Abstract:

Objective To explore the protective effect of epicatechin （EC） on acetaminophen （APAP）- induced liver injury in the mice， and to clarify its possible mechanisms. Methods Sixty C57BL/6J mice were randomly divided into blank control group， APAP model group， low dose of EC group （10 mg·kg^-1）， medium 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 the mice in blank control group， the mice in other groups were intraperitoneally injected with APAP （200 mg·kg^-1） to induce the liver injury models. One hour before APAP injection， the mice in low， medium， and high doses of EC groups were intraperitoneally injected with 10， 20， and 40 mg·kg^-1 EC， respectively.Thirty-six nuclear factor erythroid 2-related factor 2 deficitent mice （Nrf2^-/- mice） were randomly divided into control group， APAP group and APAP+EC group. After 24 h， the mice were sacrificed， and blood and liver tissue were collected for subsequent analysis. The pathomorphology of live tissue of the mice in various groups were assessed by HE staining， while the serum levels of aspartate aminotransferase （AST） and alanine aminotransferase （ALT）， the activities of myeloperoxidase （MPO） in liver tissue， and the levels of tumor necrosis factor-α（TNF-α）， interleukin-1β （IL-1β）， malondialdehyde （MDA）， adenosine triphosphate （ATP）， and glutathione （GSH），and ferrous ions （Fe²⁺） in liver tissve of the mice were determined with kits. The expression levels of nuclear factor-kappa B （NF-κB） and Nrf2 signaling pathway-related proteins in liver tissue of the mice in various groups were detected by Western blotting method. Results Compared with APAP model group， the liver injuries induced by APAP in different doses of EC groups were improved. Compared with blank control group， the serum ALT and AST levels of the mice in APAP model group were significantly increased （P<0.01）； compared with APAP model group， the serum ALT and AST levels in low， medium 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 TNF-α and IL-1β levels in liver tissue of the mice in APAP model group were significantly increased （P<0.01）； compared with APAP model group， the MPO activities and the levels of TNF-α and IL-1β in liver tissue in low， medium 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<0.01）， and the levels of ATP and GSH in liver tissue were decreased （P<0.01）； compared with APAP model group， the levels of MDA and Fe²⁺ in liver tissue of the mice in different doses of EC groups were significantly decreased （P<0.05 or P<0.01）. Compared with blank control group， the GPX4 and xCT protein expression levels in liver tissue of the mice in APAP model group were significantly decreased （P<0.01）； compared with APAP model group， the expression levels of GPX4 and xCT in liver tissue of the mice in different doses of EC were significantly increased（P<0.05 or P<0.01）. Compared with blank control group， the expression levels of NF-κB p-p65 and phosphorylated inhibitor of NF-κB alpha（p-IκBα） in liver tissue of the mice in APAP mode group were significantly increased expression（P<0.01）. Companed with blank control group， the levels of NF-κB p-p65 and p-IκBα proteins in liver tissue of the mice in low， medium and high doses of EC groups were significantly reduced（P<0.01）. Compared with blank control group， the levels of Nrf2 and HO-1 in liver tissue of the mice in APAP model group were increased （P<0.01）； compared with APAP model group， the levels of Nrf2 and HO-1 in liver tissue of the mice in low， medium and high doses of EC groups were significantly increased（P<0.01）. Compared with control group， the serum AST and ATL and level 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 of the mice were significantly decreased（P<0.01）. Conclusion EC can alleviate APAP-induced liver injury， and the mechanism may be related to the activation of Nrf2/GPX4 signaling pathway by EC to inhibit iron death.

Key words: Epicatechin, Acetaminophen, Liver injury, Ferroptosis, nuclear factor erythroid 2-related factor 2