Study design
The study protocol was approved by the Medical ethics committee of Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (2017LCSY069) and registered in Chinese Clinical Trial Registry (http://www.chictr.org.cn/edit.aspx?pid=24555&htm=4) with number ChiCTR1800014364 on 01 January 2018.
This study will be a 16-week, randomized, multi-center, double-blinded, three-arm, dose-optimization, placebo-controlled clinical trial. A total of 243 NAFLD patients will be recruited through recruitment advertisements and doctors’ introduction from the following three community healthcare centers in Shanghai, namely Zhangjiang community healthcare center, Beicai community healthcare center, Sanlin community healthcare center. All three centers are based on Pudong New Area, Shanghai and no obvious difference will be supposed to exist in population and socioeconomic aspects. Eligible NAFLD patients who agree to participate in the study will be randomly assigned to standard dose Lingguizhugan decoction (SLGD) group, low dose Lingguizhugan decoction (LLGD) group, or the control group according to ratio of 1:1:1. On the basis of behavioral intervention therapy, patients in the SLGD and LLGD groups will be treated with different doses of LGZG, and the control group will receive placebo. LLGD group will be made of half of the standard dose, and the placebo will be one-tenth of the standard dose but with soluble starch, mixed colorant and bitter principle to achieve comparable appearance, smell and taste. The course of treatment will last 12 weeks. A 4-week follow-up will also be arranged to assess the continued effect. Participants will be assessed at week 0 (baseline), week 4,week 8, week 12 (end of treatment), week 16 (end of follow-up). All participants will be asked to provide written informed consent before entering the trial. The study flow chart is shown in Fig. 1, and participant timeline is presented in Table 1. The Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) Checklist is presented in Additional file 1.
Participants
Diagnostic criteria
All participants should meet the diagnostic criteria of NAFLD and TCM pattern differentiation criteria of spleen-yang deficiency. Diagnostic criteria of NAFLD will be based on the American Association for the Study of Liver Diseases (AASLD) in 2017 [12]. To be specific, four following criteria should be all met: (a) Imaging (ultrasound, computed tomography or magnetic resonance imaging) or histological evidence of hepatic steatosis; (b) No significant alcohol consumption, defined as >21 standard drinks per week in men and >14 standard drinks per week in women; (c) Exclusion of other reasons inducing hepatic steatosis, including but not limited to hepatitis C, specific drugs induced, parenteral nutrition and severe malnutrition; (d) No coexistence of other chronic liver disease, including but not limited to hemochromatosis, autoimmune liver disease, chronic viral hepatitis, alpha-1 antitrypsin deficiency, hepatolenticular degeneration and drug-induced liver disease.
The TCM pattern differentiation criteria of spleen-yang deficiency will refer to the expert consensus of spleen deficiency by China Association of Traditional Chinese Medicine in 2017, previous systematic review, and the clinical guidelines for the new Chinese medicine [29-31]. Ten symptoms and signs will be assessed by continuous 100-point scale, Higher score means severer degree. Every symptom or sign possesses with certain weight. The scoring will be calculated through multiplying the rating score by the weight. Spleen-yang deficiency pattern will be defined as total score of ten dimensions equal or greater than twenty. The detailed rating scale is shown in Table 2. Inclusion criteria
Participants who meet all of the following criteria will be included: (a) aged between 18 and 80 years, males or females; (b) confirmed diagnosis of NAFLD; (c) confirmed diagnosis of spleen-yang deficiency pattern; (d) voluntary informed content and agreement of participating in every visit, examinations and treatment according to the protocol.
Exclusion criteria:
Participants who meet any of the following criteria will be excluded: (a) combination with other specific liver diseases which would induce fatty liver, including but not limited to alcoholic liver disease, chronic hepatitis C, autoimmune liver disease, and hepatocellular degeneration; (b) fatty liver induced by drugs (e.g. tamoxifen, ethylamine iodifurone, valproate, methotrexate, glucocorticoid), total parenteral nutrition, inflammatory bowel disease, hypothyroidism, Cushing syndrome, abetalipoproteinemia, and other syndromes related to insulin-resistance (e.g. lipid atrophic diabetes, Mauriac syndrome); (c) combination with serious primary diseases and mental diseases, including but not limited to cardiovascular and cerebrovascular diseases, hepatic diseases, renal diseases, hematologic diseases, cancers, and schizophrenia; (d) combination with diabetes or currently receiving anti-diabetic medicine treatment; (e) currently receiving treatments for NAFLD (including Chinese herbal decoction, Chinese patent medicine, and chemical agents); (f) antibiotics administration in recent one month; (g) allergy to compositions of experimental agents, or possessing with allergic constitution; (h) pregnancy and lactation women, and women who are likely to be pregnant but refuse to keep predefined contraception measures during the study; (i) participation in other clinical trials in recent three months or currently joining other trials; (j) mental or legal disability; (k) cannot obey medical advice for therapeutic lifestyle modifications; (l) suspicious of drug abuse or possessing other forbidden criteria.
