The anti-cholesterolaemic effect of a consortium of probiotics: An acute study in C57BL/6J mice

Abstract

Hypercholesterolaemia is a major risk factor for cardiovascular disease and it has been found that some probiotic bacteria possess cholesterol-lowering capabilities. In this study, the ability of the Lab4 probiotic consortium to hydrolyse bile salts, assimilate cholesterol and regulate cholesterol transport by polarised Caco-2 enterocytes was demonstrated. Furthermore, in wild-type C57BL/6J mice fed a high fat diet, 2-weeks supplementation with Lab4 probiotic consortium plus Lactobacillus plantarum CUL66 resulted in significant reductions in plasma total cholesterol levels and suppression of diet-induced weight gain. No changes in plasma levels of very low-density lipoprotein/low-density lipoprotein, high-density lipoprotein, triglycerides, cytokines or bile acids were observed. Increased amounts of total and unconjugated bile acids in the faeces of the probiotic-fed mice, together with modulation of hepatic small heterodimer partner and cholesterol-7α-hydroxylase mRNA expression, implicates bile salt hydrolase activity as a potential mechanism of action. In summary, this study demonstrates the cholesterol-lowering efficacy of short-term feeding of the Lab4 probiotic consortium plus L. plantarum CUL66 in wild-type mice and supports further assessment in human trials.

Figure 1

BSH activity and cholesterol assimilation by Lab4. (a) De Man, Rogosa and Sharpe (MRS) agar plates (control, top and bottom left-sided panels) or MRS agar plates containing 0.05% taurodeoxycholic acid (TDCA, top and bottom right-sided panels) that were inoculated with Lab4 on filter discs (top panels) or as bacterial streaks (bottom panels, n = 1) for 48 hours under anaerobic conditions. (b) Cholesterol concentration in MRS broth containing 0.3% (w/v) ox-bile and 200 µg/ml cholesterol (control) or in MRS broth containing 0.3% (w/v) ox-bile and 200 µg/ml cholesterol that were inoculated with Lab4 for 18 hours under anaerobic conditions. The data are presented as a representative image from 3 identical experiments (unless stated, Fig. 1a) or the mean ± SD from three independent experiments (Fig. 1b). Statistical analysis was performed using Student’s t-test and values of p are stated where appropriate.

Figure 2

The effect of Lab4 on cholesterol homeostasis in Caco-2 enterocytes. (a) Gene transcript levels of NPC1L1, ABCG-5, ABCG-8, ABCA-1 and HMGCR in Caco-2 cells that were treated with 70 µg/ml cholesterol (Control) or cholesterol (70 µg/ml) and Lab4 (1 × 10⁸ cfu/ml) for 6 hours. Gene transcript levels were calculated using the comparative cycle threshold (Ct) method and normalised to β-actin levels with the control given an arbitrary value of 1.0. (b) Cholesterol uptake by untreated (control) Caco-2 cells or those incubated with Lab4 (1 × 10⁸ cfu/ml) for 5 hours prior to the addition of radiolabelled cholesterol for an additional hour. Intracellular radioactivity (disintegrations per minute) was normalised to total protein content and presented as a percentage of the control that has been arbitrarily assigned as 100%. Efflux of intracellular radiolabelled cholesterol to apolipoprotein-AI (Apo-AI, 10 μg/ml) in the basolateral compartment (c) or TDCA micelles (1 nM) in the apical compartment (d) by untreated (control) Caco-2 cells or those treated with Lab4 (1 × 10⁸ cfu/ml) for 6 hours. The percentage of intracellular cholesterol effluxed from the cells was determined by dividing the radioactivity of the apical media or basolateral media by the combined radioactivity in the apical media, basolateral media and cell fraction. The data are presented as the mean ± SD from at least three independent experiments. Statistical analysis was performed using Student’s t-test where *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 3

Effect of probiotics on body weight. Body weights of mice in the HFD and HFD + P groups were recorded throughout the intervention period at the indicated time points and the percentage change in body weight since day 0 was calculated for each mouse. Data is presented as the mean ± SD for 6 mice in each group. Statistical analysis was performed using Student’s t-test where *p < 0.05.

Figure 4

Principle component analysis (PCA) and heatmap analysis of plasma and faecal bile acid profiles. PCA score plots of bile acid signatures from the (a) plasma or (c) faeces of BL, HFD and HFD + P mice. Heatmaps of the bile acid relative intensity from (b) plasma or (d) faeces of each mouse.