Astaxanthin Alleviated Hepatic Steatosis in Obese Mice and Decreased Lipid Accumulation in Hepatocytes

To assess the effect of astaxanthin on hepatic steatosis, high-fat diet (HFD)-induced obese (DIO) and genetically obese (ob/ob) mice were treated with astaxanthin. Figure 1a shows the chemical structure of astaxanthin. After 10 weeks of feeding, astaxanthin administration reduced hepatic steatosis and triglyceride (TG) accumulation significantly in both DIO and ob/ob mice, even though weight and adiposity were not affected by astaxanthin (Fig. 1b,d). To further clarify the effect of astaxanthin on lipid accumulation in vitro, primary hepatocytes were incubated with either astaxanthin or α-tocopherol, a lipophilic antioxidant that is efficacious when used to treat NAFLD, in the presence of oleic acid. Incubation with astaxanthin, but not α-tocopherol, resulted in dose-dependent decrease in TG accumulation, as assessed by Oil Red O staining and the cellular TG content in lipid-loaded primary hepatocytes (Fig. 1e). Next, quantitative real-time PCR (qPCR) analysis was performed to elucidate the mechanism by which astaxanthin decreased lipid accumulation in hepatocytes (Fig. S1a). Astaxanthin treatment did not affect the mRNA levels of lipogenic and fatty acid oxidation genes, while expression of Cd36, a key regulator of lipid uptake, was increased significantly by oleic acid treatment and decreased by astaxanthin in a dose-dependent manner (Fig. S1a). Moreover, the phosphorylation levels of p38 MAPK and c-Jun in oleic-acid-loaded hepatocytes were unaffected by astaxanthin (Fig. S1b). To assess whether the attenuation of lipid accumulation by astaxanthin is associated with protection against lipotoxicity, hepatocytes were co-incubated with palmitic acid and astaxanthin. However, astaxanthin had little effect on apoptosis, as assessed by cleavage of caspase-3 and viability in palmitic acid-loaded hepatocytes (Fig. S2). Taken together, these results suggest that astaxanthin reduced lipid accumulation by decreasing lipid uptake and therefore improved simple fatty liver.

Figure 1 Astaxanthin reduced hepatic steatosis in DIO and ob/ob mice and decreased lipid accumulation in vitro. (a) The chemical structure of astaxanthin. (b) The body weights and tissue weights of mice (n = 5–8). NT, no treatment; AX, astaxanthin treatment. (c) Representative hematoxylin and eosin (H&E)-stained liver sections. Scale bars = 100 μm. (d) Hepatic TG content (n = 5–8). *P < 0.05 vs. control group. (e) Oil Red O staining of cultured primary hepatocytes and cellular TG levels (n = 6). *P < 0.01, vs. control incubation; #P < 0.05, ##P < 0.01 vs. oleic acid (OA)-treated cells. Full size image

Astaxanthin Improved Dyslipidemia and Liver Dysfunction in a NASH Model

To determine the most effective doses of astaxanthin on diet-induced NASH, C57BL/6J mice were fed different diets for 12 weeks (normal chow [NC], NC containing 0.0067% or 0.02% astaxanthin, high-fat, cholesterol and cholate diet [CL], or CL containing 0.0067% or 0.02% astaxanthin). Treatment with astaxanthin ameliorated liver pathology and decreased plasma aspartate aminotransferase (AST) and ALT levels in diet-induced NASH in a dose-dependent manner (Fig. S3a and S3b). Astaxanthin accumulated in various tissues after consumption of the CL diet containing 0.02% (w/w) of this material (Fig. S3c); the astaxanthin concentrations were higher in the spleen, heart and liver than in other tissues, suggesting that astaxanthin accumulates in the liver and ameliorates NASH. The effects were more prominent in the 0.02% astaxanthin-treated group; therefore, subsequent experiments were performed using that dose (named the CL+AX group).

Next, we compared the effects of astaxanthin and vitamin E as similar lipophilic antioxidants on the prevention of NASH. The metabolic parameters of mice after 12 weeks on the CL diet are shown in Table 1. Astaxanthin decreased plasma TG, total cholesterol (TC), non-esterified fatty acid (NEFA), AST and ALT levels significantly in CL mice. In contrast, vitamin E tended to decrease TG and TC levels, but did not affect NEFA, ALT and AST levels. Bodyweight, food intake and liver weight were unaffected by astaxanthin or vitamin E in both NC and CL mice. These results suggest that astaxanthin improved dyslipidemia and liver dysfunction in NASH mice.

