Ergothioneine Attenuates CCl 4 ‐Induced Acute Liver Injury by Inhibiting Oxidative Stress and Apoptosis via Regulating LKB1/Nrf2 Pathway

ABSTRACT Boletus edulis is a traditional medicinal and edible fungus with hepatoprotective, anticancer, and cough‐relieving effects. Ergothioneine (EGT) has been shown to be one of the active ingredientswith a variety of biological activities, including antioxidant, anti‐apoptotic, and anti‐inflammatory effects. Our previous research showed that EGT can alleviate CCl 4 ‐induced liver fibrosis. However, the impact on CCl 4 ‐induced acute liver injury is still unclear. In this study, we used a CCl 4 ‐induced acute liver injury model in mice and evaluated the effect of EGT on mouse liver using liver appearance, HE staining, liver index, and liver function indicators. We evaluated key cellular proteins associated with oxidative stress and cell apoptosis through WB and immunofluorescence staining. In vitro, AML‐12 cells were stimulated with CCl 4 and analyzed by immunoblotting, flow cytometry, and biochemical assays. In animal models, the results of HE staining and liver function indexes showed that EGT significantly improved CCl 4 ‐induced ALI. In vitro, detection of Bcl‐2‐associated X protein (Bax), B‐cell lymphoma‐2 (Bcl2), and ROS expression levels showed that EGT significantly reduced the damage caused by CCl 4 by inhibiting apoptosis and oxidation. Both in vivo and in vitro, EGT significantly increased the activity of Nuclear factor erythroid2‐related factor 2 (Nrf2) and the expression of downstream antioxidative factors. Meanwhile, EGT also increased the transcription of antioxidant factors, ultimately inhibiting the levels of oxidative stress and apoptosis. Mechanistically speaking, EGT promotes the entry of Nrf2 into the nucleus to exert antioxidant effects. Liver kinase B1 (LKB1)/AMP‐activated protein kinase (AMPK)/Glycogen synthase kinase‐3beta (GSK3β) is a key upstream regulatory signal of Nrf2. EGT can promote the phosphorylation of LKB1, AMPK, and GSK3β both in vivo and in vitro. In AML‐12 liver cells, knocking out LKB1 eliminates the effects on AMPK/GSK3β and abolishes the protective effect of EGT on CCl 4 . This indicates that LKB1 activation plays a crucial role in EGT regulation of LKB1/Nrf2 signaling. Our research results indicate that EGT can improve mouse ALI induced by CCl 4 and support its use as a hepatoprotective drug.