Raw Bowl Tea (Tuocha) Polyphenol Prevention of Nonalcoholic Fatty Liver Disease by Regulating Intestinal Function in Mice

Abstract

A high-fat diet-induced C57BL/6N mouse model of non-alcoholic fatty liver disease (NAFLD) was established. The effect and mechanism of Raw Bowl Tea polyphenols (RBTP) on preventing NAFLD via regulating intestinal function were observed. The serum, liver, epididymis, small intestine tissues, and feces of mice were examined by biochemical and molecular biological methods, and the composition of RBTP was analyzed by HPLC assay. The results showed that RBTP could effectively reduce the body weight, liver weight, and liver index of NAFLD mice. The serum effects of RBTP were: (1) decreases in alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), D-lactate (D-LA), diamine oxidase (DAO), lipopolysaccharide (LPS), and an increase of high density lipoprotein cholesterol (HDL-C) levels; (2) a decrease of inflammatory cytokines such as interleukin 1 beta (IL-1β), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor alpha (TNF-α), and interferon gamma (INF-γ); (3) a decrease the reactive oxygen species (ROS) level in liver tissue; and (4) alleviation of pathological injuries of liver, epididymis, and small intestinal tissues caused by NAFLD and protection of body tissues. qPCR and Western blot results showed that RBTP could up-regulate the mRNA and protein expressions of LPL, PPAR-α, CYP7A1, and CPT1, and down-regulate PPAR-γ and C/EBP-α in the liver of NAFLD mice. In addition, RBTP up-regulated the expression of occludin and ZO-1, and down-regulated the expression of CD36 and TNF-α in the small intestines of NAFLD mice. Studies on mice feces showed that RBTP reduced the level of Firmicutes and increased the minimum levels of Bacteroides and Akkermansia, as well as reduced the proportion of Firmicutes/Bacteroides in the feces of NAFLD mice, which play a role in regulating intestinal microecology. Component analysis showed that RBTP contained seven polyphenolic compounds: Gallic acid, (-)-epigallocatechin, catechin, L-epicatechin, (-)-epigallocatechin gallate, (-)-gallocatechin gallate, and (-)-epicatechin gallate (ECG), and high levels of caffeine, (-)-epigallocatechin (EGC), and ECG. RBTP improved the intestinal environment of NAFLD mice with the contained active ingredients, thus playing a role in preventing NAFLD. The effect was positively correlated with the dose of 100 mg/kg, which was even better than that of the clinical drug bezafibrate.

Keywords: intestinal function; mice; nonalcoholic fatty liver disease; polyphenols; raw Bowl tea.

Figure 1

Polyphenol constituents of raw Bowl tea. (A) Standard chromatograms; (B) Polyphenols of raw Bowl tea chromatograms. 1: gallic acid, 2: (-)-epigallocatechin (EGC); 3: catechin; 4: caffeine; 5: L-epicatechin (EC); 6: (-)-epigallocatechin gallate (EGCG); 7: (-)-gallocatechin gallate (GCG); 8: (-)-epicatechin gallate (ECG).

Figure 2

The ROS level of liver tissue in mice. ^a–e Mean values with different letters in the bar are significantly different (p < 0.05) according to Duncan’s multiple-range test. RBTP-L: mice treated with low concentrations of polyphenols of raw Bowl tea (50 mg/kg); RBTP-H: mice treated with high concentrations of polyphenols of raw Bowl tea (100 mg/kg); Bezafibrate: mice treated with bezafibrate (100 mg/kg).

Figure 3

H&E pathological observation of hepatic tissue in mice. Magnification 100×. RBTP-L: mice treated with low concentrations of polyphenols of raw Bowl tea (50 mg/kg); RBTP-H: mice treated with high concentrations of polyphenols of raw Bowl tea (100 mg/kg); Bezafibrate: mice treated with bezafibrate (100 mg/kg).

Figure 4

H&E pathological observation of epididymal tissue in mice. Magnification 100×. RBTP-L: mice treated with low concentrations of polyphenols of raw Bowl tea (50 mg/kg); RBTP-H: mice treated with high concentrations of polyphenols of raw Bowl tea (100 mg/kg); Bezafibrate: mice treated with bezafibrate (100 mg/kg).

Figure 5

H&E pathological observation of small intestine tissue in mice. Magnification 100×. RBTP-L: mice treated with low concentrations of polyphenols of raw Bowl tea (50 mg/kg); RBTP-H: mice treated with high concentrations of polyphenols of raw Bowl tea (100 mg/kg); Bezafibrate: mice treated with bezafibrate (100 mg/kg).

Figure 6

The PPAR-α, PPAR-γ, CYP7A1, CPT1, LPL, and C/EBPα mRNA (A) and protein (B) expression in hepatic tissue of mice. ^a–e Mean values with different letters in the bar are significantly different (p < 0.05) according to Duncan’s multiple-range test. RBTP-L: mice treated with low concentrations of polyphenols of raw Bowl tea (50 mg/kg); RBTP-H: mice treated with high concentrations of polyphenols of raw Bowl tea (100 mg/kg); Bezafibrate: mice treated with bezafibrate (100 mg/kg).

Figure 7

The CD36, occludin, ZO-1 and TNF-α mRNA (A) and protein (B) expression in small intestine tissue of mice. ^a–e Mean values with different letters in the bar are significantly different (p < 0.05) according to Duncan’s multiple-range test. RBTP-L: mice treated with low concentrations of polyphenols of raw Bowl tea (50 mg/kg); RBTP-H: mice treated with high concentrations of polyphenols of raw Bowl tea (100 mg/kg); Bezafibrate: mice treated with bezafibrate (100 mg/kg).

Figure 8

The mRNA expression in microorganisms in feces of mice. ^a–e Mean values with different letters in the bar are significantly different (p < 0.05) according to Duncan’s multiple-range test. RBTP-L: mice treated with low concentrations of polyphenols of raw Bowl tea (50 mg/kg); RBTP-H: mice treated with high concentrations of polyphenols of raw Bowl tea (100 mg/kg); Bezafibrate: mice treated with bezafibrate (100 mg/kg).