Interventions
All clinical investigators responsible for diagnosis and treatment will be registered TCM practitioners. Before the start of the trial, a 3-day training session will be held for all clinical investigators involved in the study. All participants with NAFLD will receive treatment for 12 weeks according to the random assignment. LGZG has been utilized for a long history in clinical practice. Participation in this trial will not anticipate to bring any harms. Hence, no post-trial care will be arranged.
Behavioral intervention
Behavioral interventions will be given to all three groups and the prescription will be based on the Guidelines for Prevention and Control of Overweight and Obesity in Chinese Adults [32]. In brief, participants will be required to limit calorie intake (approximate 1660 kcal per day) and guarantee physical exercises (moderate aerobic exercise at least 150 minutes per week). Subjects will be informed to complete daily dietary and exercise records during treatment (additional file 2).
Drug intervention
LGZG is a traditional formula which is composed of Poria (Fulin), Ramulus Cinnamomi (Guizhi), Rhizoma Atractylodis Macrocephalae (Baizhu), and Radix Glycyrrhizae (Gancao). The standard dose for each herb is 12g, 9g, 6g, and 6g, respectively. LLGD group will be made of half of the standard dose, and the placebo will be one-tenth of the standard dose. The course of treatment last 12 weeks. Participants will be required to take one dosage (one pack) of granules 30 minutes after breakfast, once daily on weekdays. A total of 60 packs per patient will be administrated. The granules used in this study will be produced by Sichuan neo-green pharmaceutical technology development co., LTD (Sichuan, China). The crude herbs will be extracted and made into granules. The production process will be under the standard of Good Manufacturing Practice. No significant difference was found among granules samples from three groups in color, appearance, shape, smell and weight (Fig. 2).
Concomitant treatment regulations
Except for research products, other drugs (including Chinese herbal decoction, Chinese patent drug and Chemical Drugs) and interventions (including acupuncture, cupping and massage) which may interfere effect will be forbidden during the study. For participants with other diseases combined who cannot stop relevant therapies, they should record concomitant interventions in details. If the disease progresses during the study period, patients could withdraw from the study and use other treatment methods. The case will be regarded as an invalid case, and the patient will be required to complete the relevant examinations and evaluations as much as possible.
Drug dispensation and compliance assessment
Drugs will be packed as required in double-blinded study. During the treatment period, a certain amount of packs will be distributed in week 0, week 4 and week 8 (22 packs). The additional two packs will be the reservation for unforeseen circumstances. After taking the medicine, the patients will be informed to return all the remaining products and packages at next visit. The clinical investigators will count the number of remaining products and calculate the compliance. The research team will establish a WeChat group for health guidance and periodically contact subjects by telephone or WeChat to check safety and improve compliance.
Outcome measures
Schedule for all outcome measures is shown in table 1. All demographic data will be determined before treatment, including the date of birth, gender, body weight, height, medication history, past medical history, smoking and drinking status and course of disease.
The primary outcome will be the proportion of participants with at least one-unit decrease of HOMA-IR from baseline to treatment endpoint (week 12) [33]. Secondary outcomes will include body measurements (body weight and body mass index), hepatic function (alanine aminotransferase (ALT), aspartate transaminase (AST), gamma-glutamyl transpeptidase (GGT), and alkaline phosphatase (ALP)), lipid metabolism profiles (total cholesterol (TC), TG, low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), apolipoprotein A (apoA), apolipoprotein B (apoB) and non-high density lipoprotein cholesterol (non-HDL-C)), glucose metabolism profiles (fasting blood glucose (FPG), fasting insulin and glycosylated hemoglobin (HbA1c)), inflammatory responses (blood cell count (WBC), C- reactive protein (CRP) and inflammatory cytokines), liver-kidney echo ratio by ultrasound, and scorings of various questionnaires (TCM spleen-yang pattern scale, the MOS item short from health survey (SF-36), self-rating depressive scale (SDS) and self-rating anxiety scale (SAS)). All secondary outcomes will also be evaluated from baseline to treatment endpoint.
Safety variables will contained vital signs (including blood pressure, respiratory, heart rate and body temperature) evaluated in every visit, electrocardiogram (ECG) and blood tests (including blood routine test and renal function) evaluated at baseline, week 4 and week 12. Adverse events (AEs) will be observed and recorded throughout the whole trial. LGZG is a classical herbal formula and possesses with satisfactory safety profiles. Potential AEs may be mild gastrointestinal reactions, which will be alleviated spontaneously without any additional medical interventions. Clinical investigators should document any AEs in case report form (CRF), containing following information: occurrence time, symptoms and signs, severity, duration, laboratory examinations, interventions, outcomes and follow-up time. If an AE occurs, clinical investigators should adopt appropriate measures to guarantee the safety of participants based on the actual situation. If serious AEs happen, clinical investigators should inform principal investigator in two hours. Then principal investigator should report to medical ethics committee and data monitoring committee within 24 hours. Whether termination of the trial will be decided after discussion.
