Theabrownin inhibits obesity and non-alcoholic fatty liver disease in mice via serotonin-related signaling pathways and gut-liver axis

Abstract

Introduction: Non-alcoholic fatty liver disease (NAFLD) with obesity seriously threats public health. Our previous studies showed that dark tea had more potential on regulating lipid metabolism than other teas, and theabrownin (TB) was considered to be a main contributor to the bioactivity of dark tea.

Objectives: This in vivo study aims to reveal the effects and molecular mechanisms of TB on NAFLD and obesity, and the role of the gut-liver axis is explored.

Methods: The histopathological examinations, biochemical tests, and nuclear magnetic resonance were applied to evaluate the effects of TB on NAFLD and obesity. The untargeted metabolomics was used to find the key molecule for further exploration of molecular mechanisms. The 16S rRNA gene sequencing was used to assess the changes in gut microbiota. The antibiotic cocktail and fecal microbiota transplant were used to clarify the role of gut microbiota.

Results: TB markedly reduced body weight gain (67.01%), body fat rate (62.81%), and hepatic TG level (51.35%) in the preventive experiment. Especially, TB decreased body weight (32.16%), body fat rate (42.56%), and hepatic TG level (42.86%) in the therapeutic experiment. The mechanisms of action could be the improvement of fatty acid oxidation, lipolysis, and oxidative stress via the regulation of serotonin-related signaling pathways. Also, TB increased the abundance of serotonin-related gut microbiota, such as Akkermansia, Bacteroides and Parabacteroides. Antibiotics-induced gut bacterial dysbiosis disrupted the regulation of TB on serotonin-related signaling pathways in liver, whereas the beneficial regulation of TB on target proteins was regained with the restoration of gut microbiota.

Conclusion: We find that TB has markedly preventive and therapeutic effects on NAFLD and obesity by regulating serotonin level and related signaling pathways through gut microbiota. Furthermore, gut microbiota and TB co-contribute to alleviating NAFLD and obesity. TB could be a promising medicine for NAFLD and obesity.

Keywords: Anti-obesity; Gut microbiota; Non-alcoholic fatty liver disease; Serotonin; Theabrownin.

Graphical abstract

Fig. 1

The preventive effect of TB on NAFLD and obesity. (A) The simplified flow chart. (B) Hepatic H&E and Oil red O staining (black scale bar = 200 μm and blue scale bar = 50 μm), and body fat distribution scanned by NMR (the adipose tissue is highlighted. (C) The changes in body weight (the P-values were calculated by comparing different groups with CD group). (D) The level of TG in serum and liver. (E) The level of TC in serum. (F) The level of LDL-C in serum. (G) The level of ALT in serum. (H) Body fat rate. (I) The average daily food intake of a mouse per week. Data are shown as mean with SD or median with interquartile range, n = 10. Data were analyzed by either ANOVA or Kruskal-Wallis test. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001. The symbols of triangle, inverted triangle, square and circle on the bars represent the samples of CD group, CD + TB group, HFD group, and HFD + TB group, respectively. Abbreviation: ALT, alanine aminotransferase; ANOVA, analysis of variance; CD, control diet; H&E, hematoxylin and eosin; LDL-C, low-density lipoprotein cholesterol; HFD, high-fat diet; NMR, nuclear magnetic resonance; TB, theabrownin; TC, total cholesterol; TG, triglyceride; SD, standard deviation.

Fig. 2

The therapeutic effect of TB on NAFLD and obesity. (A) The simplified flow chart. (B) The changes in body weight. (C) The photographic records, hepatic H&E and Oil red O staining (black scale bar = 200 μm and blue scale bar = 50 μm), and body fat distribution scanned by NMR (the adipose tissue is highlighted). (D) Body fat rate. (E) The level of TG in serum and liver. (F) The level of TC in serum and liver. (G) The levels of HDL-C and LDL-C in serum. (H) The levels of ALT and AST in serum. (I) The ROS fluorescence images of liver (bright green area is ROS fluorescence, yellow scale bar = 1,000 μm and white scale bar = 20 μm). (J) The relative ROS fluorescence intensity and MDA level in liver. (K) The activities of SOD and CAT in liver. Data are shown as mean with SD or median with interquartile range, n = 5. Data were analyzed by either two-tailed Student’s t-test or Mann-Whitney U test. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001. The symbols of square and circle on the bars represent the samples of HFD group and HFD + TB group, respectively. Abbreviation: ALT, alanine aminotransferase; AST, aspartate aminotransferase; CAT, catalase; HDL-C, high-density lipoprotein cholesterol; H&E, hematoxylin and eosin; HFD, high-fat diet; LDL-C, low-density lipoprotein cholesterol; MDA, malonaldehyde; NMR, nuclear magnetic resonance; ROS, reactive oxygen species; SD, standard deviation; SOD, superoxide dismutase; TB, theabrownin; TC, total cholesterol; TG, triglyceride.

