This trial adopted a multicenter, randomized, double-blind, placebo-controlled design with balanced allocation (1:1) across 2 arms, enrolling a total of 110 participants (55 per study group). This protocol is version 02, dated March 25, 2023. The study is registered with the Chinese Clinical Trial Registry (ChiCTR2400084357) on May 15, 2024. This protocol is described according to the SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) 2013 and SPIRIT-TCM (Standard Protocol Items for Clinical Trials with Traditional Chinese Medicine: Recommendations, Explanation and Elaboration) 2018 [21].

Recruitment is conducted through multiple channels, including public advertisements (posters, social media, and community outreach) and the identification of potential participants from gastroenterology outpatient and previous inpatient lists. Eligible patients are comprehensively informed about the study’s purpose, procedures, treatment duration, potential benefits, and risks. A gastroenterology specialist confirms the diagnosis for all patients who express interest. Written informed consent must be obtained from each participant prior to randomization. All patient information will be kept confidential. Patients may withdraw at any trial stage, and the reasons for patient withdrawal will be well documented. The flowchart of this trial is shown (Figure 1).

Participant enrollment is conducted within a multicenter framework involving 4 tertiary hospitals, each with an established gastroenterology service and access to a defined patient population with CAG. Recruitment is carried out through parallel approaches, including outpatient screening, inpatient record review, and community-based outreach. Screening and enrollment procedures are standardized across centers, with unified protocols applied at each site. Recruitment progress is monitored throughout the study period in relation to the planned timeline. As of the time of manuscript submission, patient enrollment is ongoing. The first patient was enrolled on April 20, 2025, and the study is projected to conclude in October 2026.

The superiority test of the outcome measure Operative Link on Gastritis Assessment (OLGA) improvement rate was used as a categorical variable, and the sample size of the control group was estimated by n1. n2 represents the sample size of the control group, k represents the ratio of the number of cases between the 2 groups (k=1), and the minimum clinically meaningful difference is set △=10%. According to the results of the team’s previous research, π1=45%, π2=10% [12]. By further incorporating a 2-sided significance level (α) of 0.05 and a statistical power of 80%, the calculation yielded n2=43 and n1=43. Considering the 20% clinical dropout rate, the sample size was determined to be 108 cases, that is, 54 cases in the experimental group and 54 cases in the control group.

For the superiority test of categorical variables with the improvement rate of the outcome measure Operative Link on Gastric Intestinal Metaplasia Assessment (OLGIM), in which △=10%, k=1, π1=42%, and π2=8% [12], using the same significance level (α=0.05) and statistical power (80%), the calculation yielded n2=44 and n1=44. Considering the 20% clinical dropout rate, the sample size was determined to be 110 cases, that is, 55 cases in the experimental group and 55 cases in the control group.

The larger calculated sample size was chosen to ensure adequate statistical power. Therefore, the sample size was 110 cases, 55 cases in the experimental group, and 55 cases in the control group.

In this study, case collection is being carried out in 4 institutions (Wangjing Hospital of China Academy of Chinese Medical Sciences, Shanxi Provincial Hospital of Traditional Chinese Medicine, Second Affiliated Hospital of Baotou Medical College, and Hubei Provincial Hospital of Traditional Chinese Medicine). The study uses a stratified block randomization method. Stratification is based on both the research center and the baseline OLGA/OLGIM stage. The third-party statistical agency used SAS software (SAS Institute Inc) to generate a random number list, and each clinical research center randomly allocates the qualified participants to the experimental group and the control group according to the ratio of 1:1. The trial is also designed as a double-blind trial. The blinding is conducted by independent personnel, and the blind record is formed. To ensure the integrity of the blinding, all medications and placebos are uniformly packaged. According to the Standard for the Use of Food Additives in the National Standard for Food Safety, the placebos are prepared from starch, dextrin, food coloring, bitters, etc, based on the type and volume of the raw materials. In appearance, color, dosage form, quality, taste, and odor, it is the same as or basically similar to the experimental drug, but does not contain the active ingredient of the experimental medicine.

All randomization code envelopes must remain strictly sealed, with any unblinding requiring full documentation of personnel, date/time, and rationale, immediately recorded in the case report form (CRF). Emergency unblinding is only permitted for life-threatening situations requiring knowledge of the investigational product, and must involve joint authorization by the clinician and study director, followed by participant discontinuation and detailed CRF documentation. Post study, all unsealed envelopes undergo verification and final blinded review by an independent audit team to ensure compliance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use-Good Clinical Practice and regulatory standards.

