Study design
The study protocol was approved by the Medical ethics committee of Longhua Hospital to Shanghai University of Traditional Chinese Medicine (2017LCSY069). We have register it at the Chinese Clinical Trial Registry (http://www.chictr.org.cn/edit.aspx?pid=24555&htm=4) under number ChiCTR1800014364.
This study is a 12-week, randomized, multi-center, double-blinded, three-arm, dose-optimization, placebo-controlled clinical trial. A total of 243 NAFLD patients will be recruited at the following three community healthcare center in shanghai: Zhangjiang community healthcare center, Beicai community healthcare center, Sanlin community healthcare center. Patients meet the inclusion criteria who agree to participate in the study will be randomly assigned at a randomization of 1:1:1 ratio to to standard dose Lingguizhugan decoction (SLGD) group, low dose Lingguizhugan decoction (LLGD) group, or the control group. 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 is one-tenth of the standard dose as well as soluble starch, mixed colorant and bitter principle. The course of treatment last 12 weeks, taking 1 dosage of granules every time, once daily on weekdays, a total of 60 packs per patient. Participants will be assessed at baseline, 4-week,8-week and 12-week after treatment, respectively with another 4-week of follow-up. All participants will be asked to provide written informed consent. Recommendations for Interventional Trials (SPIRIT) Checklist is presented in Additional file 1. The study flow chart is shown in Fig. 1
Participates
Diagnostic criteria
All participants must be diagnosed with NFLAD, and have to a diagnosis of spleen-yang deficiency pattern. Diagnostic criteria of this trial for NAFLD will be based on the American Association for the Study of Liver Diseases (AASLD) in 2017.[12] The syndrome differentiation standard of spleen-yang deficiency pattern syndrome refer to the consensus from relative experts TCM diagnosis and treatment of spleen-yang deficiency pattern by the spleen-stomach Branch of China Association of Traditional Chinese Medicine in 2017, and the study of systematic evaluation on the literature of spleen deficiency syndrome diagnostic standard, as well as the clinical guidlines for the new Chinese medicine.[29-31] The NAFLD with spleen-yang deficiency pattern clinical signs and symptoms grated rating scale see in table 1.
Inclusion criteria
- Aged between 18 and 80 years, males or females
- Meet the diagnostic criteria of NAFLD
- Accord with the pattern differentiation criteria of spleen-yang deficiency pattern
- Sign the informed content and agree to participate in every visit, examinations and treatment according to the protocol
Exclusion criteria:
- Combine 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
- 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)
- Combine with serious primary diseases, including cardiovascular and cerebrovascular diseases, liver diseases, kidney diseases and hematological diseases, cancer, and other severe complications and mental diseases
- Combine with diabetes and participants who are on anti-diabetic medicine treatment
- Currently receive treatments for non-alcoholic fatty liver disease (including Chinese herbal decoction, Chinese patent medicine, and chemical agents)
- Received antibiotics in recent one month or currently receive antibiotics
- Participants who are known to be allergic to the composition of product or possess allergic constitution
- Pregnant and lactating women, and the women who are likely to be pregnant but refuse to keep predefined contraception measures during the study
- Participated in other clinical trials in recent three months or currently join another trial
- Mentally or legally disabled
- Cannot obey medical advice for therapeutic lifestyle modifications
- Suspicious of drug abuse or possess other forbidden criteria
Interventions
All participants with NAFLD will receive treatment for 12 weeks according to the random assignment.
Behavioral intervention
Behavioral interventions will be given to all three groups and the prescription is based on the Guidelines for Prevention and Control of Overweight and Obesity in Chinese Adults.[32] Subjects need to have dietary and exercise records during treatment in table. 2.
Drug intervention
LGZG is a tradition formula which is composed of Poria cocos, Cassia Twig, Atractylodes macrocephala Koidz, and Glycyrrhiza uralensis Fisch. LLGD group will be made of half of the standard dose, and the placebo is one-tenth of the standard dose. The course of treatment last 12 weeks, taking 1 dosage of granules every time, once daily on weekdays, a total of 60 packs per patient. Participants will be assessed at baseline, 4-week,8-week and 12-week after treatment and 4 weeks following visit. The granules used in this study will be produced by Sichuan neo-green pharmaceutical technology development co., LTD (Sichuan, China). The drugs were no significant differences in the color, appearance, shape, smell and weight in the three groups. The production process will be quality controlled to guarantee the external packaging and microorganism limitation test are up to standard. Certificates of quality for the manufacturing of drugs will be provided by the manufacturer. The herbs will be extracted and made into granules, when dissolved in hot water, the appearance, smell, and taste are identical. Comparation demonstration are shown in Fig. 2.
Concomitant treatment regulations
- In addition to research products, the drugs (including Chinese herbal decoction, Chinese patent drug and Chemical Drugs) and other treatments which could affect NAFLD were forbidden during the study.
- For participates with other diseases combined who cannot stop other drugs and therapeutic method must be recorded in detail in the combined medication list.
- But as the disease progresses during the study period, patients can withdraw from the study and use other treatment methods. The case is regarded as an invalid case, and the patient should complete the relevant examination and evaluation as much as possible.
