Introduction
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease caused by excess fat accumulation and is closely associated with obesity and metabolic syndrome (Tessari, Coracina, Cosma, & Tiengo, 2009). The NAFLD often progresses to non-alcoholic steatohepatitis (NASH) accompanied by chronic liver injury, fibrosis, and inflammation, a part of which occurs liver cirrhosis and hepatocellular carcinoma (Bellentani, 2017). During progression to NASH, excess lipid accumulation in the liver results in the activation of hepatic macrophages and hepatic stellate cells and induces the infiltration of immune cells. These activated cells release active oxygen along with various cytokines and chemokines, thereby enhancing immune cell migration, hepatic inflammation, and fibrosis. Recently, a single-cell RNA-seq (scRNA-seq) analysis in NASH mice showed the presence of several types of macrophages in different active states, such as resident Kupffer cells and monocyte-derived Kupffer cells in NASH (Daemen et al., 2021; Remmerie et al., 2020; Seidman et al., 2020). Thus, activated macrophages appear to play an essential role in NASH progression.
Nuclear factor erythroid 2-related factor (Nrf)2, a transcription factor that induces antioxidant-associated gene expression under oxidative stress, might function as a therapeutic target for NASH (Bataille & Manautou, 2012). Kelch-like ECH-associated protein (Keap)1 binds the Neh2 domain of Nrf2 in the cytoplasm and prevents its translocation to the nucleus (Itoh et al., 2013). Active oxygen produced during inflammation induces the dissociation of Nrf2 from Keap1, leading to the nuclear translocation of Nrf2 and its transcriptional activity for antioxidant gene expression. Notably, NASH caused by a methionine- and choline-deficient diet is considerably aggravated in Nrf2-deficient mice (Chowdhry et al., 2010). In contrast, Nrf2 activation by Keap1-knockdown attenuates steatosis in NASH (Zhang, Yeager, Tanaka, & Klaassen, 2010). Further, several pharmacological approaches for activating Nrf2 are also known to attenuate NASH progression (Sharma et al., 2018; Shimozono et al., 2013). Bardoxolone (CDDO), a potent pharmacological activator of Nrf2, is reported to inhibit the induction of various cytokines such as interferon-1 and interleukin-1 in mouse peritoneal macrophages and rat brain microglia (Tran, McCoy, Sporn, & Tansey, 2008) and to induce monocyte differentiation from myeloid leukaemia cells (Suh et al., 1999) These findings suggest that CDDO may inhibit macrophage-related inflammation. Notably, CDDO-Me markedly improves obesity-induced inflammatory diseases in the kidney and heart and has also been used in clinical trials for treating chronic kidney disease and pulmonary arterial hypertension (Chin et al., 2018). However, the hepatoprotective effects of CDDO-Me on NASH remain unknown.
Therefore, in this study, we investigated the hepatoprotective effects of CDDO-Me in a diet-induced NASH mouse model and its pharmacological mechanisms using whole-transcriptome analysis with RNA-seq.