Table 1 Effects of astaxanthin (AX) and vitamin E (VE) on metabolic parameters after 12 weeks of treatment. Full size table

Astaxanthin Prevented the Development of Hepatic Steatosis by Suppressing Lipogenic Gene Expression

Mice in each group had similar bodyweights (Fig. 2a) and consumed similar quantities of food (Table 1). However, liver size was increased significantly by CL diet feeding and was unaffected by astaxanthin and vitamin E administration (Fig. 2b). Histological analysis revealed severe lipid accumulation in the livers of CL mice, which was decreased markedly by astaxanthin and decreased slightly by vitamin E at 20 weeks of age (Fig. 2c). Consistent with these histological findings, CL mice exhibited significantly increased hepatic TG, TC and NEFA levels compared with NC-fed mice, whereas astaxanthin administration reduced lipid accumulation significantly in the CL group (Fig. 2d). However, vitamin E treatment did not reduce hepatic lipid levels. The levels of thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation and oxidative stress, in the liver were increased by the CL diet feeding, revealing exaggerated lipid peroxidation in the livers of NASH mice. Both astaxanthin and vitamin E treatment decreased hepatic lipid peroxidation (Fig. 2d).

Figure 2 Astaxanthin prevented the development of hepatic steatosis in NASH mice. (a) Weight gain in mice. (b) Representative photographs of liver. Scale bars = 1 cm. (c) Representative H&E-stained liver sections. Scale bars = 100 μm. (d) Hepatic TG, TC, NEFA and TBARS contents (n = 5–8). *P < 0.05, **P < 0.01 vs. the NC diet; #P < 0.05, ##P < 0.01 vs. the CL-diet-fed group. (e) mRNA expression of lipogenic and fatty acid oxidation genes in the livers of mice (n = 8). *P < 0.05, **P < 0.01 vs. the CL group. Full size image

During the development of steatohepatitis, the expression of lipogenic regulator genes, including Srebp1c, Lxra, Chrebp and fatty acid synthesis genes, including Fasn and Scd1, was increased significantly in the livers of CL compared with NC mice (Fig. 2e, Fig. S4a). Treatment with astaxanthin suppressed the expression of these lipogenic genes, whereas vitamin E did not alter or slightly decreased gene expression (Fig. 2e). By contrast, astaxanthin had little effect on the expression of genes related to fatty acid oxidation, whereas vitamin E administration increased the expression of Ppara and Lcad in CL mice (Fig. 2e, Fig. S4a). On the other hand, the upregulated expression of Cd36 by CL diet was downregulated by astaxanthin and unaffected by vitamin E (Fig. 2e, Fig. S4a). These results suggest that astaxanthin suppressed lipogenesis and lipid uptake to reduce lipid accumulation in the liver of NAFLD/NASH mice.

Astaxanthin Improved Glucose Intolerance and Insulin Resistance

To determine whether astaxanthin affected glucose tolerance or insulin resistance in NASH mice, glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were performed (Fig. 3). GTTs indicated that the administration of astaxanthin decreased blood glucose levels at 180 min in NC-fed mice, whereas vitamin E had no effect (Fig. 3a). However, CL diet-induced glucose intolerance and hyperinsulinemia in both the fasting and fed states were suppressed significantly by astaxanthin (Fig. 3b,c). Vitamin E treatment also reduced plasma insulin levels. ITTs demonstrated that CL+AX mice had slightly increased insulin sensitivity compared with CL mice (Fig. 3d). These results were associated with enhanced insulin-stimulated phosphorylation of the insulin receptor (IR)-β subunit (p-IRβ) and Akt (p-Akt) in the livers of CL+AX mice compared with CL mice, whereas vitamin E had little effect on hepatic insulin signaling (Fig. 3e). Furthermore, insulin signaling was enhanced by astaxanthin in palmitic-acid-loaded primary hepatocytes (Fig. S5a). At the cellular level, palmitic-acid-induced insulin resistance was associated with a pro-inflammatory response, such as increased phosphorylation of p38 MAPK, NF-κB p65 and ERK. These pro-inflammatory signals were slightly decreased by astaxanthin treatment (Fig. S5b). Therefore, astaxanthin protected mice against diet-induced hepatic insulin resistance and glucose intolerance.

