Effects of Short-term Perioperative Daidzein Intervention on Liver Histopathology in Patients With MASLD

Short-Term Daidzein Intervention in MASLD

This clinical trial aims to learn whether daidzein can improve liver tissue changes in adults with metabolic dysfunction-associated steatotic liver disease, also called MASLD. MASLD is a liver condition linked to extra fat in the liver and metabolic problems such as obesity, diabetes, abnormal blood lipids, or high blood pressure.

Daidzein is a natural compound found in soy. Earlier laboratory studies suggest that daidzein may help protect the liver. This study will test whether taking daidzein for a short time before surgery can improve liver tissue findings in people with MASLD.

The main questions this study aims to answer are:

Does short-term daidzein treatment improve liver tissue injury in people with MASLD? Is daidzein safe and well tolerated before surgery? Are changes in blood or urine equol levels related to the effects of daidzein? Equol is a substance made by gut bacteria after some people take daidzein.

Researchers will compare people who take daidzein before surgery with people who receive standard care without daidzein.

Participants will:

Be adults with MASLD who are scheduled for elective gallbladder surgery or another benign biliary surgery.

Be randomly assigned to take daidzein or to receive standard care without daidzein.

Take daidzein by mouth for 28 days before surgery if assigned to the daidzein group.

Avoid soy foods during the study period. Provide blood and urine samples. Have a small liver tissue sample collected during surgery. Be followed for safety and recovery after surgery.

The liver tissue sample will be used to check liver fat, inflammation, and liver cell injury. Researchers will also study markers related to liver injury, immune activity, and how the body responds to daidzein.

Metabolic dysfunction-associated steatotic liver disease, or MASLD, is one of the most common chronic liver diseases. It is closely associated with obesity, type 2 diabetes, dyslipidemia, hypertension, and other cardiometabolic risk factors. MASLD is diagnosed when hepatic steatosis is present together with at least one cardiometabolic risk factor. Its disease spectrum ranges from simple steatosis to steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. Because of its high prevalence and potential for disease progression, MASLD has become an important public health problem.

Current treatment strategies for MASLD mainly include weight reduction, dietary control, physical activity, and management of metabolic abnormalities. However, these approaches may be difficult to standardize and maintain over time, and treatment responses vary among individuals. There remains a need for safe, practical, and mechanism-based interventions that may improve liver injury and disease activity in MASLD.

MASLD is not only a metabolic liver disease but also an immunometabolic disease. Its development and progression involve hepatic lipid accumulation, oxidative stress, hepatocellular injury, ferroptosis-related biological changes, and immune-inflammatory responses. However, direct evidence from human liver tissue remains limited. In particular, it is unclear whether a short-term, mechanism-oriented intervention can induce measurable changes in liver histology, ferroptosis-related markers, and intrahepatic immune-inflammatory features in patients with MASLD.

Daidzein is an important bioactive component of soy isoflavones. It has been reported to have metabolic regulatory, antioxidant, and anti-inflammatory properties. Previous experimental studies by the investigators suggest that daidzein can improve metabolic dysfunction and reduce liver injury in MASLD models. These studies also suggest that the beneficial effects of daidzein may depend, at least in part, on gut microbiota-mediated metabolic conversion.

The investigators' preclinical work combining metagenomic and metabolomic analyses identified equol as a key gut microbiota-derived metabolite that increases after daidzein intervention. Mechanistic findings suggest that the daidzein-equol axis may improve MASLD-related liver injury by coordinating hepatocyte protection and restraint of immune-inflammatory responses. These findings provide a scientific basis for evaluating daidzein in a clinical translational setting.

Several important questions remain unanswered. First, the effects of daidzein in humans may be influenced by the metabolic capacity of the gut microbiota. Not all individuals can efficiently convert daidzein into equol. Therefore, the response to daidzein may vary between equol producers and non-producers. Second, whether equol-producing capacity is associated with the magnitude of response to daidzein has not been prospectively evaluated in patients with MASLD. Third, although preclinical studies suggest that the daidzein-equol axis may regulate ferroptosis and immune-inflammatory responses, it remains unknown whether short-term daidzein intervention can produce detectable changes in human liver tissue.

This study is designed to address these questions using a perioperative window-of-opportunity approach. Adult patients with MASLD who are scheduled to undergo elective laparoscopic cholecystectomy or other benign biliary surgery will be enrolled. This clinical setting provides a feasible opportunity for short-term preoperative intervention without delaying or replacing standard surgical care. It also allows intraoperative collection of a small liver tissue sample for direct histological and biomarker assessment.

This is a prospective, open-label, randomized controlled study. Eligible participants will be randomly assigned in a one-to-one ratio to a daidzein intervention group or a control group. Participants in the daidzein group will receive oral daidzein capsules before surgery. Participants in the control group will receive standard perioperative care without daidzein intervention. Both groups will undergo their planned surgical procedure according to routine clinical practice.

The overall objective of the study is to evaluate whether short-term perioperative daidzein intervention can improve liver histological changes in patients with MASLD and to explore its biological effects on ferroptosis-related markers, intrahepatic immune-inflammatory features, and equol-related metabolic heterogeneity. The primary scientific focus is the difference in liver histological activity between the daidzein group and the control group based on intraoperative liver tissue assessment.