The primary outcome, OLGA improvement, is evaluated based on assessments at baseline (visit 1) and posttreatment (visit 8). The severity of gastric atrophy is assessed at baseline and post-treatment according to the OLGA staging system, with the overall stage at each time point determined by the most severe atrophic grade across all biopsy sites. Patients are categorized into three groups based on changes in OLGA stage: regression, defined as a reduction of ≥1 stage after treatment; stable, defined as no change in OLGA stage after treatment; and progression, defined as an increase of ≥1 stage after treatment.

Secondary outcomes include the improvement of gastric mucosal atrophy and IM (assessed at visits 1 and 8), as well as changes in TCM syndrome scores (assessed at visits 1, 2, 3, 4, 5, 6, 7, and 8). Based on the histopathological characteristics of CAG, a graded scoring system will be used for the pathological assessment. The severity of gastric mucosal atrophy and IM will be classified into 4 grades—absent, mild, moderate, and severe—which will be assigned scores of 0, 2, 4, and 6, respectively. In cases where multiple biopsy specimens or histological sections from a single patient exhibit varying degrees of lesions, the score corresponding to the most severe lesion will be assigned. The improvement in these pathological scores from baseline to posttreatment will be compared between the 2 groups. Additionally, clinical symptoms will be quantitatively evaluated using a standardized scoring method. The primary symptoms include stomach pain and fullness, while secondary symptoms encompass gastric discomfort, poor appetite, dry mouth, bitter taste in the mouth, belching, acid reflux, heartburn, and fatigue. The symptom grading criteria are formulated according to the Guiding Principles for Clinical Research of New Traditional Chinese Medicines (2002 edition). Specifically, primary symptoms will be comprehensively assessed based on their severity and frequency, and categorized into 4 grades (absent, mild, moderate, and severe), scoring 0, 2, 4, and 6 points, respectively. Secondary symptoms will be evaluated using the same 4-tier scale but will be scored as 0, 1, 2, and 3 points, respectively. The total symptom scores will be recorded at baseline and at 4-week intervals throughout the treatment period. Changes in the total symptom scores relative to baseline will be compared between the 2 groups to comprehensively assess symptom improvement.

Vital signs examination, including temperature, respiration, heart rate, and blood pressure, is recorded at baseline and posttreatment (visits 1, 2, 3, 4, 5, 6, 7, and 8). The routine blood, urine, and stool tests, liver function tests (alanine aminotransferase and aspartate aminotransferase), and renal function tests (blood urea nitrogen and creatinine) are recorded at baseline (visit 1) and at the end of treatment (visit 8). Throughout the study, adverse events (AEs) will be continuously monitored and documented following treatment administration. Any participant exhibiting abnormalities post treatment—despite baseline normalcy—will undergo periodic assessments until values stabilize. All AEs will be actively tracked until complete resolution is achieved for each participant.

To clarify the mechanism of WW-1 in treating patients with CAG, gastric mucosa, saliva, tongue coating, and stool are collected at baseline (visit 1) and at the end of the treatment (visit 8), followed by the Infinium MethylationEPIC BeadChip assay and metagenomic analysis. All sample collection, storage, and transportation processes are carried out in accordance with the standard operating procedure, which has been developed by the expert committee.

Pathological histology is the gold standard for diagnosing CAG. However, consistency among pathologists in clinical practice is often inadequate, particularly in the assessment of atrophy. High-quality international studies related to CAG emphasize the necessity of multiple pathologists’ diagnoses. This study uses a centralized blind pathology approach, using a remote pathology consultation platform for the diagnosis and evaluation of CAG. Pathology experts from the pathology departments of Wangjing Hospital of the China Academy of Chinese Medical Sciences, the Cleveland Clinic in the United States, and Peking Union Medical College Hospital conduct blind diagnoses and reach a consensus. This process ensures the accuracy of pathological results before and after treatment, thereby enhancing the objectivity, rigor, and authenticity of efficacy assessments.

Prior to the trial, all study personnel received protocol-specific training. Investigators must accurately complete CRFs as unalterable primary records, with original laboratory/imaging reports attached. Clinically significant abnormal data require physician verification. Monitors conduct regular site visits, perform source data verification, and provide feedback through reports and meetings to ensure data quality.

To ensure data accuracy, investigators at all participating sites must complete the CRFs with complete honesty. Regular audits are conducted by monitoring personnel to verify the recorded information. When making corrections, the original entries must remain legible, with all modifications accompanied by the investigator’s signature and the date of amendment.

Once reviewed by the monitors, the CRFs are forwarded to the clinical trial data management team. The entire process—including communication between researchers, monitors, and data administrators—is meticulously documented. Prior to data entry, the administrators perform another round of validation. Any discrepancies or clarifications will be recorded as query forms and archived for future review. This structured approach ensures traceability and maintains the integrity of the clinical trial data throughout the study.

