Objectives and hypothesis
Objectives:
Main research objective is to explore the underlying effects of Jianpi Qinghua Decoction on gut microbiota and intestinal permeability in newly diagnosed T2DM patients.
Secondary objective of this trial are to compare the changes in glycemic and lipid control between the control and treatment groups.
Hypothesis:
Jianpi Qinghua Decoction improves the metabolism of diabetes patients by regulating the structure and function of the microbiota as well as the intestinal permeability of newly diagnosed T2DM patients.
Overview
This study is a randomized, double-blind, placebo-controlled clinical trial designed following the CONSORT SPIRIT 2013 guidelines (see Additional file 1) [18], will be conducted over a period of 3 years from 2020, at Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine in Shanghai, China. 120 patients with newly diagnosed T2DM as determined by 1999 World Health Organization criteria (FBG 7 mmol/l or greater and / or 2-h OGTT 11.1 mmol/l or greater) [19] will be randomly allocated in a 1:1 ratio into a placebo or treatment group (n = 60 / group) and will receive either placebo (1/10 Jianpi Qinghua Decoction + maltodextrin) and granule (Jianpi Qinghua Decoction) twice daily, respectively, for 12 weeks. The eligibility criteria for the subjects are presented in Table 1. The participants will be recruited on a voluntary basis from the outpatient clinic of Shuguang Hospital and sign informed consent. The progression of trial enrollment, interventions and assessments is presented in Fig. 1.
Interventions and Lifestyle guidance
Interventions
The period of intervention will last for 3 months, the visits and the evaluations will be done as follows: screening (visit 0), 1 month (visit 1), 2 months (visit 2), 3 months (visit 3) (Fig. 1). The treatment drug is Jianpi Qinghua Decoction granule composed of Pueraria lobata 30g, Scutellaria 9g, Coptis chinensis 6g, Euonymus alatus 15g, Codonopsis pilosula 30g, Astragalus 30g, Dioscorea polystachya 30g, Polygonatum 30g, oral administration, twice daily, for 12 weeks. The placebo medication comprises 1/10 Jianpi Qinghua Decoction and dextrin, oral administration, twice daily, for 12 weeks. Patients with hyperlipidemia and / or hypertension should be treated with corresponding hypolipidemic and / or antihypertensive drugs according to specialist' s advice.
Jianpi Qinghua Decoction granule and placebo will be prepared by Lei Yun Shang Pharmaceutical Group Co., Ltd according to a standard production process. All of the drugs will be quality controlled throughout the trial. All herbs used in this study are from the same place and in the same batch.
Lifestyle guidance
Participants in both groups of the study will receive lifestyle advice from a specialist. This advice is according to Guideline for the prevention and treatment of type 2 diabetes mellitus in China for Chinese adults [20]. The lifestyle intervention of the guideline focus on introduction of T2DM – related knowledge, daily nutrition management and physcial exercise.
Anthropometric parameters and blood pressure measurement
Anthropometric parameters will be measured at visit 0 (baseline visit) and visit 3 (at 12 weeks). Height (cm) will be measured using tape measure to the nearest 0.1 cm. Body weight (kg) will be measured in light, indoor clothing to the nearest 0.1 kg using a universal weight scale. Calculating Body Mass Index (BMI, kg / m2) according to the formula: BMI = Body weight / Height2. Waist circumference (WC, cm) will be measured at midway between the iliac crest and the lower costal margin at the end of normal expiration using a tape measure to the nearest 0.1 cm. Similarly, the hip circumference (HC, cm) also will be measured at widest part of the buttocks at the intertrochanteric level to the nearest 0.1 cm using a tape measure. Calculating Waist-to-hip ratio according to the formula:Waist-to-hip ratio =WC / HC. All anthropometric parameters will be made by a same researcher.
Blood pressure for each participant will be measured by a same researcher at visit 0 (baseline visit) and visit 3 (at 12 weeks) using a mercury sphygmomanometer in both of the hands and the mean blood pressure readings. Participants will be required to rest for 10 minutes prior to the first blood pressure measurement with a minimum of 15 minutes between the two occasions.
Biochemical assays
Blood samples will be collected for fasting blood glucose (FBG, mmol/L), 2-h plasma glucose (2hPG, mmol/L), glycated hemoglobin (HbA1c, %), fasting insulin (FINS, mU/L), total cholesterol (TC, mmol/L), total triglycerides (TG, mmol/L), high-density lipoprotein cholesterol (HDL, mmol/L), and low-density lipoprotein cholesterol (LDL, mmol/L). These parameters will be measured in a clinical laboratory of Shuguang Hospital. The homeostatic model assessment of insulin resistance (HOMA - IR) index will be used to determine insulin resistance using the following formula:
HOMA ‐ IR = FBG × FINS / 22.5.
