Editor: Jian-Gao Fan, Shanghai Jiaotong University School of Medicine, China
These authors equally contributed.
Nonalcoholic fatty liver disease (NAFLD) is closely related to gut-microbiome. There is a paucity of research on which strains of gut microbiota affect the progression of NAFLD. This study explored the NAFLD-associated microbiome in humans and the role of
The gut microbiome was analyzed via next-generation sequencing in healthy people (n=37) and NAFLD patients with elevated liver enzymes (n=57). Six-week-old male C57BL/6J mice were separated into six groups (n=10 per group; normal, Western, and four Western diet + strains [109 colony-forming units/g for 8 weeks;
Compared to healthy subjects (1.6±4.3), NAFLD patients showed an elevated
Ingestion of
- Ingestion of
-
- The use of
Nonalcoholic fatty liver disease (NAFLD) is characterized by liver lipid accumulation or inflammation. Also, it is one of the most common chronic liver diseases and can lead to the development of liver diseases, such as cirrhosis and hepatocellular carcinoma [
Gut microbiome plays an important role in human physiology with significant impacts on inflammation and disease [
A previous study reported that cholesterol is a significant risk factor for NAFLD [
This prospective observational study was carried out between April 2017 and March 2020 (
Baseline evaluation was performed on complete blood count, liver function test, and viral markers. Patients with NAFLD underwent abdominal ultrasound or computed tomography. AST, ALT, creatine, total cholesterol, gamma glutamyl transferase (γGT), triglyceride (TG), fasting blood sugar (FBS), and high density lipoprotein cholesterol were included as serum biochemical parameters. Tests for hepatitis viruses and human immunodeficiency were conducted in all subjects. Enrolled patients and control groups underwent stool sampling and clinical analysis. Clinical data were simultaneously matched with metagenomics data. Fecal samples were obtained in a plastic collection kit at various times during the day. All samples were stored at −80°C. In the case of healthy controls, the patients collected the stool samples at home and kept them at −20°C in a refrigerator. The patients then sent the stool box to the hospital, where the samples were kept at −80°C in a refrigerator.
Metagenomic DNA was extracted using a QIAamp stool kit (cat. no. 51504; QIAGEN, Hilden, Germany). Barcoded universal primers were utilized in the amplification of the V3–V4 region of the bacterial 16S rRNA gene. Polymerase chain reaction (PCR) has conducted in the following steps: denaturation at 95°C for 5 minutes, 20 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, followed by extension at 72°C for 10 minutes. Agencourt AMPure XP system (Beckman Coulter, Pasadena, CA, USA) was employed for the purification of amplicons. PicoGreen and quantitative PCR was utilized for the quantification of the purified amplicons. Following the pooling of the barcoded amplicons, MiSeq sequencer on the Illumina platform (ChunLab Inc., Seoul, Korea) was used for sequencing according to the manufacturer’s specifications.
The 16S-based Microbial Taxonomic Profiling platform of EzBioCloud Apps (ChunLab Inc.) was used for microbiome profiling. Following the taxonomic profiling of each sample, a comparative analysis of the samples was performed by a comparative of EzBioCloud Apps. ChunLab’s 16S rRNA database (DB ver. PKSSU4.0) [
Using 16S rRNA sequencing, primers were prepared according to the constant region, and base sequence of the variable region was confirmed through PCR amplification.
Six-week old specific-pathogen-free C57BL/6J male mice were sourced from Doo-Yeol Biotech (Seoul, Korea). All mice were housed in individual steel micro isolator cages that were maintained at 22±2°C in a 12/12-hour light/dark cycle. Throughout the experiment, the mice had free access to water and food, and were monitored daily. The experiment design included an adaptation period for all groups, during which the mice were fed a normal diet for a week, and the groups receiving a westernized diet were given a 3-day intake adaptation period. The mice were treated humanely, and all aspects of the animal study was conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. This experiment was designed by selecting a candidate for the treatment of liver disease among the probiotics approved by South Korea’s Ministry of Food and Drug Safety. All of the procedures were licensed by the Institutional Animal Care and Use Committee of the College of Medicine, Hallym University (2018–04).
