1. Quality and Quantity Analyses of Weifuchun
WFC tablet was kindly provided by Huqingyu-tang Pharmaceutical Co., Ltd. (Hangzhou, China). Quality and quantity analyses of the aqueous extract were performed with UPLC TOF-MS. HPLC-grade acetonitrile, methanol, and formic acid were purchased from Fisher Scientic (Santa Clara, USA). Naringin, ginsenoside Rb1, and oridonin were identified in WFC by UPLC TOF-MS. The following conditions were used to analyze naringin, ordionin, and ginsenoside Rb1: system, Acquity UPLC system (Waters, USA), which consists of a solvent degasser, a binary pump, an auto-sampler and a column oven; column, Acquity UPLC BEH C18 RP column (1.7 μm, 100 mm × 2.1 mm i.d.; Waters, USA); mobile phase A, 0.1% formic acid in water; mobile phase B, 100% acetonitrile; flow rate, 0.3 mL/min; wavelengths, 210 nm for ginsenoside Rb1, 254 nm for ordionin and 280 nm for naringin; injection volume, 10 μL; MS/MS detector, Acquity Synapt G2 Q-TOF tandem mass spectrometer connected to the UPLC system by an ESI interface and controlled by MassLynx version 4.1 (Waters, UK). Samples were analyzed in the positive model. Data were collected and analyzed by Waters MassLynx version 4.1.
2. Trial Oversight
This randomized and controlled trial was conducted in the outpatient clinics of Shuguang Hospital and Shanghai TCM-Integrated Hospital affiliated to Shanghai University of Traditional Chinese Medicine. All subjects (patients and health volunteers) provided written informed consent before enrollment. The trial was approved by the institutional review board at Shuguang Hospital and was conducted in accordance with the provisions of the Declaration of Helsinki and the CONSORT guidelines. An independent data and safety monitoring board reviewed the progress of the trial.
The study protocol, which describes the study in more detail, can be found in the clinical trial registry (https://register.clinicaltrials.gov) with the identifier NCT03814629. The study was approved by the Ethics Committees of Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine (No. 2016-478-29-01). Recruitment and data collection occurred between October 2015 and September 2017. Patients with a previous histological diagnosis of CAG with or without IM/dysplasia were selected as potential subjects. Health volunteers were those never had stomach trouble and other serious disease. The trial was not registered until all the patients had been enrolled because registration was not mandated after the trial had started.
3. Participants and Eligibility Criteria
Only those who fulfilled the diagnosis of both CAG and IM or dysplasia, and H.pylori(-) were considered eligible subjects, male or female, between 18 and 70 years. Participants with H.pylori positive infection without radical treatment, peptic ulcer or severe dysplasia (suspected malignant transformation), severe systemic diseases such as cardiovascular and cerebrovascular disease, hepatic diseases, kidney or lung disease, or with other tumors, were excluded. Participants were excluded if they had an allergic constitution or allergies to any known ingredients in WFC. Finally, patients with other diseases interfering with the study or patients unwilling to undergo repeated endoscopy after treatment were also not included.
The TCM standard for diagnosing syndromes was worked out with reference to the standard for diagnosing the type of spleen and stomach weakness in the guidelines of diagnosing and treating CAG. Major symptoms are stomach pain or discomfort or stomach symptoms remission after warm or press operation. Minor symptoms include: (1) anorexia; (2) loose stools; (3) physical and mental fatigue; (4) shortness of breath and lazy speech; (5) stomach distention after eating; (6) belching; (7) chest distress; (8) stomach pain refused to press; and (9) light-colored tongue with small and wiry pulse. Patients with one the major symptom and two or more minor symptoms were diagnosed as suffering from the syndrome of spleen and stomach weakness.
4. Trial Design and Treatment
Before endoscopy, patients were randomly assigned in a 1:1 ratio to receive either WFC therapy or vitacoenzyme. Computer-generated randomization was performed in a blinded manner, with status concealed from all the patients and the primary physician, endoscopist, pathologist and statistician. After randomization, endoscopy was performed. The patients started the assigned trail medication within 1 week after endoscopy.
Each subject received either WFC tablets (1.44g) (Hangzhou huqingyutang pharmaceutical co. LTD. Hangzhou, China, lot number 16066129) or vitacoenzyme tablets (0.8g) (Beihai sunshine pharmaceutical co. LTD, Guangxi, China, lot number 102029), taken orally after meal 3 times a day for 6 months. Before randomization, H. pylori status was determined by rapid urea test (RUT) or by pathology. Positive subjects received standard eradication therapy. H. pylori status was re-evaluated at the end of the 6th month. If required, the status would be re-evaluated by 13C-urea breath test at 4 weeks after the cessation of therapy.
5. Screening Measures
The demographics of participants were collected, including age, gender, course of disease, and current and past gastric disease treatment. The histological diagnosis and grading was made according to the Consensus on Chronic Gastritis in China (Shanghai, 2012), the updated Sydney system and expert panel consensus.
6. Histological scores
Each gastric tissue was evaluated separately for the following items: chronic inflammation (CI), acute inflammation (AI), atrophy, IM, and dysplasia. Atrophy was defined as loss of glands and graded as absent (0), mild (1), moderate (2), or severe (3). IM was graded as absent (0), mild(1), moderate (2), or severe (3) according to the proportion of the gastric mucosa replaced by the metaplastic issue. Presence and severity of dysplasia defined by atypical cytological and architectural derangement, subcategory of mild (1), moderate (2) and severe (3) grade adhered to the diagnosis for gastric neoplasia.
7. High-throughput sequencing
Total DNA was extracted using the QIAamp DNA Stool Mini Kit. All extractions yielding>2ng/μ of total DNA, as indicated by NanoDrop 2000 UV-Vis Spectrophotometer measuring. Each DNA sample was amplified for the V3 region of 16S rRNA gene and libraries were sequenced in a single run of the Illumina MiSeq sequencing platform at NovelBio Biomedical technology Co.,LTD.
8. Statistical analysis
According the clinical report and previous research results, we calculated the sample size of 35 patients in each group for the trial population. We allowed for a 15% initial dropout rate, and a further 10% loss to follow-up, resulting in the enrollment of 47 patients in each group. An interim analysis was not planned.
The statistical analyses were performed by using IBM SPSS Statistics 21.0. Data are expressed as mean±standard or median (range) for continuous variables, and frequencies (percentages) for categorical variables. Student's t-test or Mann-Whitney test or Chi square test was used to compare baselines including demographic data and basic evaluating variables. For comparison of variations from baseline to endpoint, paired t-test was performed on variables with normal distributions, and wilcoxon signed-ranks test on non-normal variables. For comparison microbial abundances between groups, the ANOVA was used. Chi-square test or Fisher's exact test was used for atrophy and intestinal metaplasia disappearance rate and symptom disappearance rate. All statistical tests were two-sided and assumed to be statistically significant at a level of P<0.05.