Water extract of earthworms mitigates mouse liver fibrosis by potentiating hepatic LKB1/Nrf2 axis to inhibit HSC activation and hepatocyte death

Abstract

Ethnopharmacological relevance: When left untreated, liver fibrosis (LF) causes various chronic liver diseases. Earthworms (Pheretima aspergillum) are widely used in traditional medicine because of their capacity to relieve hepatic diseases.

Aim of the study: This study aimed to explore the anti-LF effects of water extract of earthworms (WEE) and the underlying molecular mechanisms.

Materials and methods: A CCl₄-induced mouse model of LF was used to study the impact of WEE on LF in vivo. The anti-LF activity of WEE in mice was compared with that of silybin, which can be clinically applied in LF intervention and was used as a positive control. Activation of LX-2 hepatic stellate cells (HSCs) and apoptosis and ferroptosis of AML-12 hepatocytes induced by TGFβ1 were used as in vitro models.

Results: WEE drastically improved LF in mice. WEE reduced markers of activated HSCs in mice and inhibited TGFβ1-induced activation of LX-2 HSCs in vitro. Additionally, WEE suppressed CCl₄-induced apoptosis and ferroptosis in mouse hepatocytes. Mechanistically, WEE induced Nrf2 to enter the nuclei of the mouse liver cells, and the hepatic levels of Nrf2-downstream antioxidative factors increased. LKB1/AMPK/GSK3β is an upstream regulatory cascade of Nrf2. In the LF mouse model, WEE increased hepatic phosphorylated LKB1, AMPK, and GSK3β levels. Similar results were obtained for the LX-2 cells. In AML-12 hepatocytes and LX-2 HSCs, WEE elevated intracellular Nrf2 levels, promoted its nuclear translocation, and inhibited TGFβ1-induced ROS accumulation. Knocking down LKB1 abolished the impact of WEE on the AMPK/GSK3β/Nrf2 cascade and eliminated its protective effects against TGFβ1.

Conclusions: Our findings reveal that WEE improves mouse LF triggered by CCl₄ and supports its application as a promising hepatoprotective agent against LF. The potentiation of the hepatic antioxidative AMPK/GSK3β/Nrf2 cascade by activating LKB1 and the subsequent suppression of HSC activation and hepatocyte apoptosis and ferroptosis are implicated in WEE-mediated alleviation of LF.

Keywords: Hepatic stellate cell activation; Hepatocyte death; Liver fibrosis; Nrf2 signaling; Water extract of earthworms.