Figure 3 Astaxanthin ameliorated diet-induced glucose intolerance and hepatic insulin resistance. (a,b) Glucose tolerance tests (GTTs; n = 5–8). *P < 0.05, **P < 0.01 NC+AX group vs. NC group or CL+AX group vs. CL group. (c) Plasma insulin levels (n = 5–8). *P < 0.05 vs. mice fed a NC diet; #P < 0.05, ##P < 0.01 vs. mice fed a CL diet. (d) Insulin tolerance tests (ITTs) in CL-diet fed mice (n = 8). *P < 0.05 vs. CL group. (e) Hepatic insulin signaling (n = 4). *P < 0.05 vs. CL group. Full size image

Astaxanthin Reduced the Activation of Both Kupffer and Stellate Cells and Attenuated Hepatic Inflammation and Fibrosis

We confirmed previously that the number of F4/80+ macrophages/Kupffer cells was increased significantly in the livers of CL mice, suggesting that the CL diet induced intense inflammation in the liver5. Astaxanthin and vitamin E treatment markedly and slightly reduced the number of F4/80+ cells, respectively, as assessed by immunostaining and the analysis of mRNA expression (Fig. 4a,b). In addition, astaxanthin decreased the expression of proinflammatory cytokines, including Tnf, Il6 and Il1b, which were upregulated by the CL diet, to extents greater than did vitamin E (Fig. 4b, Fig. S4b). These findings were also associated with the attenuated phosphorylation of JNK, p38 MAPK and NF-κB p65 (Fig. 4c). Therefore, astaxanthin reduced the infiltration and activation of Kupffer cells to attenuate hepatic inflammation in NASH mice.

Figure 4 Astaxanthin attenuated hepatic inflammation and fibrosis in NASH mice. (a) F4/80 immunostaining, Azan and Sirius Red staining, α-SMA immunostaining; scale bars = 100 μm. (b) mRNA expression of F4/80 and inflammatory cytokines in mouse livers. (c) Immunoblots and quantification of p-p38MAPK, p-JNK and p-NF-κB p65 levels in the liver. (d) Hydroxyproline content and immunoblotting and quantification of α-SMA expression in mouse livers. (e) mRNA expression of fibrogenic genes in the livers. n = 5–8, *P < 0.05, **P < 0.01 vs. NC or CL group; #P < 0.05, ##P < 0.01 vs. the CL group. Full size image

Histological analyses using Azan and Sirius Red staining revealed that the CL diet alone induced fibrosis, as described previously5. Astaxanthin prevented the development of hepatic fibrosis (Fig. 4a). In addition, hydroxyproline content, a biochemical marker of hepatic collagen content, increased significantly in CL mice vs. NC mice. Importantly, astaxanthin lowered the hydroxyproline content significantly, whereas vitamin E had only a small effect (Fig. 4d). Immunohistochemical staining for α-SMA showed that the CL diet-induced increase in α-SMA-positive HSC numbers was markedly decreased by astaxanthin (Fig. 4a) and slightly by vitamin E; these observations were confirmed by immunoblotting and qPCR (Fig. 4d,e). In addition, astaxanthin inhibited the increased expression of the fibrogenic genes Tgfb1, Col1a1 and PAI-1 caused by consumption of the CL diet, whereas vitamin E suppressed PAI-1 mRNA expression (Fig. 4e, Fig. S4c). Combined, these results suggest that astaxanthin decreased the accumulation of collagen by inhibiting the activation of HSCs in the liver, thereby attenuating hepatic fibrosis.

Reciprocal Decrease in M1-type Macrophages and Increase in M2-type Macrophages in the Livers of Astaxanthin-fed Mice

To further quantify hepatic macrophage subsets, FACS was used to analyze macrophages/Kupffer cells isolated from mice (Fig. S6). Consistent with the results of immunohistochemistry, the total number of hepatic macrophages increased by 1.9-fold in mice fed the CL diet compared with the NC diet (Fig. S7a and S7b). However, CL+AX mice exhibited a slightly decreased total macrophage content compared with CL and CL+VE mice (Fig. 5a,b). Specifically, CL+AX and CL+VE mice exhibited a 56% and 33% reduced CD11c+ CD206− (M1-type) macrophage count, respectively, whereas the number of CD11c− CD206+ (M2-type) macrophages was increased by 3.7- and 1.5-fold, respectively. In addition, the percentage of M1-type and M2-type macrophages was decreased and increased significantly, respectively, by both astaxanthin and vitamin E treatment (Fig. 5b). These effects resulted in a predominance of M2 rather than M1 macrophage population in the livers of both astaxanthin- and vitamin E-fed mice (Fig. 5c). These results were associated with a reduction in the expression of M1 macrophage markers (Cd11c, iNOS, Mcp1 and Ccr2) and an increase in the expression of M2 macrophage markers (Cd163, Cd206, Il10, Chi3l3 and Mgl1) mRNA expression (Fig. 5d, Fig. S4d). However, a predominance of a Ly6C− over Ly6Chi monocyte population was not observed in either the peripheral blood or bone marrow of CL+AX and CL+VE mice (Fig. S7c and S7d). This suggests that astaxanthin and vitamin E caused a dynamic shift to an M2-dominant macrophage phenotype in the livers of NASH mice.