Within the drug clinical trial facilities of DHABUCM, the data management software EpiData (version 3.1; EpiData Association) is used, and a database is set up. Data entry personnel carry out independent double data entry. Once the data entry process is completed, a number of CRFs are sampled to assess the entry quality, and any existing issues are addressed. The database is reviewed by data administrators in collaboration with principal researchers. The original CRFs are filed and saved after data entry and verification as required. Electronic data files containing data banks, inspection procedures, analysis results, codebooks, and description files have several backups and are saved on different record media properly. All original files are saved according to “China’s Drug Clinical Trial Quality Management Standard” within the specified time.

The approach of this study is to use an intention-to-treat analysis, ensuring that all participants are included in the analysis as originally assigned. Specialized statisticians will handle the statistical analysis in a blinded manner to maintain the integrity of the results. Regarding missing data, particularly for the primary histopathological end point, we will address this issue using the Last Observation Carried Forward method to impute missing data. This approach will help mitigate the potential bias introduced by incomplete data and ensure that the analysis remains robust. Using SAS software (version 6.12; SAS Institute Inc.), all statistical tests will be 2-sided, with a P value of ≤.05 considered to indicate statistical significance. Initially, we will assess the baseline balance to determine the comparability of the intervention and control groups in terms of their fundamental characteristics. For quantitative data, we will provide a comprehensive description including the count, mean, SD, median, and range (minimum-maximum), and use either a t test or a Wilcoxon rank sum test to compare the 2 groups. Qualitative data will be presented in terms of frequency and percentage, with a chi-square test or Fisher exact test used to evaluate differences between the groups. In the analysis of combined medication use, we will enumerate the number and percentage of occurrences and then apply a chi-square test or Fisher exact test to analyze any disparities between the groups. For the assessment of treatment compliance, we will enumerate the cases where the administered dosage of the experimental drug falls below 80%, exceeds 120%, or lies within the 80%-120% range and subsequently perform a chi-square test or Fisher exact test to identify any significant differences.

Regarding safety analysis, we will detail the incidence of AEs and serious AEs that occur during the treatment period. We will also present detailed tables outlining the specifics of AEs and serious AEs to provide a thorough understanding of the safety profile of the treatment.

For the high-dimensional and compositional microbiome data, we will adopt a genome-specific and interaction-focused analytical framework. Briefly, high-quality metagenome-assembled genomes will be reconstructed, and global shifts in the gut microbiota structure will be evaluated using permutational multivariate analysis of variance with permutations. Rather than relying solely on traditional differential abundance testing, coabundance networks will be constructed using Fastspar to account for complex microbial interactions. Subsequently, connected components clustering and weighted correlation network analysis will be applied to identify stably correlated genome clusters, or guilds [14].

This study adheres to the ethical standards described in the Declaration of Helsinki and relevant Chinese clinical research norms and regulations. The trial protocol has been formally reviewed and approved by the Ethics Committee of Wangjing Hospital, China Academy of Chinese Medical Sciences (approval WJEC-KT2024-022-P002, April 2, 2024). Written informed consent must be obtained from each participant prior to enrollment and randomization. Furthermore, strict measures are implemented to maintain the privacy and confidentiality of all research participants; personal identifying information is fully anonymized, and all participant data are securely stored and accessible only to authorized study personnel.

This study hypothesizes that the WW-1 will significantly improve the histopathological outcomes of CAG, specifically by reversing gastric mucosal atrophy and IM, in patients who have achieved successful H pylori eradication. Furthermore, we anticipate that the mechanistic evaluation will demonstrate that WW-1 exerts these therapeutic effects by modulating gastric mucosal DNA methylation profiles and restoring the homeostasis of the digestive tract microbiota.