Safety Assessment Index
Safety indices will be tested on visit 0 (baseline visit) and visit 3 (at 12 weeks). Renal function tests for serum creatinine (Scr, μmol/L), serum uric acid (μmol/L), serum carbamide (mmol/L). Hepatic function tests for aspartate aminotransferase (AST, U/L), alanine aminotransferase (ALT, U/L), AST / ALT, γ-glutamyltransferase (U/L), total bilirubin (μmol/L). Blood routine and urine routine also will be tested. Adverse events will be recorded in the Case Report Forms (CRF), the specific treatment measures of adverse events see the supplementary materials. All of parameters will be measured in a clinical laboratory of Shuguang Hospital.
Intestinal permeability
Intestinal permeability will be assessed by using ELISA detecting serum Lipopolysaccharide (LPS)( Lipopolysaccharide ELISA Kit, Cloud – Clone Corp, China), serum intestinal fatty acid binding protein 2 (IFABP2) (intestinal fatty acid binding protein 2 ELISA Kit, Cloud – Clone Corp, China), serum Zonulin (Human Haptoglobin ELISA Kit, BOSTER, China) concentrations.
Gut microbial profiling
Stool samples will be collected at baseline and 3months. Microbial community genomic DNA will be extracted from stool samples using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, U.S.) according to manufacturer’s instructions and quantify using NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA). The hypervariable region V3-V4 of the bacterial 16S rRNA gene will be amplified by an ABI GeneAmp® 9700 PCR thermocycler (ABI, CA, USA). The PCR product will be extracted from 2% agarose gel and purify using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to manufacturer’s instructions and quantify using Quantus™ Fluorometer (Promega, USA). Purified amplicons will be pooled in equimolar and paired-end sequenced on an Illumina MiSeq PE300 platform/NovaSeq PE250 platform (Illumina, San Diego,USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).
Data analysis
Sample size
In this study, the sample size was estimated based on data from a previous study [21]. A mean difference in HbA1c level of 1.3% between the two groups is aimed to be detected for a specified α of 0.05 and a study power of 90%. Thus the calculated sample size was 45 cases in the Jianpi Qinghua Decoction group and 45 cases in the control group. It is estimated that the drop-out rate in this study is 20%, so the final sample size of this study included 60 cases in the Jianpi Qinghua Decoction group and 60 cases in the placebo group, total 120 cases.
Randomization
An independent statistician will use IBM SPSS Statistics version 26.0 (SPSS Inc., Chicago, USA) to generate random sequences according to the central block randomization method. Eligible patients will be randomly assigned to the treatment group and Placebo group at a ratio of 1:1.
Blinding
Both of the investigators and patients are blind to the treatment allocations. The medication will be delivered in analogical packages and labels, each one with its own serial number. The allocation serial number will be generated by a indenpedent statistician. Envelopes containing a monthly supply of either placebo or Jianpi Qinghua Decoction will be prepared according to the randomization sequence and supplied to the patients by the research assistants when they are randomized to the trial.
Data collection, management and monitoring
Questions in the CRF collect a series of of data including basic information of the patient, outcome and adverse events. Without the consent of the investigator and the patient, the CRF data shall not be released to the public. The same research investigator throughout the study standardize the data collection procedure. CRF will be used to record data for all participants, and data will enter an electronic database to preservation.
Establishing a special data entry system for this study based on the CRF. The data entry clerk will perform entry of the data and check it. After checked, the data manager will inspect data and manufacture a database list. The data manager confirm that the contents on the list are completed, the researcher will approve the database lock and sign the name and date. The data manager should save the relevant documents of data management as required, and transfer the locked database and related materials to the statistician for statistical analysis. All samples will be kept in a constant temperature and humidity storage and saved by expert staff. The main researcher will have access to the final trial dataset.
Statistical analysis
All data were presented as the mean ± standard deviation. the measurement data which not conforming to the normal analysis are described by the median, and the count data are described by the composition ratio. Data will be processed and analyzed using SPSS 26.0 software, Parametric (e.g., ANOVA) and non-parametric (e.g., Kruskal-Wallis test) statistical tests will be used for comparison between groups. P < 0.05 will be considered to indicate statistical significance.