The male C57BL/6J mice had free access to water and food, and were monitored daily (n=10 mice per each group). The mice were distributed into six groups: normal (n=10), Western diet (n=10), and Western diet with strain groups (n=10 per group). The Western diet (TD88137, Seoul, Korea) was sourced from Doo-Yeol Biotech, and it consisted of 42.7% carbohydrate, 42% fat, and 15% protein. Probiotics were suspended in distilled water maintaining a concentration of 109 CFU/g for 8 weeks. The strains used were
From animal serum, the total cholesterol, AST, and ALT were quantified using a biochemical blood analyzer (KoneLab 20; Thermo Fisher Scientific, Waltham, Finland). The AST/ALT ratio is calculated as a method to elucidate the etiology of liver damage [
Triglyceride Quantification Kit (Sigma, St-Louis, MO, USA) was used to determine liver TG by colorimetric method. Liver tissue samples were homogenized in 5% Nonidet P 40 Substitute (Roche, Mannheim, Germany). The samples were heated to 100℃ in water bath for 5 minutes or until the Nonidet P 40 became cloudy, and then cooled to room temperature. Heating was reported once more to solubilize all TG. The samples were centrifuged for 2 minutes at maximum speed to remove insoluble material, and then diluted 100-fold with deionized water before assay.
A 10% formalin was used in the fixation of specimens, which were embedded in paraffin. The tissue sections underwent staining with hematoxylin and eosin. The liver was categorized in accordance with the clinical research network scoring system for NAFLD, which ranges from grade 0 to 3 (0: <5%, 1: 5–33%, 2: 34–66%, and 3: >66% of steatosis). Inflammation was categorized according to grades 0 to 3 (0: none, 1: 1–2 foci per ×20 field, 2: 2–4 foci per ×20 field, and 3: >4 foci per ×20 field). A hepato-pathologist (S.H.H.) analyzed all of the biopsy specimens.
Liver tissue samples were homogenized in TRIzol reagent (Invitrogen, Gaithersburg, MD, USA). High Pure RNA Isolation Kit (Roche) was used to isolate RNA from liver tissue. Total RNA isolation from tissue was used a cDNA reverse transcription kit (applied Biosystems, Foster City, CA, USA), and aliquots of total RNA (2 μg) were transformed into cDNA. The cDNA subsequently underwent amplification for quantitative PCR using the Luna® Universal Probe qPCR Master Mix (New England Biolabs, Beverly, MA, USA) and target-specific probe-primer (applied Biosystems).
Continuous variables were expressed as means and standard deviations. One-way analysis of variance (ANOVA) and independent sample
This study was performed between April 2017 and March 2020. A total of 94 patients comprising normal controls (n=37) and NAFLD with elevated liver enzyme (n=57) groups were classified according to their conditions and then analyzed (
The mice liver specimen confirmed that NAFLD was induced in the Western diet group (
Several biochemical tests were performed to evaluate the liver function status. In the liver function test, the Western diet group showed remarkably increased liver enzyme (AST) levels compared to the normal control group (
Triglyceride is a type of lipid that circulate in blood. In the analysis of the liver TG level (ng/µL), all of the groups with the strain
Real-time reverse transcription-polymerase chain reaction showed elevated mRNA expressions of TNF-α, IL-6, and IL-1β in the Western diet group. However, the mRNA expression levels of these inflammatory genes were decreased in the
In the phylum level, the composition of
NAFLD can progress to various liver diseases such as cirrhosis, liver failure, and hepatocellular carcinoma in varying proportion of patients [
There is evidence that cholesterol accumulation contributes to NAFLD, and that lowering cholesterol can be used therapeutically. Also, studies have shown that cholesterol-lowering diet can improve NASH, and adding cholesterol to one’s diet may cause liver inflammation and inflammation of extrahepatic tissue [
In our study, there was a significant difference in the proportion of phylum level in stool microbes between normal and NAFLD patients. Many studies showed that various factors, including age, genetic, diet, antibiotics, and host immune system can modify the gut microbiome from the normal state [
Recent studies have shown the role of gut microbiome in the prevention and treatment of liver disease [
Currently, some microbiota can be used as a functional raw material for probiotics, and a total of 21 probiotic strains have been recognized as probiotics by South Korea’s Ministry of Food and Drug Safety, including
In previous studies,
In a previous study, patients with hypercholesterolemia who consumed
Previous studies have shown that the Western diet contributes to changes in the composition of gut microbiome [
Overall, according to the results of our animal experiment, the strains decreased the body weight along with lower serum total cholesterol, triglyceride, and steatosis levels. Pro-inflammatory cytokines were decreased and improved, but only showed a decreasing trend, probably as a result of not being given the Western diet for a long time. It is considered a one-shot state in which only fat is formed among the two-shot theory of fatty liver progression. Cytokines are known to play an important role in coordinating the production of many other mediators associated with chronic liver disease and affecting all liver cell types [
The purpose of this study was to analyze the intestinal bacteria of normal and NAFLD subjects based on the fecal results of clinical patients, as well as to confirm the difference and determine how the strains showing the difference affect the fatty liver. In conclusion, this study suggests that gut-microbiota-liver interaction plays an important role in NAFLD. It also showed that probiotics reduced liver steatosis and cholesterol in NAFLD.