Currently, the internationally recognized approach to intervening in the cancer transformation process of CAG primarily focuses on eradicating H pylori. However, research has demonstrated that, among individuals infected with H pylori, the number of patients with gastric atrophy continues to significantly increase even 1 year after eradication therapy [20]. In comparing changes in gastric mucosal lesions before and after eradication therapy, scholars often regard IM as a “point of no return” in the cascade model of GC. For instance, a meta-analysis involving 12 studies and 2658 patients revealed that eradicating H pylori significantly improved atrophy in the gastric body but did not alleviate atrophy or IM in the gastric antrum. A Japanese study found that gastric body IM, regardless of extent, was an independent predictor of GC development after H pylori eradication [22]. A long-term randomized controlled trial following 800 patients with CAG in a region of Colombia with high GC incidence for 20 years found that gastric mucosal lesion scores began to decline only 3 years after achieving H pylori negativity [6]. Therefore, delaying the inflammation-carcinoma transformation induced by H pylori represents a critical and challenging step in the prevention of GC. Actively and effectively conducting intervention studies targeting this transformation process is of great significance for GC prevention and control. Currently, there is a lack of specific therapeutic agents supported by high-quality evidence-based medicine. Existing treatment strategies are mainly focused on symptom relief and regular endoscopic and histopathological surveillance, with limited clinical efficacy. TCM, based on a holistic perspective, demonstrates multidimensional advantages in managing gastric precancerous lesions. Our previous multicenter trial, conducted in collaboration with Professor Barry Marshall’s team, indicated that a 24-week intervention with WW-1 significantly improved atrophy scores in the gastric body and angle compared to placebo [12]. Given its proven efficacy in reversing general gastric atrophy, we postulate that WW-1 holds substantial therapeutic potential for the treatment of CAG following H pylori eradication.

Crucially, the microbiota of multiple parts of the digestive tract plays an important role in the occurrence and development of CAG and precancerous lesions. The stomach constitutes a hostile environment for most microorganisms, as potent gastric acid efficiently eliminates indigenous microbes [17,18]. Consequently, traditional culture methods detect only a minimal microbial biomass in the highly acidic stomach. However, when gastric acidity is reduced, such as in cases of mucosal atrophy, the abundance of viable gastric microbiota increases substantially, accompanied by a shift in microbial composition [20]. Early studies on CAG and precancerous lesions focused on the microbiota of the stomach, but more recently, more research has begun to focus on the intestinal microbiota. Some studies have even shown that there is a stronger potential relationship between intestinal microbiota disorder and CAG and precancerous lesions than gastric microbiota. When the intestinal microbiota is out of balance, pathogenic bacteria can release a large number of cytotoxins, and related metabolites such as bile acids, polyamines, and specific enzymes can also promote CAG and precancerous lesions [23].

Moreover, the tongue coating microbiome could also serve as a potential noninvasive biomarker suitable for long-term monitoring of CAG and precancerous lesions. The findings of one study suggest that 21 tongue-coating microbiomes were differentially abundant by metagenomic sequencing between gastritis patients, including superficial gastritis, CAG, IM, and healthy individuals [19]. Notably, the study highlights Campylobacter concisus as a key microbial marker associated with the precancerous cascade of gastritis, a finding that was further validated through its detection in gastric fluid. The observed reduction in microbial diversity and the specific microbial correlations with symptoms such as bitter taste and dry mouth further emphasize the complexity and individuality of the microbiome’s role in gastritis. This discovery provides a scientific foundation for the TCM practice of tongue diagnosis. There is also obvious dysbiosis of the oral microbiota of CAG and other precancerous lesions patients, which might involve the pathogenesis of CAG and other precancerous lesions. Sung et al [20] have demonstrated that following the eradication of H pylori, a distinct cluster of oral bacteria within the gastric mucosa is associated with the development and persistence of CAG and IM, including Peptostreptococcus, Streptococcus, Parvimonas, Prevotella, Rothia, and Granulicatella. From the above, it is apparent that microbial communities throughout the entire gastrointestinal tract can exert a substantial influence on the pathogenesis of CAG and precancerous lesions.

The important mechanism of action of TCM formulas in treating diseases, especially digestive diseases, is through intervention in the digestive tract microbiota. The constituents of TCM formulas modulate the composition and structure of gastrointestinal microbiota, thereby influencing the distant functions of affected organs and tissues through the systemic actions mediated by gastrointestinal microbiota. Following oral administration, TCM formulas interact with gastrointestinal microbiota in several ways: (1) TCM can modulate the composition of gastrointestinal microbiota, (2) TCM can influence the metabolic activities of gastrointestinal microbiota, and (3) gastrointestinal microbiota is capable of transforming compounds found in TCM formulas [24]. In this study, we focus on the regulatory effects of WW-1 on 2 core microbiota groups—keystone functional microbiota and pathogenic functional microbiota. Research suggests that these 2 microbiota groups interact with each other, forming a “seesaw” model that influences human health [14]. WW-1 may help restore the dynamic balance of the microbiota by modulating these core groups, thereby improving both the digestive system and overall health.

