and waiting-list-controlled trial will help to investigate the influence ACE on clinical variables as well as visceral fat area, which will provide high-quality evidence on the of ACE for treatment of AEs: adverse events; FDA: and BMI; body index; RCT: randomized study; WC: waist circumference; VFA: visceral fat area; SFA: subcutaneous fat area; SD: standard deviation; WT: STRICTA: Standards MRI: Imaging; WHR: waist hip rate; BFP: fat percentage; HC: circumference; WHOQOL: World Quality of Life; BP: blood pressure; HR: heart rate; cholesterol; TG: HDL: lipoprotein.

dizziness, nausea and vomiting, insomnia and so on. According to the report by the U. S. Food and Drug Administration (FDA) in 2010, the antiobesity drugs, orlistat [14] and sibutramine [15], were warned and prohibited to use because of its serious liver damage and high risk of CVD, respectively. Additionally, rimonabant [16] was found to induce anxiety, depression and other mental disorders. Consequently, identification of effective and low-risk interventions is urgently needed for obesity. Acupoint catgut embedding (ACE) method, as one of complementary and alternative therapies, has been used for several decades for the treatment of many conditions, such as perimenopausal syndrome, chronic urticaria, depressive disorder, refractory insomnia, obesity, sciatica, etc. [17]. It is a method involving infix self-absorptivly surgical chromic catgut sutures into the the subcutaneous tissue at the region of acupoints with a specialised needle under aseptic precautions. Owing to the continuous acupoint stimulation with inplanted sutures, ACE was considered to be more effective than ordinary acupuncture or electroacupuncture for obesity [18]. In past researches, scholars have found that the body weight and body mass index (BMI) of obesity could obviously decrease after ACE therapy [19]. Morever, ACE could adjust the unbalance of obesity-related hormones, including leptin, ghrelin and adiponectin, to reduce the body weight [19,20]. In addition, visceral fat accumulation are often considered to be accompanied with obesity, which has been rencently found to play a vital role in the development of metabolic syndrome, a cluster of diabetes, dyslipidemia, and hypertension [21]. A randomized controlled study (RCT) conducted by Lei H [22] found that electroacupuncture treatment could reduce BMI and waist circumference (WC) as well as visceral fat area (VFA) of obesity. However, whether the ACE can improve the VFA of obesity and what the relationship between VFA and ordinarily obesity-related indices are still unknown, as well as there have been little RCT directly to investigate the VFA changes in the researches of ACE for obesity.
Hence, this study is designed as a RCT trial to investigate the effectiveness of ACE on obesity, and the safety of ACE will be also examined.

Study design
This single-center, randomized, sham-controlled trial will be conducted at Shenzhen Traditional Chinese Medicine Hospital. The study protocol has been approved by the ethics committee on Shenzhen Traditional Chinese Medicine Hospital. The protocol follows the Declaration of Helsinki and will be reported based on SPIRIT guidelines (Additional file 1).
The flow diagram of the trial has been shown in the Fig. 1.

Population and recruitment
Patients meeting the inclusion criteria of obesity will be recruited mainly through outpatient clinics, advertisements, online or offline (such as newspaper, poster, websites), Wechat public account of Shenzhen Traditional Chinese Medicine Hospital. Informed consent will be required to write before randomisation for all patients. The schedule of enrolment, intervention and assessments is shown in Fig. 2.

Inclusion criteria
The participants in this study have to: (1) be diagnosed with obesity referred to Asian adult BMI criteria defined and proposed by the WHO Western Pacific region obesity working group in 2000; (2) be WC of male ≥ 90 cm, or WC of female ≥ 80 cm; (3) be aged 18 to 65 years old; (4) be without the taboo of catgut embedding therapy; (5) be written informed consent.

Exclusion criteria
Patients with any one of the following conditions will be excluded from this trial: (1) obesity caused by certain endocrine, genetic and neurological diseases or druginduced; (2) merge other metabolic diseases such as diabetes, hypertension, dyslipidemia and so on; (3) other methods are being used to control body mass and abdominal circumference, such as surgery, drugs, etc; (4) women in pregnancy, nursing, perimenopause; (5) with some severe diseases of the heart, liver, kidney, or tumor; (6) severe audio-visual handicap; (7) diagnosed with a psychiatric disorder.
(8) participated in other tests within 3 months;

Dropout criteria
Patients meeting any of the following criteria will be dropout: (1) do not meet the inclusion criteria but mistakenly enrolled; (2) patients with poor compliance, withdrawal during the course of treatment, or receive others treatment; (3) severe adverse events or complications occur, which is not appropriate to continue treatment and have to discontinue the trial.

