Bile salt hydrolase-overexpressing Lactobacillus strains can improve hepatic lipid accumulation in vitro in an NAFLD cell model

Abstract

Background: Non-alcoholic fatty liver disease (NAFLD) includes a range of liver diseases that occur in the absence of significant alcohol consumption. The probiotic bacterial strains Lactobacillus casei LC2W, which overexpresses the bile salt hydrolase (BSH) gene (referred to as pWQH01), and Lactobacillus plantarum AR113, which exhibits high BSH activity, have been shown to improve hepatic lipid accumulation and may lower cholesterol levels in vivo. These effects may be BSH-dependent, as L. casei LC2W without BSH activity did not exert these beneficial effects.

Objective: This study aimed to investigate the effects of Lactobacillus with high BSH activity on cholesterol accumulation and lipid metabolism abnormalities in oleic acid (OA)- and cholesterol-induced HepG2 cell models, and to determine the mechanism underlying the effects.

Design: A HepG2 cell model of OA-induced steatosis and cholesterol-induced cholesterol accumulation was developed. OA- and cholesterol-treated HepG2 cells were incubated with L. plantarum AR113, L. casei LC2W or L. casei pWQH01 for 6 h at 37°C with 5% CO₂. Subsequently, a series of indicators and gene expressions were analysed.

Results: Both L. plantarum AR113 and L. casei pWQH01 significantly reduced lipid accumulation, total cholesterol (TC) levels and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) mRNA expression relative to the control group, whereas L. casei LC2W had no similar effect. Additionally, exposure to L. plantarum AR113 or L. casei pWQH01 significantly reduced the expression of sterol regulatory element-binding protein 1c (SREBP-1c), Acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and tumour necrosis factor-α (TNF-α) andsignificantly increased the expression of 5' adenosine monophosphate-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor alpha (PPARα).

Conclusion: Both L. plantarum AR113 and L. casei pWQH01 appear to improve steatosis in vitro in a BSH-dependent manner.

Keywords: HepG2 cell; Lactobacillus; bile salt hydrolase; cholesterol; hepatic lipid accumulation; non-alcoholic fatty liver disease.

Fig. 1

The effects of different concentrations of cholesterol and 1 mM oleic acid (OA) on the viability of HepG2 cells after treatment for 2, 4, 6 and 8 h. Data represent the means ± standard deviations (n = 3). Statistical differences between groups were determined by a one-way ANOVA. Different letters indicate statistically significant differences between groups, P < 0.05.

Fig. 2

The effects of probiotic strains on lipid accumulation and lipid contents in HepG2 cells. (a) After treatment with probiotic strains for 6 h, lipids were stained with Oil Red O (400× magnification). (b) Spectrophotometric quantification of the total lipid contents by dissolving the stained oil droplets from HepG2 cells. Data represent the means ± standard deviations (n = 3). Statistical differences between groups were determined by a one-way ANOVA. Different letters indicate statistically significant differences between groups, P < 0.05.

Fig. 3

The effects of probiotic strains on total cholesterol (TC) levels. (a) TC levels in HepG2 cells. (b) HMGCR mRNA levels. Data represent the means ± standard deviations (n = 3). Statistical differences between groups were determined by a one-way ANOVA. Different letters indicate statistically significant differences between groups, P < 0.05.

Fig. 4

The effects of probiotic strains on the mRNA expression of key lipid-regulating genes in HepG2 cells stimulated with oleic acid OA and cholesterol. (a) AMPK, (b) SREBP-1c, (c) ACC, (d) PPARα, (e) FAS and (f) TNFα. Data represent the means ± standard deviations (n = 3). Statistical differences between groups were determined by a one-way ANOVA. Different letters indicate statistically significant differences between groups, P < 0.05.