A study for evaluating the improvement effect on Metabolic dysfunction-associated steatotic liver disease (MASLD) of probiotics

Metabolic dysfunction-associated steatotic liver disease (MASLD) is associated with dysbiosis of the gut microbiota and altered host metabolic homeostasis. Probiotics have been proposed as a potential therapeutic strategy to modulate gut microbial composition and improve metabolic and hepatic outcomes in MASLD; however, clinical evidence regarding next-generation probiotic strains remains limited.

This study was designed to evaluate the effects of three next-generation probiotic strains-Lactobacillus delbrueckii subsp. lactis (LL001), Lactobacillus helveticus (LH001), and Pediococcus pentosaceus KID7 (PPKID7)-on liver function parameters and gut microbiome composition in patients with MASLD.

We conducted a randomized, double-blind, placebo-controlled, parallel-group clinical trial. A total of 110 adult patients diagnosed with MASLD were screened for eligibility. Eligible participants were randomly assigned to receive one of the three probiotic formulations (3 capsules per day, total 9×10⁹ CFU) or placebo for 8 weeks. All participants received concomitant silymarin during the intervention period.

Clinical assessments, serum samples, and stool samples were collected at baseline and at the end of the intervention. Liver function parameters were predefined as the primary outcome measure. Secondary outcomes included changes in anthropometric parameters, serum metabolic markers, gut microbiota composition assessed by 16S rRNA gene sequencing, and lipidomic profiles derived from serum and fecal samples. Compliance was monitored throughout the study period.

The study protocol was approved by the institutional review board, and written informed consent was obtained from all participants prior to enrollment.

Metabolic dysfunction-associated fatty liver disease applies to adults exhibiting hepatic steatosis identified through imaging techniques, blood biomarkers, or liver histology. This diagnosis is made in individuals who are overweight or obese, or who have type 2 diabetes mellitus, or at least two other metabolic risk abnormalities. Subsequently, in June 2023, a multi-society Delphi consensus statement was issued on a revised nomenclature for fatty liver diseases. This statement introduced the term metabolic dysfunction-associated steatotic liver disease (MASLD), effectively replacing the previous nonalcoholic fatty liver disease. Representative etiologies of MASLD are known to include insulin resistance, lipid toxicity due to excessive fat accumulation, inflammatory response, and endoplasmic reticulum stress.

The global rise in obesity has contributed to the increased prevalence of MASLD. MASLD affects approximately 30% of the global adult population, with its prevalence rising from 22% to 37% between 1991 and 2019. This increase aligns with the growing rates of obesity and related conditions worldwide. The more severe manifestation of MASLD, known as metabolic dysfunction-associated steatohepatitis (MASH), is histologically characterized by lobular inflammation and hepatocyte ballooning and is linked to a higher risk of fibrosis progression. Among individuals diagnosed with MASLD who do not meet criteria for a liver biopsy, the prevalence of MASH is approximately 7%. MASLD is one of the most common causes of chronic liver disease and liver-related death and serves as a risk factor for extrahepatic diseases such as diabetes and cardiovascular disease. As a result, the burden of social and economic costs caused by the disease is increasing.

Recently, interest in the role of the gut microbiome in the pathogenesis and treatment of liver diseases has increased, and the disease linkage caused by intestinal bacterial imbalance has been identified [16, 17]. Animal studies have demonstrated potential causal roles of the gut microbiota in MASLD [18, 19]. Human studies have described microbiome alterations in healthy individuals and patients with MASLD and have found several consistent specific taxa that differentiate between healthy individuals and patients with MASLD and advanced liver disease. Despite unknown mechanisms and lack of validation, therapeutic strategies utilizing the gut microbiome have the potential to provide beneficial effects in patients with MASLD.

Probiotics are live microorganisms that, when administered in adequate amounts, provide health benefits to the host. In our previous animal studies, Lactobacillus and Pediococcus supplementation improved MASLD by modulating gut microbiota and inflammation. Additional experiments with candidate strains have shown promising results in the prevention of MASLD progression. Several randomized controlled trials using ultrasonography and MASLD diagnostic markers of short duration have suggested that administration of probiotics may have a positive effect on hepatic steatosis and insulin resistance in patients. However, none of the previous studies reported the therapeutic effect of probiotics on the microbiome in patients with MASLD. In this clinical trial, we aimed to investigate whether administering probiotics for 8 weeks could effectively improve blood biochemical parameters and induce alterations in the gut microbiota among patients diagnosed with MASLD.

Inclusion Criteria:

1. Those who agreed to participate in this study and signed a written consent

2. Adult men and women over 20

3. Patients diagnosed with non-alcoholic fatty liver

   ※ Exclusion criteria for alcoholic liver disease

   • Those who have an alcoholic drinking ability of at least 60g per day for at least one year before visiting (30g for women)
   • 1 bottle of shochu 360 ml * 20% = 72 g, 1 bottle of beer 500 ml (330 ml for commercial use) * 5% = 25 (16.5 g)

4. Patients with higher liver numbers than normal ※ Adult normal liver level range by enzyme

   • AST, ALT: 40 or less
   • ALP: 20-130
   • GOT: 0-30, GPT: 0-38
   • GGT: 10-62 (male), 7-35 (female)

Exclusion Criteria:

1. Those who have consumed probiotics (lactic acid bacteria, etc.), prebiotics (dietary fiber, fructooligosaccharide, etc.), new biotics, fermented milk, etc. within the past month.

2. Those who have continuously taken antibiotics within the last 2 months or who are likely to take them during the study period

3. Those who have continuously consumed medicines or health functional foods that affect liver function within the past month.

4. Those who have participated in other clinical trials within the past 1 month (but not applicable to medical device clinical trials)

5. If you have any of the following

   • Alcoholic liver disease, hereditary metabolic disease, autoimmune hepatitis
   • systemic inflammatory disease or immune disease
   • Hepatocellular carcinoma
   • Uncontrolled cardiopulmonary disease
   • Other serious systemic disorders in the heart, lungs, blood, and endocrine system

6. A person with a history of malignancy diagnosis within the last 5 years

7. Pregnant or lactating women

8. Persons who have hypersensitivity to the test drug / placebo or components contained in the test drug / placebo or have severe allergic reactions

9. Those who are not suitable for the clinical trial because the investigator judges

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