Fucoidan and Fucoxanthin Attenuate Hepatic Steatosis and Inflammation of NAFLD through Modulation of Leptin/Adiponectin Axis

Abstract

Non-alcoholic fatty liver disease (NAFLD) is the emerging cause of chronic liver disease globally and lack of approved therapies. Here, we investigated the feasibility of combinatorial effects of low molecular weight fucoidan and high stability fucoxanthin (LMF-HSFx) as a therapeutic approach against NAFLD. We evaluated the inhibitory effects of LMF-HSFx or placebo in 42 NAFLD patients for 24 weeks and related mechanism in high fat diet (HFD) mice model and HepaRG^TM cell line. We found that LMF-HSFx reduces the relative values of alanine aminotransferase, aspartate aminotransferase, total cholesterol, triglyceride, fasting blood glucose and hemoglobin A1c in NAFLD patients. For lipid metabolism, LMF-HSFx reduces the scores of controlled attenuation parameter (CAP) and increases adiponectin and leptin expression. Interestingly, it reduces liver fibrosis in NAFLD patients, either. The proinflammatory cytokines interleukin (IL)-6 and interferon-γ are reduced in LMF-HSFx group. In HFD mice, LMF-HSFx attenuates hepatic lipotoxicity and modulates adipogenesis. Additionally, LMF-HSFx modulates SIRI-PGC-1 pathway in HepaRG cells under palmitic acid-induced lipotoxicity environment. Here, we describe that LMF-HSFx ameliorated hepatic steatosis, inflammation, fibrosis and insulin resistance in NAFLD patients. LMF-HSFx may modulate leptin-adiponectin axis in adipocytes and hepatocytes, then regulate lipid and glycogen metabolism, decrease insulin resistance and is against NAFLD.

Keywords: NAFLD; adiponectin; lipid metabolism; lipotoxicity; liver fibrosis; randomized controlled trial.

Figure 1

The consort diagram. Seventy patients were enrolled into the initial evaluation of the study. Sixteen patients were excluded owing to the possible bias from autoimmune hepatitis, seafood allergy and diabetes medication and twelve patients declined to participate. Forty-two patients were randomized into low-molecular weight fucoidan and high-stability fucoxanthin (LMF-HSFx) and placebo group.

Figure 2

LMF-HSFx attenuates hepatic lipotoxicity in patients with nonalcoholic fatty liver disease. The graph demonstrated the change from baseline of (A) Alanine transaminase, ALT; (B) Aspartate aminotransferase, AST; (C) total cholesterol, TC; (D) triglyceride, TG; Significant reduction of ALT, AST, TC and TG were observed at 6th month in LMF-HSFx group (* p < 0.05, ** p < 0.01, compared with placebo).

Figure 3

LMF-HSFx attenuates hepatic steatosis and fibrosis in patients with NAFLD. The graph demonstrated the change from baseline of (A) controlled attenuation parameter, CAP; (B) stiffness of patients with LMF-HSFx or placebo treatment. Significant reduction of CAP was observed at 6th month and significant reduction of stiffness was observed at 3rd and 6th month in LMF-HSFx group (* p < 0.05, ** p < 0.01, compared with placebo).

Figure 4

LMF-HSFx attenuates the NAFLD-induced inflammation and modulate adipogenesis. The graph demonstrated the change from baseline of serum (A) IL-6, (B) IFN-γ, (C) adiponectin and (D) leptin of patients with LMF-HSFx or placebo treatment. Significant reduction of IL-6 and IFN-γ change was observed at 3rd and 6th month in LMF-HSFx group (*** p < 0.001, compared with placebo). The significant increasing of leptin was observed at 6th month in LMF-HSFx group (** p < 0.01, compared with placebo).

Figure 5

LMF-HSFx reduces the insulin resistance in patients with NAFLD. The graph demonstrated the change from baseline of (A) AC, (B) HbA1c, (C) insulin, (D) HOMA-IR and (E) the insulin secretion index (beta cell function index) of patients with LMF-HSFx or placebo treatment. Significant reduction of AC, HbA1c change was observed at 3rd and 6th month in LMF-HSFx group (* p < 0.05, ** p < 0.01, compared with placebo). (D) The average HOMA-IR of placebo not LMF-HSFx group was higher than 3.5 at 6th month. (E) The increasing of beta cell function index in LMF-HSFx group during the 6 months.

