Effect of Probiotics or Berberine in Hepatic Steatosis Markers, Cardiometabolic and Microbiotic Profile in NAFL.

Study Overview
=================
Brief Summary
-----------------
Effect of oral selected Probiotics (PRO) and/or Berberine (BBR) supplementation on hepatic steatosis markers, cardiometabolic profile, and gut microbiota profile in the non-alcoholic fatty liver (NAFL) - a randomized double-blind clinical study.

Detailed Description
-----------------
Probiotics (PRO) and bioactive natural substances such as Berberine (BBR) can improve metabolic parameters in patients with obesity and metabolic disorders. In addition, they significantly affect the composition and function of gut microbiota (GM) and support anti-inflammation and antioxidant defense. These data have become the starting point for the proposed multidirectional approach, aimed at assessing the effect of PRO and/or BBR supplementation on: hepatic-related outcomes, changes in anthropometric measurements (body mass, BMI, body mass composition and fat mass % content), cardiometabolic profile (e.g. blood pressure, noninvasive markers of endothelial function, cardiometabolic biochemical parameters) microbiotic profile (gut microbiota composition, endotoxemia) the content of the minerals, in overweight/obese patients with nonalcoholic fatty liver (NAFL).

Official Title
-----------------
Effect of Probiotics or Berberine Supplementation on Hepatic Steatosis Markers, Cardiometabolic and Microbiotic Profile in NAFL - A Randomized Double- Blind Clinical Study.

Conditions
-----------------
Non Alcoholic Fatty Liver, Obesity

Intervention / Treatment
-----------------
* Dietary Supplement: Probiotic
* Dietary Supplement: Berberine
* Dietary Supplement: Placebo
* Dietary Supplement: Probiotc and Berberine


Participation Criteria
=================
Eligibility Criteria
-----------------
Inclusion Criteria: age 40 to 60 years; women ≥1 year since last menstruation; body mass index (BMI): 27.0 kg/m2 to 34.9 kg/m2; abdominal obesity-related waist circumference > 80 cm (women) and >94 cm (men) (in accordance to International Diabetes Federation); stable body weight in the 3 months prior to the trial (permissible deviation is ± 3 kg); NAFL - diagnosed based on USG in accordance with PGE-NAFLD recommendation Exclusion Criteria: history of following alternative diets within 3 months before the study; history of use of any dietary supplements in the 3 months before the study; history of intake of antibiotics, probiotics, prebiotics within 3 months before the study; secondary form of obesity, pharmacological treatment for obesity (in the 3 months before the study), history of bariatric surgery; another liver diseases: high risk of NASH (assessed on the FIB-4, according to the PGE-NAFLD recommendation), autoimmune hepatitis, hepatitis B and C, toxic hepatitis, cirrhosis, Wilson's disease, hemochromatosis; other gastrointestinal disorders, especially: IBD, celiac disease, gastritis and duodenitis, pancreatic disorders, gastrointestinal symptoms suggestive of IBS; clinically significant acute inflammatory process (elevated hsCRP); abnormal kidney function (GFR <60mL/min/1,73m2); T2D; dyslipidemia or hypertension - requiring the introduction and/or change of pharmacological treatment in the 6 months before the trial or during intervention; pump inhibitors, anticoagulants, drugs causing metabolic alteration, e.g., SFAs (second-generation antipsychotics); diseases requiring nutritional requirement and chronic supplementation; alcohol (>30g/d for men and >20g/d for women), nicotine or drug abuse; mental disorders, including eating disorders; cancer, autoimmune diseases; any other condition which may influence on final results of the study or pose a risk for subjects health.

Ages Eligible for Study
-----------------
Minimum Age: 40 Years
Maximum Age: 60 Years

Sexes Eligible for Study
-----------------
All

Accepts Healthy Volunteers
-----------------
No

Study Plan
=================
How is the study designed?
-----------------

Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: Parallel Assignment
Masking: Double