Fig. 3

The changes in serotonin distribution from TB intervention. (A) Heatmap for the representative metabolites involved in tryptophan metabolism according to the untargeted metabolomics study. (B) The relative level of serotonin in serum according to the untargeted metabolomics study. (C) The evaluation of serotonin level in liver by ELISA. (D) The evaluation of serotonin level in visceral white adipose tissue by ELISA. Data are shown as mean with SD or median with interquartile range, n = 5 or 10. Data were analyzed by either ANOVA or Kruskal-Wallis test. *P < 0.05, **P-value < 0.01, ***P-value < 0.001. The symbols of triangle, inverted triangle, square and circle on the bars represent the samples of CD group, CD + TB group, HFD group, and HFD + TB group, respectively. Abbreviation: ANOVA, analysis of variance; CD, control diet; ELISA, enzyme-linked immunosorbent assay; HFD, high-fat diet; SD, standard deviation; TB, theabrownin.

Fig. 4

TB alleviated NAFLD and obesity by regulating serotonin-related signaling pathways. (A) The western blot of HTR2A, PPARα and CYP4A14 in liver. (B) The expressions of target proteins in liver. (C) The western blot of HTR2B and HSL in visceral white adipose tissue. (D) The expressions of target proteins in visceral white adipose tissue. (E) The western blot of SERT, MAO-A and CPT-1 in hepatic tissue and hepatic mitochondria. (F) The expressions of target proteins in hepatic tissue and hepatic mitochondria. Data are shown as mean with SD or median with interquartile range, n = 5 or 10. Data were analyzed by either ANOVA, Kruskal-Wallis test, Student’s t-test or Mann-Whitney U test. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001. The symbols of triangle, inverted triangle, square and circle on the bars represent the samples of CD group, CD + TB group, HFD group, and HFD + TB groups, respectively. Abbreviation: ANOVA, analysis of variance; CD, control diet; CPT-1, carnitine palmitoyltransferase-1; CYP4A14, cytochrome P450 family of 4A14; HFD, high-fat diet; HTR2A, serotonin receptor 2A; HTR2B, serotonin receptor 2B; HSL, hormone-sensitive lipase; MAO-A, monoamine oxidase A; PPARα, peroxisome proliferator-activated receptor α; p-HSL, the phosphorylation of HSL; SD, standard deviation; SERT, serotonin transporter; TB, theabrownin; VDAC1, voltage-dependent anion-selective channel protein 1.

Fig. 5

The changes in gut microbiota from TB intervention and correlation analysis with serotonin. (A) Chao1 index for evaluating α-diversity. (B) Observed species richness for evaluating α-diversity. (C) Shannon index for evaluating α-diversity. (D) Simpson's index of diversity (also known as “1-D”, a higher Simpson index means a higher α-diversity). (E) Pielou’s evenness for evaluating α-diversity. (F) PCoA with Bray-Curtis distance for evaluating β-diversity. (G) PCoA with UniFrac distance for evaluating β-diversity. (H) The evaluation of LEfSe for gut microbiota. (I) The changes in abundances of the representative bacterial genera. (J) The Spearman correlation analysis between the representative bacterial genera and serotonin in different organs. Data are shown as mean with SD or median with interquartile range, n = 5. Data were analyzed by either ANOVA, Kruskal-Wallis test or PERMANOVA. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001. The symbols of triangle, inverted triangle, square and circle on the bars represent the samples of CD group, CD + TB group, HFD group, and HFD + TB group, respectively. Abbreviation: ANOVA, analysis of variance; CD, control diet; HFD, high-fat diet; LEfSe, linear discriminant analysis (LDA) effect size; PCoA, principal coordinate analysis; PERMANOVA, permutational multivariate analysis of variance; SD, standard deviation; TB, theabrownin.