Liver histological activity will be evaluated using the NAFLD Activity Score system. Although MASLD terminology is used in this study, the NAFLD Activity Score remains a widely used histological scoring system for assessing steatosis, lobular inflammation, and hepatocellular ballooning. These components will be used to assess liver tissue activity and injury.

The study will also assess ferroptosis-related markers in liver tissue. Core markers such as GPX4 and ACSL4 will be evaluated by methods such as immunohistochemistry or immunofluorescence. These analyses are intended to explore whether daidzein intervention is associated with changes in ferroptosis-related biological features in human MASLD liver tissue.

Intrahepatic immune-inflammatory features will also be explored. The study will evaluate CD8-positive T-cell infiltration and related inflammatory features in liver tissue. These assessments are intended to examine whether daidzein may influence the immune-inflammatory microenvironment in MASLD.

Blood and urine samples will be collected to measure equol levels and selected biochemical and metabolic indicators. Equol measurements will be used to explore whether the ability to produce equol is associated with the response to daidzein. This exploratory analysis may help identify biological factors that contribute to individual differences in response to daidzein intervention.

The study will also assess clinical laboratory indicators, including liver enzymes and metabolic parameters. These data will be used together with liver tissue findings to provide a more comprehensive assessment of the biological effects of daidzein. Safety and tolerability will be monitored throughout the study, including adverse events, perioperative complications, and postoperative recovery.

The planned sample size is forty-four participants, with twenty-two participants in each group. This sample size is intended to support an exploratory, early-phase mechanistic clinical study. The study is not designed to provide definitive evidence of long-term clinical efficacy. Instead, it is intended to provide early human tissue-level evidence on whether short-term daidzein intervention can produce measurable histological and biological changes in MASLD.

The observation period will extend from enrollment to thirty days after surgery. Data collection will include baseline clinical information, relevant medical history, imaging information, medication use, intervention adherence, adverse events, intraoperative liver tissue findings, blood and urine test results, perioperative complications, and short-term postoperative recovery.

Although the study is open-label for participants and clinical investigators, outcome assessment will include measures to reduce bias. Liver tissue samples will be coded and de-identified whenever feasible. Histological evaluation will be performed by independent pathologists or trained evaluators who are not involved in treatment allocation or intervention management. Immunohistochemistry, immunofluorescence, and related molecular assessments will also be performed under blinded or independent evaluation conditions whenever possible.

Data will be collected using case report forms and entered into a study database. Each participant will be assigned a unique study identification code. Personal information will be protected through de-identification and restricted data access. Data quality procedures will include double checking of key data, review of missing values, range checks, consistency checks, and source data verification when needed. Corrections to study data will follow predefined procedures to ensure accuracy, completeness, and traceability.

The statistical analysis will be conducted according to predefined analysis principles. Continuous variables will be summarized using means and standard deviations or medians and interquartile ranges, depending on data distribution. Categorical variables will be summarized using counts and percentages. Between-group comparisons will be performed using appropriate statistical tests based on the type and distribution of the data. Safety analyses will summarize adverse events, serious adverse events, perioperative complications, and tolerability. Exploratory analyses will evaluate whether equol-producing capacity is associated with differences in liver histology, biomarker changes, or other biological responses to daidzein.

This study is expected to provide early clinical and translational evidence for the potential role of daidzein in MASLD. By using intraoperative liver tissue assessment, the study may help determine whether short-term daidzein intervention can induce measurable improvements in liver histological activity and related biological abnormalities. The findings may also provide preliminary evidence for the role of gut microbiota-derived equol in shaping individual responses to daidzein.

Inclusion Criteria:

• Adults aged 18 to 70 years.
• Male or female participants.
• Scheduled to undergo elective laparoscopic cholecystectomy or other benign biliary surgery for benign biliary disease.
• Diagnosis of metabolic dysfunction-associated steatotic liver disease based on hepatic steatosis shown by liver biopsy or imaging, including controlled attenuation parameter, ultrasound, or magnetic resonance imaging, together with at least one cardiometabolic risk factor.
• Expected preoperative window of at least 28 days before surgery.
• Able and willing to provide written informed consent.
• Willing to provide blood samples, urine samples, and intraoperative liver tissue samples.
• Able and willing to avoid soy-containing foods during the study period, including soybeans, edamame, black soybeans, soy milk, tofu, dried tofu, tofu skin, yuba, tofu pudding, natto, miso, fermented soybeans, tempeh, and other traditional or fermented soy products.

Exclusion Criteria:

• Viral hepatitis, autoimmune liver disease, drug-induced liver injury, Wilson disease, or other clearly defined chronic liver diseases.
• Significant alcohol consumption, defined as more than 140 g per week for women or more than 210 g per week for men.
• Liver cirrhosis, decompensated liver disease, or hepatobiliary malignancy.
• Use of antibiotics, probiotics, soy isoflavone-containing products, or hormone-like dietary supplements within 4 weeks before enrollment.
• Treatment within 3 months before enrollment that may substantially affect body weight or the severity of fatty liver disease.
• Known allergy to soy products, daidzein, or isoflavones.
• Pregnancy or breastfeeding.
• Any condition that, in the opinion of the investigator, makes the participant unsuitable for this study.