Many studies are being conducted to identify the role of probiotics in various diseases. However, there is insufficient research on how probiotics affect diseases. Therefore, an analysis of animal experiments based on the human gut microbiome is needed to elucidate the underlying mechanisms. Microbiome identifies the effects of metabolic disorders in the gut microbiome on insulin resistance [
The
NYL and KTS designed the study; NYL, SHH, BYK, BKK and KTS performed the experiments and collected data; KTS selected the patients, provided the samples; NYL and KTS analysis data and drafting of the manuscript; All other authors assisted the experiments; NYL, MJS and KTS reviewed and refined the manuscript.
This research was supported by Hallym University Research Fund, Korea National Research Foundation (NRF2018M3A9F3020956 and NRF-2018M3A9F3020942), Basic Science Research Program (2020R1A6A1A03043026) through the NRF funded by the Ministry of Education, and Hallym University Research Fund 2018 (HURF-2018-67). The experiment was conducted with support from CKD bio, and Chunlab helped with the analysis.
alanine aminotransferase
aspartate aminotransferase
body mass index
colony-forming units
fasting blood sugar
interleukin
multi touch point
de Man, Rogosa and Sharpe
nonalcoholic fatty liver disease
National Center for Biotechnology Information
polymerase chain reaction
Sequence Read Archive
triglyceride
tumor necrosis factor
gamma glutamyl transferase
Patient enrollment diagram. Ninety-four patients participated in this experiment. According to their conditions, patients were divided into either normal control or NAFLD with elevated liver enzyme groups. NAFLD, nonalcoholic fatty liver disease; AST, aspartate aminotransferase; ALT, alanine aminotransferase; BMI, body mass index.
Human gut microbiome was analyzed for 16s rRNA. (A) Phylum composition of normal controls and NAFLD patients with elevated liver enzymes. (B) The relative abundance of
Result of NAFLD model induced by Western diet. (A) Flow chart of the animal experiment. (B) Gross specimen of mice liver. (C) Effect of strains on body weight gain. (D) Body weight, Liver weight, and L/B ratio in each group. L/B, liver/body; NAFLD, nonalcoholic fatty liver disease. *
Effects of strains on serum cholesterol level and steatosis of the liver. (A) Effect of strains on liver function test by serum biochemistry analysis. (B) Hematoxylin and eosin staining showed steatosis grade. There were large fat vacuoles displacing the nuclei to the edges of the cells (Western diet).
Animal stool analysis. (A) Phylum composition of animal stool in normal control, Western diet, and
Baseline characteristics of patients
Variable | Normal controls (n=37) | NAFLD patients with elevated liver enzyme (n=57) | |
---|---|---|---|
Sex, male | 14 (39) | 23 (43) | NS |
Age (years) | 61 (8) | 53 (14) | 0.004 |
AST (IU/L) | 23 (5) | 52 (22) | <0.001 |
ALT (IU/L) | 19 (7) | 68 (37) | <0.001 |
Creatine (mg/dL) | 0.9 (0.2) | 1.6 (0.7) | NS |
Total cholesterol (mg/dL) | 176 (36) | 181 (39) | NS |
γGT (IU/L) | 27 (18) | 75 (67) | <0.001 |
TG (mg/dL) | 104 (61) | 238 (280) | 0.022 |
FBS (mg/dL) | 98 (11) | 126 (26) | <0.001 |
HDL cholesterol (mg/dL) | 57 (17) | 47 (11) | NS |
BMI (kg/m2) | 21 (2) | 38 (20) | <0.001 |
Values are presented as mean (standard deviation or %).
NS, not significant; AST, aspartate aminotransferase; ALT, alanine aminotransferase; γGT, gamma glutamyl transferase; TG, triglyceride; FBS, Fasting blood sugar; HDL, high density lipoprotein cholesterol; BMI, body mass index.
Information of used probiotics strains
Strain | Number of bacteria (CFU/g) | Characteristics | Known roles in disease |
---|---|---|---|
2.80E+11 | Gram-positive | Prevent and reduce diarrhea [ |
|
Reduce total cholesterol [ |
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1.50E+11 | Gram-positive | Ameliorate colitis [ |
|
Increase bile acid excretion [ |
|||
4.80E+11 | Gram-positive | Decrease body fat [ |
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Inhibit lipid deposition [ |
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1.00E+12 | Gram-positive | Fortifies the intestinal barrier [ |
|
Decrease cholesterol levels [ |
CFU, colony forming unit.