Fig. 6

The changes in gut microbiota and hepatic target proteins in the antibiotic interference experiment. (A) The simplified flow chart. The feces used for FMT were collected from the TB group. (B) The PCR amplification for 16S rRNA gene in feces. (C) PCoA with Bray-Curtis distance for evaluating β-diversity. (D) PCoA with UniFrac distance for evaluating β-diversity. (E) The changes in abundances of the representative bacterial genera. (F) The level of serotonin in liver. (G) The Spearman correlation analysis between the representative bacterial genera and serotonin in liver. (H) The western blot of HTR2A, PPARα, CYP4A14 and SERT in liver. (I) The relative quantification of target proteins in liver. Data are shown as mean with SD or median with interquartile range, n = 5. Data were analyzed by either ANOVA, Kruskal-Wallis test or PERMANOVA. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001. The symbols of circle, square, and inverted triangle on the bars represent the samples of TB group, TB + AB group, and TB + FMT group, respectively. Abbreviation: AB, antibiotic cocktail; ANOVA, analysis of variance; CYP4A14, cytochrome P450 family of 4A14; FMT, fecal microbiota transplant; HFD, high-fat diet; HTR2A, serotonin receptor 2A; PCoA, principal coordinate analysis; PCR, polymerase chain reaction; PERMANOVA, permutational multivariate analysis of variance; PPARα, peroxisome proliferator-activated receptor α; SD, standard deviation; SERT, serotonin transporter; TB, theabrownin.

Fig. 7

The effects of fecal microbiota transplant on NAFLD and obesity. (A) The simplified flow chart. The feces used to FMT were collected from the HFD and HFD + TB groups in the 14-week preventive experiment. (B) Hepatic H&E and Oil red O staining (black scale bar = 200 μm and blue scale bar = 50 μm), and body fat distribution scanned by NMR (the adipose tissue is highlighted). (C) The level of TG in serum. (D) The level of TG in liver. (E) The level of LDL-C in serum. (F) The level of ALT in serum. (G) Body fat rate. Data are shown as mean with SD or median with interquartile range, n = 10. Data were analyzed by either ANOVA or Kruskal-Wallis test. *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001. The symbols of square, rhombus, and inverted triangle on the bars represent the samples of Model FMT group, TB FMT group, and TB FMT + TB group, respectively. Abbreviation: AB, antibiotic cocktail; ALT, alanine aminotransferase; ANOVA, analysis of variance; FMT, fecal microbiota transplant; H&E, hematoxylin and eosin; HFD, high-fat diet; LDL-C, low-density lipoprotein cholesterol; NMR, nuclear magnetic resonance; SD, standard deviation; TB, theabrownin; TG, triglyceride.

Fig. 8

The proposed mechanism of TB to alleviate NAFLD and obesity. The HFD was used to induce NAFLD and obesity. TB could alleviate NAFLD and obesity based on the following mechanisms: (1) TB decreased the level of serotonin in liver while increased the level of serotonin in blood circulation and visceral white adipose tissue. (2) TB promoted fatty acid oxidation in liver by decreasing the expression of HTR2A and increasing the expressions of downstream molecules, including PPARα, CYP4A14 and CPT-1. (3) TB reduced the excessive degradation of serotonin to mitigate oxidative stress in liver by decreasing the expression of SERT and MAO-A. (4) Serotonin could be circulated to visceral white adipose tissue and promote lipolysis by activating HTR2B and HSL. (5) TB increased the abundances of gut bacterial genera Akkermansia, Bacteroides and Parabacteroides. (6) The regulation of TB on HTR2A, PPARα, CYP4A14 and SERT needed the participation of gut microbiota (such as Akkermansia, Bacteroides and Parabacteroides). Moreover, fecal microbiota transplant indicated that TB and gut microbiota co-contributed to alleviating NAFLD and obesity. Together, TB alleviated NAFLD and obesity by regulating serotonin-related signaling pathways in a gut microbiota-dependent manner. Abbreviation: CPT-1, carnitine palmitoyltransferase-1; CYP4A14, cytochrome P450 family of 4A14; HFD, high-fat diet; HSL, hormone-sensitive lipase; HTR2A, serotonin receptor 2A; HTR2B, serotonin receptor 2B; NAFLD, non-alcoholic fatty liver disease; MAO-A, monoamine oxidase A; SERT, serotonin transporter; TB, theabrownin; PPARα, peroxisome proliferator-activated receptor α.