Shikonin Attenuates Concanavalin A-Induced Acute Liver Injury in Mice via Inhibition of the JNK Pathway

Abstract

Objective: Shikonin possesses anti-inflammatory effects. However, its function in concanavalin A-induced acute liver injury remains uncertain. The aim of the present study was to investigate the functions of shikonin and its mechanism of protection on ConA-induced acute liver injury.

Materials and methods: Balb/C mice were exposed to ConA (20 mg/kg) via tail vein injection to establish acute liver injury; shikonin (7.5 mg/kg and 12.5 mg/kg) was intraperitoneally administered 2 h before the ConA injection. The serum liver enzyme levels and the inflammatory cytokine levels were determined at 3, 6, and 24 h after ConA injection.

Results: After the injection of ConA, inflammatory cytokines IL-1β, TNF-α, and IFN-γ were significantly increased. Shikonin significantly ameliorated liver injury and histopathological changes and suppressed the release of inflammatory cytokines. The expressions of Bcl-2 and Bax were markedly affected by shikonin pretreatment. LC3, Beclin-1, and p-JNK expression levels were decreased in the shikonin-pretreated groups compared with the ConA-treated groups. Shikonin attenuated ConA-induced liver injury by reducing apoptosis and autophagy through the inhibition of the JNK pathway.

Conclusion: Our results indicated that shikonin pretreatment attenuates ConA-induced acute liver injury by inhibiting apoptosis and autophagy through the suppression of the JNK pathway.

Figure 1

Effects of 2% DMSO and shikonin 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 = 4, P > 0.05). The serum levels of TNF-α, IL-1β, and IFN-γ of four groups were evaluated in each group with ELISAs (n = 4, P > 0.05). (b) Representative hematoxylin-and-eosin-stained sections of the liver. Original magnification, ×200.

Figure 2

Effects of shikonin on liver function and pathology of mice with ConA-induced acute hepatitis. (a) The levels of serum ALT and AST changed depending on the shikonin dose, 7.5 mg/kg or 12.5 mg/kg. Data are given as means ± SD (n = 8, ^∗ P < 0.05 for NC versus ConA, ^# P < 0.05 for ConA + shikonin (7.5) versus ConA, and ⁺ P < 0.05 for ConA + shikonin (12.5) 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 NC versus ConA, ^# P < 0.05 for ConA + shikonin (7.5) versus ConA, and ⁺ P < 0.05 for ConA + shikonin (12.5) versus ConA).

Figure 3

Effects of shikonin on the production of IL-1β, TNF-α, and IFN-γ in mice with ConA-induced acute hepatitis. (a) The levels of serum TNF-α, IL-1β, and IFN-γ, measured with ELISAs were reduced by shikonin pretreatment in mice at doses of both 7.5 mg/kg and 12.5 mg/kg. Data are presented as means ± SD (n = 8, ^∗ P < 0.05 for NC versus ConA, ^# P < 0.05 for ConA + shikonin (7.5) versus ConA, and ⁺ P < 0.05 for ConA + shikonin (12.5) versus ConA). (b) The mRNA levels of IL-1β, TNF-α, and IFN-γ were evaluated in each group with real-time PCR (n = 8, ^∗ P < 0.05 for NC versus ConA, ^# P < 0.05 for ConA + shikonin (7.5) versus ConA, and ⁺ P < 0.05 for ConA + shikonin (12.5) versus ConA). (c) The expression levels of the IL-1β, TNF-α, and IFN-γ proteins were determined with western blotting and the gray values were calculated (n = 8, ^∗ P < 0.05 for NC versus ConA, ^# P < 0.05 for ConA + shikonin (7.5) versus ConA, and ⁺ P < 0.05 for ConA + shikonin (12.5) versus ConA). (d) Immunohistochemistry staining (×200) showing the expression of IL-1β, TNF-α, and IFN-γ in liver tissue at 6 h. The ratio of brown area to total area was analyzed with Image-Pro Plus 6.0 (n = 8, ^∗ P < 0.05 for NC versus ConA, ^# P < 0.05 for ConA versus ConA + shikonin).

Figure 4

Effects of shikonin on apoptosis and autophagy in mice with ConA-induced acute hepatitis. (a) cDNA levels of LC3, P62, Bcl-2, and Bax were measured with real-time PCR (n = 8, ^∗ P < 0.05 for NC versus ConA, ^# P < 0.05 for ConA + shikonin (7.5) versus ConA, and ⁺ P < 0.05 for ConA + shikonin (12.5) versus ConA). (b) Protein expression of LC3, Beclin-1, P62, Bcl-2, Bax, and caspase 9 was detected with western blotting and the gray values were calculated (n = 8, ^∗ P < 0.05 for NC versus ConA, ^# P < 0.05 for ConA + shikonin (7.5) versus ConA, and ⁺ P < 0.05 for ConA + shikonin (12.5) versus ConA). (c) Immunohistochemistry staining (×200) showed the expression of Bcl-2, Bax, and LC3 protein in liver tissue at 6 h. The ratio of brown area to total area was analyzed with Image-Pro Plus (v 6.0) (n = 8, ^∗ P < 0.05 for NC versus ConA, ^# P < 0.05 for ConA versus ConA + shikonin).

Figure 5

Effects of shikonin on the regulation of the IL-1β/JNK/p-JNK pathway in mice with ConA-induced acute hepatitis. (a) The levels of proteins IL-1β, total JNK, and p-JNK in liver tissue are shown as western blot bands and the gray values were calculated (n = 8, ^∗ P < 0.05 for NC versus ConA, ^# P < 0.05 for ConA + shikonin (7.5) versus ConA, and ⁺ P < 0.05 for ConA + shikonin (12.5) versus ConA). (b) The expression of p-JNK in hepatic tissues was determined with immunohistochemistry at 6 h (original magnification, ×200). The ratio of brown area to total area was analyzed with Image-Pro Plus (v 6.0) (n = 8, ^∗ P < 0.05 for NC versus ConA and ^# P < 0.05 for ConA versus ConA + shikonin). (c) The generation of ROS was detected with ROS Fluorescent Probe-DHE. And ROS were measured and analyzed in three random vision fields by Image-Pro Plus 6.0 (n = 8, ^∗ P < 0.05 for NC versus ConA and ^# P < 0.05 for ConA + shikonin versus ConA).

Figure 6