Sample size calculation
The sample size was comfirmed on a basis of the results of a previous clinical trial [19].
The primary efficacy parameter in the present study is also BMI from baseline to the treatment after 2 month. According to previous study [19], in the ACE group, the BMI decreased 1.65 and the standard deviation (SD) was 1.24, additionally, in sham ACE group, the BMI decreased 0.38 and the SD was 1.51. Considering a two-sided significant level of 0.05 and a power of 0.90, 26 participants in each group are required which is calculated by t test in G*Power software (Version 3, Institute for Experimental Psychology, Heinrich-Heine-University, Germany). A dropout rate of 15% was considered, a total of 99 participants will be recruited in this trial and 33 participants per group.

Randomization and allocation concealment
A total of 99 eligible paticipants will be randomly assigned to verum ACE group, sham ACE group and waiting-list (WT) group at a ration of 1:1:1. The random allocation sequence number will be generated with the Strategic Applications Software (SAS, version 9.1.3, SAS Institute Inc, Cary, NC, USA) by an independent statistician, who is not involved in the treatment or data collection. The randomization allocation numbers will be sequential, and written on cards and sealed in an opaque envelope by an independent assistant. The three groups will be concealed from the researchers until completion of the statistical analysis.

Blinding
The acupuncturists could not be blinded for the entire process. For eliminating potential bias, outcome assessors and statisticians will be blinded to group assignment. Patients' allocation is only revealed under some special cases, such as severe allergy, serious infection, uncontrolled pain and so on.

Interventions
The ACE intervention conforms with the Standards for Reporting Interventions in Clinical Trials of Acupuncture (STRICTA) guidelines. Patients in verum ACE group, sham ACE group and WT group will recieve real ACE therapy, sham ACE therapy and delayed active ACE therapy 20 weeks later, respectively. The course of two ACE groups will contain 8 sessions over 8 weeks (one session per two weeks).

Acupoints:
ACE treatment is semi-standardisd in this study. According to the clinical practice,  Table 1 and Fig. 3.

Catgut embedding preparation:
The catgut (Suzhou medical Co., Ltd., Jiangsu, China) size 3-0 with the length of 1.5 cm will be placed in front of a embedding needle (Zhengjianggaoguan medicine Co., Ltd., Shenzhen, China) for the operation of each acupoint. All the catgut embedding preparation processes were performed under the sterile conditions.

Operation Procedures of ACE:
Firstly, the acupuncturists have to disinfect hands with 75% alcohol and dress medical gloves, medical mask and medical hat. Simultaneously, let patients lie on the bed, supine or prone position depended on the location of acupoints, and fully expose the skin of the acupoint area. Secondly, the acupoints regions will be sterilized with iodophor and alcohol twice by the acupuncturists. Thirdly, the acupuncturists will locate the acupoint, then, stab the embedding needle into the skin at the acupoint regions, in result, the catgut will be embedded into the acupoints with the depth of about 1.5 cm, 1cm or 1cm at the abdomen, both feet or back, respectively. The embedding needle will be withdrawn until the patient has a feeling of sourness, meanwhile catgut will be left under the tissue.
Following this, sterile cotton balls will be pressed on the acupoints for hemostasis, and then band-aids will be pasted on the acupoints to prevent wound infection. Finally, warn the patients don't touch water on embedded acupoints within 24 hours.
The frequency of the catgut embedding treatment is once per week for a duration of 8 weeks.

Sham ACE group
Patients in the sham ACE group will undergo similar procedures as the verum ACE group except that nothing is put into the catgut embedding needles before operation, so no catgut leave under the acupoints tissue of patients after needle extraction. The acupuncture prescriptions are also as same as that in the verum ACE group.

Waiting-list control group
The WT group will donnot have any intervention. The patients will be required to receive delayed ACE therapy for free after a waiting period of 20 weeks.

