A polyphenol-rich plant extract prevents hypercholesterolemia and modulates gut microbiota in western diet-fed mice

Abstract

Introduction: Totum-070 is a combination of five plant extracts enriched in polyphenols to target hypercholesterolemia, one of the main risk factors for cardiovascular diseases. The aim of this study was to investigate the effects of Totum-070 on cholesterol levels in an animal model of diet-induced hypercholesterolemia.

Methods: C57BL/6JOlaHsd male mice were fed a Western diet and received Totum-070, or not, by daily gavage (1g/kg and 3g/kg body weight) for 6 weeks.

Results: The Western diet induced obesity, fat accumulation, hepatic steatosis and increased plasma cholesterol compared with the control group. All these metabolic perturbations were alleviated by Totum-070 supplementation in a dose-dependent manner. Lipid excretion in feces was higher in mice supplemented with Totum-070, suggesting inhibition of intestinal lipid absorption. Totum-070 also increased the fecal concentration of short chain fatty acids, demonstrating a direct effect on intestinal microbiota.

Discussion: The characterization of fecal microbiota by 16S amplicon sequencing showed that Totum-070 supplementation modulated the dysbiosis associated with metabolic disorders. Specifically, Totum-070 increased the relative abundance of Muribaculum (a beneficial bacterium) and reduced that of Lactococcus (a genus positively correlated with increased plasma cholesterol level). Together, these findings indicate that the cholesterol-lowering effect of Totum-070 bioactive molecules could be mediated through multiple actions on the intestine and gut microbiota.

Keywords: cholesterol; intestine; lipoproteins; microbiota; triglycerides.

Figure 1

Effect of supplementation with Totum-070 (1 g/kg and 3 g/kg) in Western diet-fed mice on body weight, body composition and caloric intake during the study period. (A) Starting at day 1, male C57BL/6JOlaHsd mice were fed a normal diet (ND), a Western diet (WD) or a WD plus daily gavage with Totum-070 (WD-T070, 1 g/kg or 3 g/kg) (n = 14 per group). Body weight was recorded during the study. (B) Body weight gain (difference between the last body weight at day 43 and the body weight at the study start, day 1). (C) Fat mass and (D) lean mass measured by EchoMRI at the study end. (E) Caloric intake during the study period. (F) Cumulative caloric intake during the study. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. ND. ## p < 0.01 Totum-070 groups vs. WD. Data are the mean ± SEM.

Figure 2

Effect of Totum-070 supplementation on plasma lipid concentration. (A) Plasma concentration of cholesterol (n = 14 per group) and (B) of triglycerides (n = 14 per group) at week 0 (before starting the WD), during the study (week 3), and at the study end (week 6). **** p < 0.0001 vs. ND. # p < 0.05 and ## p < 0.01 Totum-070 (T070) groups vs. WD. Data are the mean ± SEM.

Figure 3

Effect of Totum-070 on liver lipid metabolism. (A) Frozen mouse liver tissue sections stained with Oil Red O and counterstained with hematoxylin (upper panels). Scale bars, 50 µm. Paraffin-embedded sections of mouse livers stained with hematoxylin and eosin (H&E) (lower panels). Scale bar, 100 µm. (B) Quantification of Oil Red O in liver sections (at least three images per liver, n = 14 per group) shows reduction of neutral lipid accumulation in mice supplemented with Totum-070. (C) Quantification of hepatic triglyceride content (n = 14 per group). (D) Relative expression in liver of selected genes encoding proteins implicated in cholesterol metabolism (n = 14 per group). (E) Immunoblot to monitor LDL receptor (LDL-R) expression in liver samples from the indicated groups and quantification. (F) Relative expression in mouse liver samples from the indicated groups of selected genes encoding proteins implicated in lipid metabolism. (G) Relative expression in mouse liver samples from the indicated groups of selected genes encoding proteins implicated in lipoprotein metabolism. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. ND. #p < 0.05, ##p < 0.01, ###p < 0.001 and ####p < 0.001 Totum-070 (T070) groups vs. WD. Data are the mean ± SEM.

Figure 4

Effect of Totum-070 on intestinal lipid metabolism. (A) Total lipid content in feces (n = 5 per group). (B) Quantification of different lipid classes in feces. CE, cholesterol ester; CER, ceramides; DAG, diacylglycerides; DCER, dihydroceramides; FFA, free fatty acids; HCER, hexosylceramides; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin; and TG, triacylglycerides. (C,D) Relative expression of selected genes implicated in cholesterol and lipoprotein metabolism in jejunum (C), and ileum samples (D) (n = 14 samples per tissue and per group). (E) Quantification of different bile acid classes in feces (n = 5 per group). αMCA, α-muricholic acid; βMCA, β-muricholic acid; CA, cholic acid; UDCA, ursodeoxycholic acid; HDCA, hyodeoxycholic acid; DCA, deoxycholic acid. (F) Inhibition of lipase activity by Totum-070, n = 4–11 replicates. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. ND. #p < 0.05, ##p < 0.01, ###p < 0.001 and ####p < 0.0001 Totum-070 (T070) groups vs. WD. Data are the mean ± SEM.

Figure 5

Effect of Totum-070 on richness, diversity and composition of fecal microbiota. (A) Shannon diversity index at the phylum level. (B) Rarefied Chao index (richness index) at the genus level. (C) Shannon diversity index at the genus level. (D) and (E) Principal coordinate analysis based on the Bray–Curtis dissimilarity index at the phylum and genus levels, with 95% confidence standard deviation ellipses. (F) Principal coordinate analysis based on the Jaccard dissimilarity index at the genus level, with 95% confidence standard deviation ellipses. Boxplots show the median, 25th and 75th percentile, and adjacent values. n = 11 ND, n = 10 WD, n = 8 WD-T070 3 g/kg. * p < 0.05 vs. ND.

Figure 6

Effect of Totum-070 on composition of fecal microbiota at the genus level. Figures represent relative abundance values of genera that differed significantly between two groups or more. Boxplots show the median, 25th and 75th percentile, and adjacent values. n = 11 ND, n = 10 WD, n = 8 WD-T070 3 g/kg. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs. ND. #p < 0.05, ##p < 0.01, ###p < 0.001 and ####p < 0.0001 Totum-070 (T070) group vs. WD.

Figure 7

Effect of Totum-070 on fecal short chain fatty acid (SCFA) composition. (A) Quantification of SCFA species content in feces (n = 14 per group) at the study end. (B) Relationship between plasma cholesterol levels and fecal SCFA content in the WD and WD-T070 3 g/kg groups. #p < 0.05, Totum-070 (T070) group vs. WD. Data are the mean ± SEM.