Figure 5 Decreased M1-type and increased M2-type macrophages in NASH livers after astaxanthin administration. (a,b) A representative plot and quantitation of M1/M2 macrophages in the livers of mice. (c) M1/M2 ratios. (d) mRNA expression of M1 and M2 macrophage markers in the livers. (e,f) A representative plot of CD3+ T cells and quantitation of CD3+, CD8+, CD4+ T cells in the livers of mice (n = 8). *P < 0.05, **P < 0.01 vs. the CL group, #P < 0.05, CL+AX group vs. the CL+VE group. Full size image

Since T cells are involved in the pathogenesis of NASH, we next assessed the effects of astaxanthin and vitamin E on T cell recruitment. The total number of CD3+, CD4+ and CD8+ T cells in the liver was increased significantly by CL diet feeding (Fig. S7e) and decreased by either astaxanthin (by 50%, 54% and 52%, respectively) or vitamin E (43%, 54% and 40%) (all P < 0.05; Fig. 5e,f). Therefore, astaxanthin and vitamin E suppressed the accumulation of helper and cytotoxic T cells. In addition, astaxanthin (25–100 μM) decreased the expression of LPS-induced M1 markers (Tnf, Il1b and Ccl5) in RAW264.7 macrophages, but augmented IL-4-induced M2 marker expression (Il10, Cd209a and Chi3l3) in a dose-dependent manner (Fig. S8a and S8b). This suggests that astaxanthin improved hepatic insulin resistance and inflammation via an M2-dominant shift in macrophages/Kupffer cells and a subsequent reduction in T cell accumulation in NASH.

Astaxanthin Reversed Advanced NASH More Potently than Vitamin E in Mice

Next, we compared the therapeutic effects of astaxanthin and vitamin E on advanced-stage NASH in mice. After NASH was induced by feeding CL diet for 12 weeks, the CL diet with or without either astaxanthin or vitamin E was administered for an additional 12 weeks (Fig. 6a). Treatment with astaxanthin decreased plasma TG, TC, NEFA, AST, ALT and insulin levels in CL mice significantly without affecting body and liver weight (Table S1), whereas vitamin E treatment had little effect on these metabolic parameters. Astaxanthin significantly improved glucose intolerance and insulin resistance, whereas Vitamin E was less effective (Table S1, Fig. S9a and S9b). Histologically, astaxanthin treatment markedly ameliorated the macrovascular steatosis, macrophage/Kupffer cell infiltration and fibrosis associated with HSC activation, but Vitamin E only minimally affected these histological changes (Fig. 6b,d). Potent hepatic inflammation, as characterized by increased stress or inflammatory signaling and upregulated inflammatory genes, was attenuated significantly by astaxanthin (Fig. 6e,f). Astaxanthin suppressed fibrogenic gene expression markedly, whereas vitamin E decreased hepatic inflammation and fibrogenesis only slightly (Fig. 6f). Together, these results suggest that astaxanthin was more effective at reversing advanced NASH than was vitamin E.

Figure 6 Astaxanthin reversed advanced NASH in mice. (a) Study design to assess the therapeutic effects of astaxanthin and vitamin E. (b) Histological analysis of liver sections; scale bars = 100 μm. (c) Hepatic TG, TC, NEFA and TBARS levels. (d) Hydroxyproline content (left) and immunoblotting for α-SMA (right) in the livers. (e) Immunoblotting and quantification of p-p38MAPK, p-JNK and p-NF-κB p65 levels in the livers. (f) mRNA expression of inflammatory cytokine and fibrogenic genes in the livers (n = 8). *P < 0.05, **P < 0.01 vs. the CL group; #P < 0.05, CL+AX group vs. CL+VE group. Full size image

Astaxanthin Alleviated NASH in Humans

Since our results revealed promising preventative and therapeutic effects of astaxanthin on NASH in mice, we next extended our studies to humans. Twelve biopsy-confirmed NASH patients were treated orally with placebo (n = 5) or astaxanthin (n = 7) for a total of 24 weeks. The clinical background and plasma parameters of patients are shown in Table S2. After 24 weeks of astaxanthin treatment, plasma parameters involved in glucose and lipid metabolism and liver functions were unaffected by astaxanthin treatment. Moreover, no significant difference was observed in the changes from baseline between the placebo- and astaxanthin-treated patients (Table S2). However, astaxanthin treatment improved hepatic steatosis markedly in NASH patients (Fig. 7a). In addition, placebo treatment did not affect NAFLD activity score, while astaxanthin treatment reduced the grade of steatosis and tended to alleviate lobular inflammation, but did not alter the presence of ballooning or the stage of fibrosis (Fig. 7b). Together, these results suggest that astaxanthin reduces the total NAS score and alleviates human NASH.