MRI data acquisition
In this study, the MRI images of subcutaneous adipose tissue and visceral adipose tissue will be acquired by MRI scanner (MAGNETOM, SIEMENS) with a matrix body coil 18 channel at the MRI Center at Shenzhen Traditional Chinese Medicine Hospital of China. Before scannning, the participants will be trained for deep-breathing exercise. Through the duration of the scan, they need to hold their breath for about 15s. The parameters for the sequence are flip angle, 65°; TR/TE, 195/3.69 ms; number of excitation (NEX), 1; matrix, 256×131; slice thickness, 7 mm and echo train length, 4. In order to guarantee the image plane pass through the centre of the vertebral disc between L4 and L5, the MRI datas will be obtained from a sagittal scout. Qualitative image analysis was performed by two independent reviewers.

Outcome measurement
The clinical outcomes will measure patients' obesity levels and quality of life. All measurements will be performed at baseline; after 8-week treatments; at 12-week followup.
The secondary outcomes include waist circumference (WC), hip circumference (HC), waist hip rate (WHR), body fat percentage (BFP), and World Health Organization Quality of Life (WHOQOL). WC will be measured by a stretch -resistant tape at the midpoint between the top of the iliac crest and the lower margin of the least palpable rib. Hip circumference will be measured around the widest portion of the buttocks using a tape parallel to the floor [23]. The BFP will be measured with bioelectrical impedance. Other outcome parameters, including basal metabolic rate, blood pressure (BP) and heart rate (HR), and blood total cholesterol (TC), triglyceride (TG), and high density lipoprotein (HDL) levels also will be tested at each timepoint.

Statistical analysis
The statistical analysis will be conducted by independent statisticians who are blinded to group allocation and intervention methods. Before the data analyses, the research group will draw up a statistical plan, which includes the required data and processing method.
For the clinical data, SPSS 22.0 software (IBM SPSS Statistics, New York, NY, USA) will be used. For the MRI data, the images generated will be analyzed on a workstation (Syngo Mutimodality Workpace) for the quantification of VAT and SAT.
Demographic information and levels of measured variables will be analyzed by descriptive statistics. The categorical data will be described as percentage (n%) or analyzed using the Chi-square (x 2 ) test. Additionally, for the continuous variables, t-test will be performed, and presented with mean ± standards deviations. The paired t-tests will be used to analyze the difference of evaluation scores before and after each examination. The longitudiual and repeated measured data for variables across the 3 data collection points will be analyzed by the repeated measure analysis.
SFA was quantified as the area between outline of abdominal skin and the outer abdominal muscle, while VFA was defined as enterocoelia and retroperitoneal region between the inside edge of abdominal muscles and the spinal front. Detailed methods have been described previously [26][27][28]. The correlation coefficients between two reviewers analysing the same image for SFA and VFA (n=30) were r=0.99, p<0.001 and r=0.98, p<0.001, respectively.
Finally, the Pearson's correlation between the changes of SFA and VFA and improvement of clinical variables will be performend in each group.
For the above statistical analyses, a value of P < 0.05 will be regarded as statistically significant.

Safety
To guarantee the ACE operation is standardized and safe, the acupuncturists in this study should pay attention to those announcements as follows: (1) perform aseptic operation strictly to prevent infection; (2) catgut should not be embedded in adipose tissue that could prevent fat liquefaction; (3) catgut should not be exposed to body surface to prevent infection; (4) master the angle and depth of the embedding in order to avoid injury to the internal organs, great vessels, nerve, etc.; (5) acupoints can't touch water within 24 hours after embedding; (6) inform patients acupoints embedded catgut may keep sensation of soreness, distension and numbness for one or two days, even more than three to five days.

Management of adverse events
ACE therapy may lead to different adverse events, such as fainting during operation, subcutaneous hematoma, allergy, infection, severe pain and so on. Any adverse events the participants experience should be reported to the researchers. After confirming the validity of the adverse events by the evaluator, the acupuncturists will immediately stop the treatment precedure and deal with the abverse events. All adverse events, as well as managing these events, will be carefully recorded during the treatment and the follow-up phases.

Quality control
Acupuncturists, assistants, data collectors and statisticians have made up the study group, all of whom should abide by the rules and regulations enacted by the study group.
Before the study, each researcher will take a basic study training to understand the design, purpose, and basic information of this research, as well as master their own independent division of work. The acupuncturists will be required to be at least 3 years of practical acupuncture experience, moreover, they should be familiar with the operation process and be able to cope with any possible adverse events. Data collectors will be responsible for saving and managing the various data, and strictly proofread data. Patient withdrawal and adverse events during the study will be recorded in detail. Statisticians are fully responsible for data management and statistical analysis. Regular team meetings will be held and fully documented.