Biological sample collection
In order to further explore the mechanism of LGZG on NAFLD and its effect on oral and gut microbiota, biological samples will be collected in this study. On the consent form, the collection of biological samples will be clearly stated. And the participants will have the right to refuse providing biological samples. All biological specimens will be used for auxiliary researches in the future. Blood samples will be planned for subsequent DNA methylation and metabonomics studies. Feces and coating on the tongue will be planned for gut and oral microbiota studies. Whole blood, morning urine, feces and coating on the tongue will be collected at baseline, 4 weeks, 12 weeks and 16 weeks. All samples will be transferred and stored in a 80℃ refrigerator within 24 hours.
Sample size estimation
Sample size was estimated based on the clinical primary outcome. As far as we know, this is the first RCT evaluating the efficacy of LGZG versus placebo. No data is available for reference. So, based on our previous animal studies and consultation with TCM experts, we assumed that the effect size for SLGD group is 30%, LLGD group e is15%, and placebo group is 10%. If α = 0.05, β = 0.10, the sample size can be calculated by the following formula:
[Please see the supplementary files section to access the equation.]
The λ is the degree of freedom, taking 12.65 in this case. The pmax is 30% and the pmin is 10%. Assuming the maximum dropout rate is 20%, 81 patients will be required for each group, with a total of 243 patients.
Randomization and blinding
All participants will be randomly allocated to SLGD group, LLGD group, or the control group based on the ratio of 1:1:1. The random number table will be generated by SPSS 19.0 for Windows software. A research assistant who will not participate in this clinical trial will send the random number table to the pharmaceutical company for packaging to maintain the blinding of participants and clinical investigators. According to the order of enrollment, the clinical investigators will assign the number from small to large, and the pharmacists will distribute the corresponding drug. Participants, clinical investigators and statisticians will all be blinded until the completion of whole trial. The randomization list and blinding codes will be kept strictly confidential.
Based on the requirements of medical ethics, a double-blind study should set up an emergency letter for each blind number. Emergency letters will be sealed in opaque envelopes. The contents of the letter will contain group details and emergency situations. Emergency letters should be sealed and could only be opened when necessary. The emergency letter will be read by clinical investigators in cases of severe AEs, in which knowing the experimental treatment is compulsory for further medical interventions. The case will be treated as withdrawal and the clinical investigator will record the corresponding reason in the CRF. All emergency letters will be retrieved together with CRF after the study. Twice unblinding will be used in this trial. Briefly speaking, first unblinding will uncover the codes of treatments, namely A, B and C. After the completion of statistical analysis, the second unblinding will uncover the specific drugs corresponding to the cords.
Data collection and management
The trial steering committee will be composed of the principal investigator (GJ) and coordinators from three centers. The committee will be responsible for revising study design, reviewing overall progress and communicating with medical ethics committee per six months. An independent data monitoring committee (DMC) will also be established by the trial steering committee. The DMC will include clinical epidemiologists, data monitors and statisticians. No competing interest will exist. The DMC will review the efficacy and safety data according to the participants’ schedule, and provide suggestions to the trial steering committee on revising study design or terminating the trial.
Clinical investigators will be responsible for the data collection during this study according to the schedule. CRFs should be filled in after every visit to guarantee the accuracy and timeliness. Data monitors from DMC will review the CRFs per month. When a participant finishes all treatments and follow-up, the completed CRF will be verified by the coordinator. During the research process, the trial steering committee will also assign research assistants every month from Shanghai University of Traditional Chinese Medicine to solve potential problems encountered during the trial and guarantee the quality of the study execution.
In order to improve the compliance of patients to the intervention, free tests and free medical education will be provided. On the consent form, participants will be informed that they could withdraw the trial at any time, and the withdrawal decision will not interfere the regular medical care. Participants will also be noticed that their personal information will be de-identified throughout the trial to guarantee the confidentiality. For participants who discontinue or deviate from intervention protocols, the researcher should contact the subjects, ask the reasons, record the last time of taking medicine, and complete the evaluation items as much as possible.
Statistical analysis
The analysis of efficacy and safety will be based on the principle of intention-to-treat (ITT) principle, namely all randomized participants will enter the final analysis. The missing value will be filled by the method of the last observation carried forward. Data will be presented as mean with standard deviation, median with range or number with percentage. Data analysis will use the SPSS 19.0 for Window software. All statistical inferences will be performed using a two-sided test with a statistically significant test level of P < 0.05, and the confidence interval of parameters will be estimated by 95% confidence interval. Difference with groups will be assessed by paired t-test, chi-square test or Wilcoxon signed rank test according to specific variables. Difference among groups will be assessed by one-way analysis of variance or Kruskal-Wallis test based on characteristics of data. Post-hoc pairwise multiple comparisons will be performed if the statistical significance is found.
No interim analysis was arranged in this trial due to short intervention period and safety profiles of LGZG. Potential sub-group analyses will be determined based on characteristics of included participants, for instance, different categories of body mass index and different combined diseases.
Publication and dissemination
Regardless of the efficacy of drugs, the results will be disseminated through publications. The future report of this clinical trial will follow the requirements of CONSORT statement [34] and Chinese herbal medicine Formulas extension [35]. Authorship for the final report will be determined based on the contributions to the trial. We will not intend to use professional writers.