Concurrently, DNA methylation plays a critical role in the initiation and progression of various tumors. Consequently, modulating DNA methylation has emerged as a promising therapeutic strategy for oncology. Accumulating evidence suggests that TCM mediates potent antitumor effects through the regulation of DNA methylation processes [25]. TCM concepts of balance and holism are consistent with the equilibrium of DNA methylation modifications within the tumor microenvironment. TCM has been demonstrated to represent a class of potential and reliable agents for the modulation of epigenetic alterations. Therefore, one of the objectives of this study is to use DNA methylation microarray technology to investigate whether the TCM formula WW-1 can regulate the DNA methylation status or levels of specific key gene loci or genes (eg, tumor suppressor genes) in human gastric mucosal tissues, thereby elucidating the underlying mechanisms of WW-1 in preventing and controlling the inflammation-carcinoma transformation process.

The digestive tract microbiota has a significant influence on epigenetic modifications, including DNA methylation in the host, which plays a key role in the development of multiple diseases. Intestinal microbiota can modulate gut homeostasis and inflammation by regulating DNA methylation [26]. Our study will provide novel insights into the potential correlation between digestive tract microbiota and DNA methylation and elucidate the specific regulatory mechanisms underlying CAG and precancerous lesions for the first time.

The collection of digestive tract microbiota strictly adheres to established protocols. Our team possesses extensive research expertise in the field of microbiota, ensuring the implementation of standardized procedures and establishing a solid foundation for obtaining high-quality data [14,27]. Simultaneously, our standardized gastric mucosal sample collection and rigorous double-blind pathological evaluation of the gastric mucosa are pivotal for assessing WW-1 efficacy and lay the foundation for further elucidating the mechanism of WW-1. Accurate and adequate gastric mucosal biopsies are essential for determining the extent of gastric mucosal lesions and for risk stratification of GC using the OLGA and OLGIM staging systems. According to the updated Sydney system gastritis classification method [28], our study takes 5 biopsies from the gastroscope, including 2 from the antrum, 2 from the body, and 1 from the angle of the stomach, so that the efficacy of TCM can be observed overall and in different parts. The mucosa of the antrum minor, the site selected for the collection of the gastric flora under endoscopy, also meets the collection standards certified by high-quality research [29].

A multicenter, randomized, double-blind, placebo-controlled trial is used, with central pathological assessment by blinding as the primary outcome indicator of pathological histological improvement. This study aims to obtain evidence-based clinical data regarding the efficacy and safety of WW-1 for patients with CAG following H pylori eradication, and to reveal its mechanism of action in the prevention of GC at the level of DNA methylation and the digestive tract microbiota. Following completion of the trial’s statistical analysis, we will communicate results to participants, health care professionals, and the public through peer-reviewed publications. If successful, the findings of this clinical trial will be valuable in the treatment of CAG and drug development, providing new ideas for the diagnosis and treatment of CAG and the prevention of GC.

This study focuses on CAG after H pylori eradication to evaluate the therapeutic potential of TCM intervention, offering new strategies for early GC prevention. In previous studies, early screening and prevention of GC have mainly concentrated on pathological indicators and clinical manifestations. However, this study adopts a more comprehensive research approach by evaluating multiple dimensions, including clinical symptoms, molecular mechanisms, and microbiome, offering a more holistic perspective.

A major strength of this trial is the use of DNA methylation profiling to elucidate the mechanisms underlying TCM-mediated GC risk reduction. We hypothesize that TCM modulates the methylation status of key genes (eg, tumor suppressor genes and oncogenes) in the gastric mucosa, thereby regulating their expression and potentially halting disease progression. Furthermore, moving beyond standard pathology and isolated biomarkers, we have established a comprehensive digestive tract microbiota assessment framework. Integrating data from the tongue coating, saliva, gastric, and intestinal microbiota provides a more complete picture of how TCM impacts the gastrointestinal microenvironment, laying the groundwork for future mechanistic research.

However, several limitations should be noted. First, despite the multicenter design, the sample size remains relatively small. Larger-scale cohorts are needed to validate the generalizability of our findings across diverse populations. Second, while we explore the impact of TCM on DNA methylation, the specific interactions between these epigenetic modifications and microbiota alterations require further investigation.

Future research should focus on the following areas: first, further validation of the therapeutic effects of WW-1 on precancerous gastric lesions in larger clinical studies. Second, future studies should consider incorporating long-term follow-up to assess the safety and effectiveness of TCM intervention with prolonged use. Finally, therapeutic strategies targeting specific methylation sites or microbiota imbalances could become a research hotspot in TCM intervention and warrant further exploration.

Following the completion of the trial and comprehensive statistical analysis, the primary findings regarding the clinical efficacy and safety of WW-1, alongside the mechanistic insights, will be submitted for publication in high-impact, peer-reviewed scientific journals. We also plan to present our key findings at major international academic conferences, such as Digestive Disease Week, to share our methodology and results with the global gastroenterology community.