Regulatory and ethics approval
This study has been approved by the Chinese Clinical Trial Registry. The registration number of this trial is ChiCTR1800020248. In addition, Our study protocol has been approved by the Ethics Review Committee on Shenzhen Traditional Chinese Medicine Hospital. All crucial protocol adjustment will be submitted to the Ethics Review Committee on Shenzhen Traditional Chinese Medicine Hospital. Before the study, patients will be informed about the potential risks of the study. Patients voluntarily participate in our study with informed consent. All personal information and medical records about patients will be properly protected and keep in specialized cabinets during the whole study.

Discussion
ACE was applied widely for weight loss. Although previous studies have shown that ACE might be effective in improving the overweight, the high quality trials with rigorous design are still urgently needed to assess the effect of ACE for obesity [29]. The results of the present RCT study will contribute to a better understanding that ACE could exert the potential therapeutic effect for obesity.
Recently, increasing attention has been paid to visceral fat of obesity, which is related to energy storage, and considered to be an endocrine and paracrine organ that influences some metabolic processes by releasing the cytokines and bioactive mediators [30][31][32][33]. The body weight can be adjusted by cytokines and mediators. Thus, VFA is a significant indicator to estimate the metabolic risk in obese populations [34,35]. However, in previous studies, the efficacy of ACE on obesity were estimated mainly by BMI or WC, and few studies have been assessed by VFA. Hence, this study will be evaluated by both ordinarily obesity-related indices (BMI, WC, ect.) and VFA, in order to illuminate the afficacy of ACE on obesity clearly.
The study, designed with three-arm trials, aims to compare the changes of measurements after 2 month treatments with three different interventions methods: verum ACE, sham ACE and no intervention (waiting-list group). Various numbers of studies [36,37] have indicated that the sham control design could separate the specific and non-specific effects, which may play an important role on evaluating the effectiveness of a treatment.
Thus, the ACE and sham ACE treatments are both applied to investigate the antiobesity effects in this study. The sham ACE is operated with needling pierced into the acupoints only but without catgut infixed, and other operations are all the same as those in the ACE group. So, it could successfully blind the patients and estimators, as a result it could minimize the placebo effect. In addition, as far as non-specific effects (sham ACE vs. no intervention) and bias caused by psychological influences concerned, the third group without any intervention is set to avoid the placebo effects of group allocation or the patients' beliefs in ACE for weight loss. A possible limitation of this trial is that the followup might be difficult to completely comply with for participants due to a long interval science the end of 2 month treatment. Some proper actions, such as telephone interview, will be taken to improve compliance.
The design and methodological rigor of this trial will hopefully provide consolidated evidence on the efficacy and safety of the ACE for treating obesity, through collecting the valuable and high-quality data, and also contribute to the future research in ACE threapy.

Funding
This work will be supported by the Shenzhen "Sanming project" (SZSM201612001). The funding plays no role in the design of the trial, collection, mangement, analysis and interpretation of data, writing of the report, or the decision to submit the report for publication.

Availability of data and materials
The datasets generated in the study are available from the corresponding author on reasonable request.

Authors' contributions
YM Z, B Y and WQ Y contributed equally to this paper. ZX Y is the corresponding author.
YM Z participate in the conception and design of the trial. YM Z and WQ Y complete to draft the manuscript. HB Y is in charge of the recruitment and treatment of patients. B Y participate in data collection. WQ Y will analyze the data. ZX Y proofread the final manuscript. All authors discussed, read the manuscript, and all approved the publication of this protocol.

Ethics approval and consent to participate
The protocol has been approved by the Ethics Review Committee on Shenzhen Traditional Chinese Medicine Hospital (ethical approval: Shenzen Chinease Medicine Ethics approve ( research) 2018 77). The study is registered at Chinese Clinical Trial Registry (registration ChiCTR1800020248). It was registered on 21 November 2018; http://www.chictr.org.cn. Before all surveys, the acupuncturists will give the participants specific details about the research. After the approval of participants, informed consent will be signed and documented.

Competing interests
The authors declare that they have no competing interests.

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