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Active Comparator: Probiotic<br>Individuals receive Probiotics (9 strains: B. bifidum W23, B. lactis W51, B. lactis W52, L. acidophilus W37, L. brevis W63, L. casei W56, L. salivarius W24, Lactococcus lactis W19, and Lactococcus lactis W58) in dose: 1x109 colony forming units (CFU), daily, for 12 weeks. Intervention: Dietary Supplement: Probiotic | Dietary Supplement: Probiotic<br>* The probiotic group will receive one capsule of the probiotic mixture (dose:1x109 colony forming units (CFU) per day in one dose (before breakfast). The PRO preparation will contain the following bacterial strains: 50% Lactococcus lactis Rosell® - 1058, 25% Lactobacillus casei Rosell® - 215, 12,5% Lactobacillus helveticus Rosell® - 52, 12,5% Bifidobacterium bifidum Rosell® - 71). Probiotics will be administered orally.<br>|
| Active Comparator: Berberine<br>Individuals receive Berberine (Berberine hydrochloride 97% extract of Berberis aristata) in dose: 1500 mg/day, for 12 weeks. Intervention: Dietary Supplement: Berberine | Dietary Supplement: Berberine<br>* The berberine group will receive 1500 mg of Berberine (Berberine hydrochloride 97% extract of Berberis aristata) per day in 3 doses. Berberine will be administered orally, before breakfast, dinner, and before supper.<br>|
| Placebo Comparator: Placebo<br>Individuals receive placebo daily, for 3 months. Intervention: Dietary Supplement: Placebo | Dietary Supplement: Placebo<br>* The placebo group will receive a placebo. Placebo will contain only the excipients and will be administered orally for 24 weeks. Placebo in no way: color, taste, smell, form of taking, the dosage will not differ from the preparations tested. However, it will not contain probiotcs or berberine. Placebo will be orally administered three times a day: before breakfast, dinner, and supper (6.00-7.00 p.m.). To meet the GCP conditions, subjects from all groups will receive the same number of capsules (six) per day.<br>|
| Active Comparator: Probiotics and Berberine<br>Individuals receive: Probiotics (9 strains: B. bifidum W23, B. lactis W51, B. lactis W52, L. acidophilus W37, L. brevis W63, L. casei W56, L. salivarius W24, Lactococcus lactis W19, and Lactococcus lactis W58) in dose: 1x109 colony forming units (CFU), daily and Berberine (Berberine hydrochloride 97% extract of Berberis aristata) in dose: 1500 mg/day, for 12 weeks. Intervention: Dietary Supplement: Probiotic and Berberine | Dietary Supplement: Probiotc and Berberine<br>* Probiotics and Berberine groupwill receive both: a probiotics mixture (as in PRO group: 1x109 CFU/day; in one dose) and 1500 mg/day of Berberine (Berberine hydrochloride 97% extract of Berberis aristata; in 3 doses). Probiotcs and berberine will be administered orally before breakfast, before dinner, and before supper.<br>|

