Abstract:

Objective To discuss the improvement effect of epicatechin（EC） on acetaminophen（APAP）- induced liver injury in the mice， and to clarify its possible mechanism. Methods A total of 60 C57BL/6J mice were randomly divided into blank control group， APAP model group， low dose of EC group （10 mg·kg^-1）， middle dose of EC group （20 mg·kg^-1） and high dose of EC group （40 mg·kg?1）， with 12 mice in each group. Except for blank control group， the mice in the other groups were given intraperitoneal injection of APAP （200 mg·kg?1） to establish the liver injury models. At 1 h before APAP injection， the mice in low， middle and high doses of EC groups were intraperitoneally injected with 10， 20 and 40 mg·kg^-1 EC， respectively. A total of 36 nuclear factor E2-related factor 2 （Nrf2） deficient mice （Nrf2^-/- mice） were randomly divided into control group， APAP group and APAP+EC group， with 12 mice in each group. After modeling 24 h， the mice were sacrificed， and the blood and liver tissue of the mice were collected for subsequent detection. HE staining was used to observe the pathomorphology of the liver tissue in the mice in various groups； kit assay was used to detect the activities of aspartate aminotransferase （AST） and alanine aminotransferase （ALT） in serum of the mice in various groups and the myeloperoxidase （MPO） activity and the levels of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， malondialdehyde （MDA）， adenosine triphosphate （ATP）， glutathione （GSH） and ferrous ion （Fe2?） in liver tissue of the mice in various groups； Western blotting method was used to detect the expression levels of nuclear factor-κB （NF-κB） and Nrf2 signaling pathway-related proteins in liver tissue of the mice in various groups. Results The HE staining results showed that compared with APAP model group， the APAP-induced liver pathology injury in the mice in different doses of EC groups was significantly improved. Compared with blank control group， the levels of ALT and AST in serum of the mice in APAP model group were significantly increased （P<0.01）. Compared with APAP model group， the activities of ALT and AST in serum of the mice in low， middle and high doses of EC groups were significantly decreased （P<0.05 or P<0.01）. Compared with blank control group， the MPO activity and the levels of TNF-α and IL-1β in liver tissue of the mice in APAP model group were significantly increased （P<0.01）. Compared with APAP model group， the MPO activity and the levels of TNF-α and IL-1β in liver tissue of the mice in low， middle and high doses of EC groups were decreased （P<0.05 or P<0.01）. Compared with blank control group， the levels of MDA and Fe2? in liver tissue of the mice in APAP model group were significantly increased （P<0.05）， and the levels of ATP and GSH were significantly decreased （P<0.05）. Compared with APAP model group， the levels of MDA and Fe2? in liver tissue of the mice in low， middle and high doses of EC groups were significantly increased （P<0.05 or P<0.01）， and the levels of ATP and GSH were significantly increased （P<0.01）. Compared with blank control group， the expression levels of amino acid exchange transporter （xCT） and glutathione peroxidase 4 （GPX4） proteins in liver tissue of the mice in APAP model group were significantly decreased （P<0.05）； compared with APAP model group， the expression levels of xCT and GPX4 proteins in liver tissue of the mice in low， middle and high doses of EC groups were significantly increased （P<0.01）. Compared with blank control group， the expression levels of nuclear factor-κB （NF-κB） p-p65 and phosphorylated NF-κB inhibitor α （p-IκBα） proteins in liver tissue of the mice in APAP model group were significantly increased （P<0.05）； compared with APAP model group， the expression levels of NF-κB p-p65 and p-IκBα proteins in liver tissue of the mice in low， middle and high doses of EC groups were significantly decreased （P<0.01）. Compared with blank control group， the expression levels of Nrf2 and heme oxygenase-1 （HO-1） proteins in liver tissue of the mice in APAP model group were significantly increased （P<0.05）； compared with APAP model group， the expression levels of Nrf2 and HO-1 proteins in liver tissue of the mice in low， middle and high doses of EC groups were significantly increased （P<0.01）. Compared with control group， the ALT level in serum and the levels of MDA and Fe²⁺ in liver tissue of the Nrf2^-/- mice in APAP group were significantly increased （P<0.01）， and the levels of ATP and GSH in liver tissue were significantly decreased （P<0.01）. Conclusion EC can improve APAP-induced liver injury in the mice， and its mechanism may be related to the inhibition of ferroptosis by activating the Nrf2/GPX4 signaling pathway.

Key words: Epicatechin, Acetaminophen, Liver injury, Ferroptosis, Nuclear factor E2-related factor 2