Drug dispense and dependence assessment
Drugs are packed as required in the study of the double-blinded study. During the treatment period, each visit will be distributed to the dose required for the next visit, and the corresponding window will be reserved (20 packs plus 2 packs). After taking the medicine, the patients should return all the remaining products and packages at each visit. The researchers count the number of remaining products and record them to calculate the compliance.
Outcome measurements
All outcome measures are shown in table 3.
Demographic indicators
All demographic data need to 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.
Primary outcome
The primary endpoint is the patients’ percentage who reaches one standard unit decrease of HOMA-IR from baseline to 12 weeks.[33]
Secondary outcomes
The Secondary outcomes will be conducted at baseline, week 4 and week 12, and as follows
- Changes of white blood cell count (WBC) and C- reactive protein (CRP) in inflammatory markers from baseline to 12 weeks.
- Changes of Alanine aminotransferase (ALT), Glutamic pyruvic transaminase (AST), gamma-glutamyl transpeptidase (GGT), and alkaline phosphatase (ALP) in liver function from baseline to 12 weeks
- Changes of total cholesterol (TC), triglycerides (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) in blood lipid metabolism from baseline to 12 weeks
- Changes of fasting blood glucose (FPG), fasting insulin and glycosylated hemoglobin (HbA1c) in blood glucose metabolism from baseline to 12 weeks.
- Changes of inflammatory cytokines from baseline to 12 weeks.
- Changes of Liver-kidney echo ratio by ultrasound from baseline to 12 weeks.
- Changes of body weight and body mass index from baseline to 12 weeks.
- Changes of total score and single score in quantitative rating of clinical symptoms and tongue and pulse condition from baseline to 12 weeks.
- Changes of the MOS item short from health survey (SF-36), self-rating depressive scale (SDS) and self-rating anxiety scale (SAS).
Safety index
- vital signs, including blood pressure, respiratory, heart rate and temperature need to be recorded at each visit
- Electrocardiogram (ECG) will be evaluated at baseline, 4 weeks and 12 weeks.
- Blood routine test and liver and renal function.
- Adverse events (AEs) will be observed and recorded. Detailed monitoring throughout the process. Whether the AE is related to research product, it should be recorded in the Case report form (CRF), including the occurrence time, symptoms, signs, degree, duration, laboratory examination indicators, treatment methods, process, results, follow-up time, etc. Analyze the correlation between AE and research product, record all the related details (name, date, etc).
Biological sample collection
In order to further explore the mechanism of LGZG on NAFLD and its effect on oral and gut microbiota, biological samples should be collected in this study. 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 in time.
Sample size estimation
Sample size was estimated based on the clinical primary outcome. In view of previous studies, 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:
Where λ is the degree of freedom, taking 12.65, Pmax is 30%, Pmin is 10%, considering the Maximum dropout rate is 20%, we calculated that 81 patients were required in each group, for a total of 243 patients.
Randomization and blinding
All participants will be randomly assigned at a randomization of 1:1:1 ratio to SLGD group, LLGD group, or the control group. The random number table use for random blinding of products was developed by statistical professionals using SPSS 19.0 for Windows software. The person who will not participate in the clinical research, according to the random number table distribute the drug blinding that is the random number, and should be responsible for the preparation of the emergency letter and hand the letter over to the person in charge of clinical research. Eligible patient will be randomly assigned to each treatment group. According to the order of each participant's enrollment, the researcher randomly assigns the product number from small to large, and distribute the product with the corresponding product number.
Based on the requirements of medical ethics, a double-blind study should set up an emergency letter for each blind number. The contents of the letter contain group details and emergency situations. Emergency letters should be sealed and must not be opened if necessary. The emergency letter will be read by researcher in the event of an emergency or patient need to rescue must know what kind of treatment the patient is receiving, meanwhile the case will be discontinued and the researcher should record the reason for the discontinuation on the CRF. All emergency for blind review letters will be retrieved together with CRF after the study. This study used a twice blindly method to uncover the results.
Data collection and management
The data from all participants will be collected at baseline, 4 weeks, 12 weeks and 16 weeks (table 3) and recorded on the CRF in time and entered into an electronic data acquisition system. After each participant completes the trail and is verified by the supervisor, the data managers import them into the designated database and send them to the statisticians for statistical analysis. All sensitive and private data which can identify patient information will be encoded. The data in this clinical trial, except for the requirements of the State Food and Drug Administration, should not be provided to anyone without the written consent of the applicant. The data will be only applicable to the retrospective analysis of the efficacy of LGZG in the future.
During the research process, the sponsor will assign a clinical monitor who come from Shanghai university of TCM to conduct regular on each site to guarantee the quality of the study and solve the problems encountered during the clinical trial.
Statistical analysis
The analysis of efficacy and safety will be based on the principle of intention-to-treat (ITT). Data analysis will use the SPSS 19 for Window software. All statistical inferences will be compared with t Test with a statistically significant test level of 0.05, and the confidence interval of parameters is estimated by 95% confidence interval. The main indicator should give a two-sided 95% confidence interval for the difference and the difference values, if the lower limit of confidence interval>0 combined with clinical judgment to determine whether it has the clinical effect. In terms of Security analysis, the chi-square test will be used to compare the incidence of adverse events in both groups.