Astaxanthin prevents and reverses diet-induced insulin resistance and steatohepatitis in mice: A comparison with vitamin E

Abstract

Hepatic insulin resistance and nonalcoholic steatohepatitis (NASH) could be caused by excessive hepatic lipid accumulation and peroxidation. Vitamin E has become a standard treatment for NASH. However, astaxanthin, an antioxidant carotenoid, inhibits lipid peroxidation more potently than vitamin E. Here, we compared the effects of astaxanthin and vitamin E in NASH. We first demonstrated that astaxanthin ameliorated hepatic steatosis in both genetically (ob/ob) and high-fat-diet-induced obese mice. In a lipotoxic model of NASH: mice fed a high-cholesterol and high-fat diet, astaxanthin alleviated excessive hepatic lipid accumulation and peroxidation, increased the proportion of M1-type macrophages/Kupffer cells, and activated stellate cells to improve hepatic inflammation and fibrosis. Moreover, astaxanthin caused an M2-dominant shift in macrophages/Kupffer cells and a subsequent reduction in CD4(+) and CD8(+) T cell recruitment in the liver, which contributed to improved insulin resistance and hepatic inflammation. Importantly, astaxanthin reversed insulin resistance, as well as hepatic inflammation and fibrosis, in pre-existing NASH. Overall, astaxanthin was more effective at both preventing and treating NASH compared with vitamin E in mice. Furthermore, astaxanthin improved hepatic steatosis and tended to ameliorate the progression of NASH in biopsy-proven human subjects. These results suggest that astaxanthin might be a novel and promising treatment for NASH.

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.

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.

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.

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.

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.

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.

Figure 7. Astaxanthin alleviated NASH in humans.

(a) Representative H&E-stained liver sections from two subjects with NASH before and after treatment revealed that astaxanthin improved steatohepatitis after 24 weeks of treatment. Scale bars = 400 μm for low magnification (upper), and 100 μm for high magnification (lower). (b) NAFLD activity score (NAS) in NASH subjects before and after treatment with placebo (n = 5) or astaxanthin (AX, n = 7). *P < 0.05 vs. pretreatment.