A Controlled Fermented Samjunghwan Herbal Formula Ameliorates Non-alcoholic Hepatosteatosis in HepG2 Cells and OLETF Rats

Abstract

Hepatosteatosis (HS), a clinical feature of fatty liver with the excessive intracellular accumulation of triglyceride in hepatocytes, is manifested by perturbation of the maintenance of liver lipid homeostasis. Samjunghwan (SJH) is an herbal formula used mostly in Korean traditional medicine that is effective against a number of metabolic diseases, including obesity. Herbal drugs, enriched with numerous bioactive substances, possess health-protective benefits. Meanwhile, fermented herbal products enriched with probiotics are known to improve metabolic processes. Additionally, current lines of evidence indicate that probiotics-derived metabolites, termed as postbiotics, produce the same beneficial effects as their precursors. Herein, the anti-HS effects of 5-weeks naturally fermented SJH (FSJH) was investigated with FSJH-mixed chow diet in vivo using Otsuka Long-Evans Tokushima Fatty (OLETF) and Long-Evans Tokushima Otsuka (LETO) rats as animal models of HS and controls, respectively. In parallel, the anti-HS effects of postbiotic-metabolites of three bacterial strains [Lactobacillus brevis (LBB), Lactococcus lactis (LCL) and Lactobacillus plantarum (LBP)] isolated from FSJH were also evaluated in vitro using the FFAs-induced HepG2 cells. Feeding OLETF rats with FSJH-diet effectively reduced body, liver, and visceral adipose tissue (VAT) weights, produced marked hypolipidemic effects on serum and hepatic lipid parameters, decreased serum AST and ALT levels, and upregulated the HMGCOR, SREBP, and ACC, and downregulated the AMPK and LDLR gene expressions levels. Additionally, exposure of FFAs-induced HepG2 cells to postbiotic metabolic media (PMM) of bacterial strains also produced marked hypolipidemic effects on intracellular lipid contents and significantly unregulated the HMGCOR, SREBP, and ACC, and downregulated the AMPK and LDLR genes expressions levels. Overall, our results indicate that FSJH enriched with fermented metabolites could be an effective anti-HS formulation.

Keywords: Lactobacillus bravis; Lactobacillus lactis; Lactobacillus plantarum; Samjunghwan; fermentation; hepatosteatosis; postbiotic.

FIGURE 1

The UPLC spectra of SJH or FSJH. The comparative UPLC chromatogram of (A) SJH profile, (B) FSJH profile, and (C) The identified compounds indicated by arrows.

FIGURE 2

Bacterial composition of SJH or FSJH. The bacterial profile identified by pyrosequencting, (A) Bacterial composition of SJH (week 0), and (B,C) Bacterial composition of FSJH (weeks 1 and 5).

FIGURE 3

Effect of FSJH extract on lipid accumulation and intracellular lipid content in FFAs-induced HepG2 cells: (A) Oil red o stained images showing lipid accumulation, indicated by arrows (200× magnification and 100 μm scale bar), (B) Spectrophotometric-based quantification of the oil red o staining of HepG2 cells, (C) Total cholesterol (TC), and (D) Triglyceride (TG). The cells were treated with 400 μg/ml SJH or FSJH extracts. Data represent the means ± SD (n = 3). Statistical differences between groups were determined by one-way ANOVA. Different letters indicate statistically significant differences between groups, P < 0.05.

FIGURE 4

Effect of bacterial strains PMM on lipid accumulation and intracellular lipid content in FFAs-induced HepG2 cells: (A) Oil red o stained images showing lipid accumulation, indicated by arrows (200× magnification and 100 μm scale bar), and (B) Spectrophotometric-based quantification of the oil red o staining of HepG2 cells, (C) Total cholesterol (TC), and (D) Triglyceride (TG). The cells were treated with 1% of PMM of bacterial strains. Data represent the means ± SD (n = 4). Statistical differences between groups were determined by one-way ANOVA. Different letters indicate statistically significant differences between groups, P < 0.05.

FIGURE 5

Effect of bacterial strains PMM on lipid-regulating gene expression in FFAs-induced HepG2 cells. The quantitative real-time PCR showing the impact of PMM on lipid-regulating gene expression, (A) HMGCOR, (B) SREBP, (C) ACC, (D) AMPK, and (E) LDLR. The gene expression of a reference protein was normalized to that of GAPDH. The cells were treated with 1% of PMM of bacterial strains. Data represent the means ± SD (n = 4). Statistical differences between groups were determined by one-way ANOVA. Different letters indicate statistically significant differences between groups, P < 0.05.

FIGURE 6

Effect of FSJH diet on glucose tolerance in rats. The oral glucose tolerance test performed on 16 h fasted rats, (A) Glucose level of each time points, and (B) Glucose AUC of glucose tolerance test. Data represent the means ± SD (n = 6). Statistical differences between groups were determined by one-way ANOVA. Different letters indicate statistically significant differences between groups, P < 0.05.

FIGURE 7

Effect of FSJH diet on the body and visceral organ weight in rats: (A) The comparative body weight of week 0 and week 10, (B) Liver weight, and (C) Visceral adipose tissue (VAT) during the sacrifice, (D) The frozen histology of liver based on oil red o staining, and (E) Paraffin histology based on H&E staining (200× magnification and 100 μm scale bar). Data represent the means ± SD (n = 6). Statistical differences between groups were determined by one-way ANOVA. Different letters indicate statistically significant differences between groups, P < 0.05.

FIGURE 8

Effect of FSJH diet on serum, liver and decal biochemical-profile in rats: The serum lipid profile, (A) Serum insulin (INS), (B) Serum total cholesterol (TC), (C) Serum triglyceride (TG), (D) Serum high-density lipoprotein HDL, (E) Liver TC, (F) Liver TG, (G) Stool TC, (H) Stool TG, (I) Serum aspartate transaminase (ALT), and (J) Serum alanine transaminase (AST). Data represent the means ± SD (n = 3 or 6). Statistical differences between groups were determined by one-way ANOVA. Different letters indicate statistically significant differences between groups, P < 0.05.

FIGURE 9

Effect of FSJH diet on lipid-regulating gene expression in the liver of rats. The quantitative real-time PCR showing the impact of FSJH diet on the lipid-metabolic genes expression, (A) HMGCOR, (B) SREBP, (C) ACC, (D) AMPK, and (E) LDLR. The gene expression of a reference protein was normalized to that of GAPDH. Data represent the means ± SD (n = 6). Statistical differences between groups were determined by one-way ANOVA. Different letters indicate statistically significant differences between groups, P < 0.05.

FIGURE 10

Effect of FSJH or PMM on hepatosteatosis. Impact of FSJH extract or diet or PMM of bacterial strains on the lipid accumulation, lipid contents, and lipid-regulated genes expression. SJH = Samjunghwan, FSJH = Fermented SJH, FFAs = Free Fatty Acids, FSJHEx = FSJH Extract, PMM = Postbiotic metbolic media of bacterial strains, RCDt = Regular Chow Diet, and FSJHDt = Fermented Samjunghwan Diet.