Figure 6

LMF-HSFx inhibits hepatic lipotoxicity in liver tissues of mice fed with high-fat diet. (A) Liver sections stained with Hematoxylin-eosin (HE) from high-fat diet (HFD) mice or normal diet (ND) mice with or without LMF-HSFx treatment. The HE staining of liver sections was performed at 16th week from ND mice or HFD mice with or without orally gavaged LMF-HSFx (400 mg/kg/BW/day). Scale bar: 100 μm. (B) LMF-HSFx decreases the volume of lipid droplet in HFD mice liver. The differential distribution of lipid droplets in HE stained-liver tissues of HFD mice with or without 400 mg/kg/BW/day LMF-HSFx treatment after 16 weeks. The X-axis is the lipid droplets diameter and Y-axis is the total lipid droplet numbers in 10 HE stained-liver images from one mouse, n = 3 mice. X-axis unit: 10³ pixel and Y-axis unit: the total lipid droplet numbers in 10 images of each area 2560 × 1922 pixel². (C) The blood glucose (mg/dL, dL: 100 mL), (D) triglyceride (mg/dL), (E) AST (U/L) and (F) ALT (U/L) concertation in ND or HFD mice with or without 200 or 400 mg/kg/BW/day LMF-HSFx treatment through oral gavage after 16 weeks (^# p < 0.05, compared with DN mice; * p < 0.05, compared with HFD mice).

Figure 7

LMF-HSFx modulates SIRT-PGC-1 axis in palmitic acid-treated HepaRG hepatocytes. LMF-HSFx restored palmitic acid-induced SIRT2, 3, 6, PGC-1β and ATGL degradation. The HepaRG cells were cultured in PA 200 or 400 μM with or without LMF-HSFx 25 μg/mL. Representative Western blots of (A) SIRT-1, 2, 3, 4, 6, control protein GAPDH; (C) PGC-1α, PGC-1β, ATGL and GAPDH. Summarized bar graphs depicting the protein level of (B) SIRT-1, 2, 3, 4, 6; (D) PGC-1α, PGC-1β, ATGL. Each column represents mean ± SEM, taking the control group as 100% (* p < 0.05 compared with control; ^# p < 0.05, compared with PA200 group).

Figure 8

LMF-HSFx ameliorates PA-induced early apoptosis and cellular mitochondrial disruption in HepaRG cells. (A) the early apoptosis by Annexin V/PI stain and (B) mitochondrial disruption apoptosis by JC-1 labeling on flow cytometer of the HepRG cells in PA 200 or 400 μM with or without LMF-HSFx 25 μg/mL, LMF-HSFx 25 μg/mL and untreated control groups. (C) summarized bars depicting the differentiation percentage of apoptotic cells in groups by Annexin V/PI stain, taking total cells as 100%. (D) summarized bars depicting the Green/red fluorescence ratio in groups by JC-1 labeling, taking control group cells as 1fold (* p < 0.05, compared with control; ^# p < 0.05, compared with PA200 group; ^$ p < 0.05, compared with PA400 group).

Figure 9

The work mechanism of LMF-HSFx for NAFLD. LMF-HSFx targets Adipocytes and hepatocytes. In adipose tissue, LMF-HSFx decreases the insulin resistance and enhances the Adiponectin and Leptin expression. In hepatocyte, LMF-HSFx directly activates SIRT-PGC1 axis and PGC-1 family expression. Combination with the stimulation effects from Adiponectin and Leptin, the SIRT-PGC1 axis mediated mitochondrial function and fatty acid oxidation are activated by LMF-HSFx but gluconeogenesis and De novo lipogenesis are inhibited by LMF-HSFx. LMF-HSFx also decreases the pro-inflammatory cytokine (IL6 and INFγ) release from hepatocyte and suppresses the fibrosis in NAFLD.