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.