What is the study measuring?
-----------------
Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Changes in Fibrosis-4 (FIB-4) - Index for Liver Fibrosis. | FIB-4 will be estimated using a medical calculator (based on parameters as: age, ALT, AST, and platelet count). | At the baseline and 12 weeks of treatment | 
| Changes in HSI - Hepatic Steatosis Index. | HSI will be estimated using a medical calculator (based on parameters as: gender, ALT, AST, BMI, and type 2 diabetes). | At the baseline and 12 weeks of treatment | 
| Changes in NAFLD-LFS (liver fat score). | NAFLD-LFS will be estimated using a medical calculator (based on serum aspartate transaminase/alanine transaminase (AST/ALT) ratio, fasting serum aspartate transaminase (AST) level, fasting serum insulin level, presence of metabolic syndrome and diabetes mellitus). | At the baseline and 12 weeks of treatment | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Changes in blood pressure. | Resting seated BP (both systolic and diastolic) will be measured using a brachial cuff (Omron Healthcare, Kyoto, Japan) three times at 2-min intervals, and the average value will be calculated. | At the baseline and 12 weeks of treatment | 
| Changes in weight. | Weight will be measured in light clothing and without metal objects (i.e., belt, jewelry). Weight will be measured to the nearest 0.1 kg. | At the baseline and 12 weeks of treatment | 
| Changes in waist circumference, hip circumference. | WC will be measured between the iliac crest and the lower rib at the end of a normal expiration, and HC will be measured around the widest portion of the buttocks. Both HC and WC will be measured using stretch-resistant medical tape (Seco) to the nearest 0.5 cm. The measurement of WC and HC will be performed according to the World Health Organization (WHO) protocol. | At the baseline and 12 weeks of treatment | 
| Changes in waist to hip ratio. | WHR will be calculated as WC to HC quotient. | At the baseline and 12 weeks of treatment | 
| Changes in BMI. | BMI will be calculated according to the formula: BMI = kg/m2 where kg is a person's weight in kilograms and m2 is their height in meters squared. | At the baseline and 12 weeks of treatment | 
| Changes in fat mass content in the body. | The fat mass content [in kg] in the body will be estimated using BIA methods (InBody 270 and Bioscan 920-2), | At the baseline and 12 weeks of treatment | 
| Changes in pulse wave velocity (PWV). | The pulse wave velocity (PWV) will be measured using the SphygmoCor Px (Atcor Medical Blood Pressure Analysis System, Sydney, Australia) in a temperature-controlled room before making anthropometric measurements and blood collection. | At the baseline and 12 weeks of treatment | 
| Changes in pulse wave analysis (PWA). | The pulse wave analysis (PWA)will be measured using the SphygmoCor Px (Atcor Medical Blood Pressure Analysis System, Sydney, Australia) in a temperature-controlled room before making anthropometric measurements and blood collection. | At the baseline and 12 weeks of treatment | 
| Gut (taxonomic and functional) microbiota analysis in stool. | Analyzed by the NGS method. | At the baseline and 12 weeks of treatment | 
| Short-chain fatty acids (SCFAs) concentration in stool. | Analyzed using gas chromatography (Agilent Technologies 1260 A GC system with a flame ionization detector (FID)) | At the baseline and 12 weeks of treatment | 
| Measurement of hair minerals (Fe, Mg, Ca, Cu, Zn) concentration. | Performed using atomic absorption spectrometry (Atomic Absorption Spectrophotometer ZA3000, Hitachi, Tokyo, Japan) | At the baseline and 12 weeks of treatment | 
| Changes in ALT, AST, GGT | The ALT, AST, GGT will be measured using standard methods. | At the baseline and 12 weeks of treatment | 
| Changes in non-esterified free fatty acids. | The NEFA will be measured using standard methods. | At the baseline and 12 weeks of treatment | 
| Changes in lipids profile (TC, HDL, TG). | The TC, HDL, TG will be measured using standard methods. | At the baseline and 12 weeks of treatment | 
| Changes in low-density lipoprotein (LDL). | The low-density lipoprotein cholesterol (LDL) will be calculated according to the Friedwald equation. | At the baseline and 12 weeks of treatment | 
| Changes in fasting glucose level. | The fasting glucose level will be measured using standard methods. | At the baseline and 12 weeks of treatment | 
| Changes in fasting insulin level. | The ELISA will be used in the estimation. | At the baseline and 12 weeks of treatment | 
| Changes in insulin resistance index (HOMA-IR) | The HOMA-IR will be calculated according to formula: HOMA-IR = (insulin * glucose) / 22.5 for the glucose concentration in mmol/L, or: HOMA-IR = (insulin * glucose ) / 405 for glycemia in mg/dL. In both cases, the insulin is in mU/L. | At the baseline and 12 weeks of treatment | 
| Changes in parameter of liver damage: cytokeratin 18. | The ELISA will be used in the estimation cytokeratin 18 (ccK18). | At the baseline and 12 weeks of treatment | 
| Changes in parameter of liver damage: Glutathione S-transferase (GST). | The ELISA will be used in the estimation GST. | At the baseline and 12 weeks of treatment | 
| Changes in parameter of liver damage: collagen IV. | The ELISA will be used in the estimation collagen IV. | At the baseline and 12 weeks of treatment | 
| Changes in parameter of liver damage: hyaluronic acid. | The ELISA will be used in the estimation hyaluronic acid. | At the baseline and 12 weeks of treatment | 
| Changes in hsCRP. | The hsCRP will be measured using ELISA. | At the baseline and 12 weeks of treatment | 
| Changes in pentraxin 3. | The pentraxin 3 (PTX3) will be measured using ELISA. | At the baseline and 12 weeks of treatment | 
| Gut barrier integrity parameter: calprotectin. | The ELISA, will be used in the estimation. | At the baseline and 12 weeks of treatment | 
| Gut barrier integrity parameters: liver fatty acid-binding protein (L-FABP), intestinal fatty acid-binding protein (I-FABP). | The ELISA, will be used in the estimation. | At the baseline and 12 weeks of treatment | 
| Gut barrier integrity parameters: lipopolysaccharide (LPS). | The ELISA, will be used in the estimation. | At the baseline and 12 weeks of treatment | 
| Cardiometabolic risk. | Cardiometabolic risk will be estimated at baseline and after 3 months of treatment using the SCORE scale. SCORE scale summarize 5 risk factors (sex, age, systolic blood pressure, total cholesterol and smoking). | At the baseline and 12 weeks of treatment | 

 Terms related to the study
=================
Keywords Provided by Centre Hospitalier Valida
-----------------
NAFL; obesity; probiotics; berberine; microbiota;