Table 1.
Nucleotide sequences of primers used for qRT-PCR.
Fig 1.
Effects of olive oil and astaxanthin on the liver function and pathology of healthy mice.
(A) The levels of serum ALT and AST in the four groups did not differ. Data are given as means ± SD (n = 6, P > 0.05). (B) The percentage of different immune cell subsets, serum levels of TNF-α, IL-6, IL-1β, and IFN-γ of four groups were evaluated in each group with ELISAs or flow cytometry (n = 6, P > 0.05). (C) Representative hematoxylin-and-eosin-stained sections of the liver. Original magnification, ×200.
Fig 2.
Effects of astaxanthin on liver function and pathology of mice with ConA-induced acute hepatitis.
(A) The levels of serum ALT and AST changed depending on the astaxanthin dose, 20 mg/kg or 40 mg/kg. Data are given as means ± SD (n = 8, *P < 0.05 for Oil versus ConA, #P < 0.05 for ConA+Astaxanthin (20) versus ConA, +P < 0.05 for ConA+Astaxanthin (40) versus ConA). (B) The necrotic and edematous area stained with hematoxylin and eosin and used for the liver sections was analyzed with Image-Pro Plus 6.0 (magnification, ×200). The results show statistically significant differences among the different groups (n = 8, *P < 0.05 for ConA+Astaxanthin (20) versus ConA, #P < 0.05 for ConA+Astaxanthin (40) versus ConA).
Fig 3.
Effects of astaxanthin on the production of NF-κB p65, IL-6, IL-1β, and IFN-γ in mice with ConA-induced acute hepatitis.
(A) The index of plasma TNF-α, IL-6, IL-1β, and IFN-γ, measured with ELISAs, was reduced by astaxanthin pretreatment in mice at doses of both 20 mg/kg and 40 mg/kg. Data are presented as means ± SD (n = 8, *P < 0.05 for Oil versus ConA, #P < 0.05 for ConA+Astaxanthin (20) versus ConA, +P < 0.05 for ConA+Astaxanthin (40) versus ConA). (B) The mRNA levels of NF-κB p65, IL-6, IL-1β, and IFN-γ were evaluated in each group with real-time PCR (n = 8, *P < 0.05 for Oil versus ConA, #P < 0.05 for ConA+Astaxanthin (20) versus ConA, +P < 0.05 for ConA+Astaxanthin (40) versus ConA). (C) The expression levels of the NF-κB p65, IL-6, IL-1β, and IFN-γ proteins were determined with western blotting.
Fig 4.
Effects of astaxanthin on apoptosis and autophagy in mice with ConA-induced acute hepatitis.
(A) cDNA levels of LC3, Beclin-1, Bcl-2, and Bax were measured with real-time PCR (n = 8, *P < 0.05 for Oil versus ConA, #P < 0.05 for ConA+Astaxanthin (20) versus ConA, +P < 0.05 for ConA+Astaxanthin (40) versus ConA). (B) Protein expression of LC3, Beclin-1, Bcl-2, Bax, and caspase 9 was detected with western blotting. (C) Immunohistochemistry was used to detect LC3, Beclin-1, Bcl-2, and caspase 9 (original magnification, 200). The integrated optical densities (IODs) of different the indices are expressed as means ± SD (n = 8, +P < 0.05 for Oil versus ConA, *P < 0.05 for ConA+Astaxanthin (20) versus ConA, #P < 0.05 for ConA+Astaxanthin (40) versus ConA). (D) Autophagosome formation was detected in liver tissues with transmission electron microscopy at 8 h (magnification, 10,000). Arrows indicate autophagosomes.
Fig 5.
Effects of astaxanthin on the regulation of the TNF-α/JNK/p-JNK pathway in mice with ConA-induced acute hepatitis.
(A) The expression of TNF-α and TRAF2 was determined with real-time PCR (n = 8, *P < 0.05 for Oil versus ConA, #P < 0.05 for ConA+Astaxanthin (20) versus ConA, +P < 0.05 for ConA+Astaxanthin (40) versus ConA). (B) The levels of proteins TNF-α, TRAF2, JNK, and p-JNK in liver tissue are shown as western blot bands. (C) The expression of TNF-α and p-JNK in hepatic tissues was determined with immunohistochemistry at 8 h (original magnification, 200) and their IODs changed significantly with astaxanthin treatment (n = 8, *P < 0.05 for ConA+Astaxanthin (20) versus ConA, #P < 0.05 for ConA+Astaxanthin (40) versus ConA). (D) The levels of proteins ERK, p-ERK, P38 MAPK, and p-P38 MAPK in liver tissue are shown as western blot bands. The relative band densities were calculated (n = 3, *P < 0.05 for Oil versus ConA, #P < 0.05 for ConA+Astaxanthin (20) versus ConA, +P < 0.05 for ConA+Astaxanthin (40) versus ConA).
Fig 6.
Effects of astaxanthin on the proliferation and apoptosis of primary hepatocytes induced by TNF-α.
(A) The proliferation of primary hepatocytes treated with astaxanthin before TNF-α induction was detected with CCK8. (B) The apoptosis of primary hepatocytes was determined with flow cytometry (n = 3, #P < 0.05 for TNF-α versus control, +P < 0.05 for TNF-α+Astaxanthin versus TNF-α). (C) The protein levels of Beclin-1, LC3, Bcl-2 and Bax proteins in primary hepatocytes are shown as western blot bands. The relative band intensities were calculated (n = 3, #P < 0.05 for TNF-α versus control, +P < 0.05 for TNF-α+Astaxanthin versus TNF-α).
Fig 7.
Mechanism of astaxanthin action.
In ConA-induced autoimmune hepatitis, astaxanthin reduces autophagy by inhibiting the JNK/p-JNK pathway. TNF-α, a proinflammatory cytokine, combined with TNFR1 and TRAF2, was expressed on the surfaces of hepatocytes after ConA injection. This led to the activation of JNK, which phosphorylated Bcl-2, thereby promoting the release of caspase 9 and caspase 3, causing apoptosis. Inactive Bcl-2 dissociated from Beclin-1, enhancing the induction of autophagy. Thus, astaxanthin successfully inhibits the release of TNF-α in stressed cells during acute liver injury and also reduces apoptosis and autophagy by reducing the phosphorylation of JNK.