Liver fibrosis (LF) is the massive deposition of intrahepatic extracellular matrix (ECM) that occurs in most chronic hepatic diseases (Roehlen et al., 2020). As an increasing amount of liver parenchyma is replaced by scar tissue mainly composed of extracellular matrix, without timely intervention, LF may progress to cirrhosis, hepatocellular carcinoma, liver failure, or other critical liver diseases (Kumar et al., 2021). Currently, only a few drugs that specifically target LF are clinically available. Thus, the search for new bioactive substances with anti-fibrotic potential is imperative. Hepatic stellate cells (HSCs) account for approximately 10% of all liver cells (Khomich et al., 2019). The primary functions of HSCs include storing vitamin A, supporting liver regeneration, and regulating immune response (Puche et al., 2013). In a normal liver, HSCs are quiescent. Upon stimulation by pathogenic factors, such as excessive xenobiotics, cholestasis, or infections, HSCs are activated to produce large quantities of ECM, resulting in LF (Iwaisako et al., 2014). Therefore, inhibiting HSC activation is a feasible strategy for ameliorating LF.

Hepatocyte death triggered by pathogenic factors is of great significance for activating HSCs and promoting LF (Guo et al., 2022). Inflammation can be provoked by dead hepatocytes or damage-associated molecular patterns released during hepatocyte death, leading to a sharp elevation of profibrotic cytokines such as transforming growth factor β1 (TGFβ1) in the liver. These cytokines induce HSC activation (Mederacke et al., 2022). HSCs are also directly activated by the phagocytosis of dead hepatocytes (Schwabe et al., 2020). Apoptosis is primarily regulated by B-cell lymphoma-2 (Bcl-2) and caspases (Green and Llambi, 2015). Persistent hepatocyte apoptosis is a critical factor in the etiology of LF (Jiang et al., 2023). In addition to apoptosis, ferroptosis is another cell death process (Dixon et al., 2012). Iron overload because of the downregulation of light and heavy chains of ferritin (FTL and FTH), which store intracellular iron, and lipid peroxidation because of glutathione peroxidase 4 (GPX4) dysfunction are the major characteristics of ferroptosis (Chen et al., 2021). Hepatocyte apoptosis as well as ferroptosis contribute to LF progression (Zhou et al., 2019). Reactive oxygen species (ROS) are products of incomplete oxygen reduction and have very high biological activity (Chen et al., 2020). ROS levels in hepatocytes increase upon stimulation with various pathogenic factors. High ROS levels directly trigger hepatocyte apoptosis or ferroptosis (Chang et al., 2021). Moreover, many profibrotic cytokines promote HSC activation by increasing intracellular ROS levels (Ramos-Tovar and Muriel, 2020).

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor expressed in most mammalian organs (Liu et al., 2023a, Liu et al., 2023b; Sha et al., 2023; Tonelli et al., 2018). As Nrf2 translocates into the nucleus, it induces the transcription of antioxidant genes, including heme oxygenase 1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), and the catalytic and modifier subunits of glutamate cysteine ligase (GCLC and GCLM), enhancing the intracellular antioxidative capacity (Jia et al., 2019; Zhang et al., 2022a, Zhang et al., 2022b). Many bioactive substances alleviate LF by potentiating hepatic Nrf2 expression (Zhou et al., 2022). Liver kinase B1 (LKB1) exerts tumor-suppressive activity, by way of Ser431 phosphorylation in mouse LKB1 (Ser 428 phosphorylation in human LKB1), which is indispensable for its catalytic function (Sapkota et al., 2001). LKB1 directly catalyzes the phosphorylation and activates the AMP-activated protein kinase (AMPK) (Zhang et al., 2013). When AMPK is phosphorylated and activated, it can induce glycogen synthase kinase 3β (GSK3β) phosphorylation to eliminate GSK3β-mediated inhibition of Nrf2. AMPK can also directly phosphorylate Nrf2 and induce nuclear translocation (Joo et al., 2016). These findings indicate that the LKB1/AMPK cascade is critically involved in regulating Nrf2 signaling.

Dried bodies of earthworms (Pheretima aspergillum), termed “dilong” in Chinese, have long been used as traditional Chinese medicine (TCM). According to the Chinese Pharmacopoeia, dilongs can pacify the liver. In the clinical practice of TCM, dilong is used to treat LF or liver cirrhosis (Jiang et al., 2021; Zhang et al., 2020). Pharmacological studies have revealed that earthworms relieve liver injury provoked by paracetamol or endoplasmic reticulum stress (Balamurugan et al., 2008; Wang et al., 2016). However, limited scientific data support the protective activities of earthworm extracts against LF. Moreover, although earthworms activate Nrf2 in the lungs (Yang et al., 2016), whether this traditional medicine affects Nrf2 signaling in the liver remains uncertain. The upstream molecular events through which earthworms activate Nrf2 are also unclear. Therefore, we prepared a water extract of earthworms (WEE) and analyzed its major components. Subsequently, we explored the anti-LF effects of WEE and its underlying molecular mechanisms.