Our LC8 Tg mice showed significantly alleviated steatohepatitis compared to WT mice when they were fed an MCD diet, as revealed by decreased hepatic triglyceride content, improved liver histology, and lower serum ALT level. LC8 overexpression decreased the expression of lipogenic genes and increased that of β-oxidation–related genes. LC8 overexpression also reduced hepatic oxidative stress, as indicated by decreased ROS and lipid peroxidation, which were associated with decline of macrophage infiltration into the liver and hepatic inflammation. Moreover, LC8 overexpression considerably protected the liver from fibrosis. Our findings suggest that LC8 can ameliorate MCD diet–induced NASH by regulating lipid metabolism and reducing lipotoxicity, oxidative stress, inflammation, and fibrosis.
LC8 has been identified as a novel NF-κB inhibitor that blocks IκBα phosphorylation by IKK through a redox-dependent interaction with IκBα [18, 19]. NF-κB is a key regulator of inflammation and plays an essential role in the development and progression of steatohepatitis [20, 33]. In the current study, LC8 was found to protect MCD diet–induced steatohepatitis by inhibiting NF-κB. LC8 overexpression efficiently reduced MCD diet–induced hepatic expression of NF-κB targets such as TNF-α, IL-1β, COX-2, iNOS, and MCP-1. In addition, LC8 overexpression attenuated the phosphorylation of IκBα and p65 and subsequent p65 nuclear translocation. Moreover, immunoprecipitation using the antibody against IκBα showed that LC8 overexpression inhibits the release of p65 from IκBα. These results suggest the underlying mechanism in which LC8 interferes with IκBα phosphorylation by binding to it, attenuating the liberation of p65 from IκBα and leading to NF-κB inhibition.
TNF-α is a key inflammatory cytokine involved in the development of NASH in human patients  and murine models [27, 35]. TNF-α is a potent NF-κB activator as well as an NF-κB target and can amplify NF-κB signaling. Therefore, TNF-α can enhance the expression of IL-1β, COX-2, iNOS, and MCP-1. During the development of steatohepatitis, COX-2 plays a role as a pro-inflammatory mediator; IL-1β, together with TNF-α, activates local immune cells and induces MCP-1, which attracts macrophages to the liver, leading to sustained hepatic inflammation [11, 36, 37]. Also, other research indicates that iNOS deficiency attenuates hepatic inflammation in mice with fructose-induced steatosis .
Hepatic triglyceride accumulation in steatosis is caused by increased uptake or synthesis of fatty acids and reduced fatty acid oxidation. In our steatohepatitis model, LC8 overexpression significantly reduced hepatic triglyceride content and steatosis. This reduction was associated with lower expression of LXR-α, SREBP-1, SCD, FASN, and ACC, which are involved in fatty acid synthesis, and higher expression of PPAR-α, CPT1-α, LCAD, ACOX1, and UCP2, which are related to fatty acid oxidation. LXR-α is a major transcription factor that regulates the expression of lipogenic regulators, SREBP-1, and lipogenic genes (Scd, Fasn and Acc) [39, 40]. PPAR-α is a key regulator of the transcription of lipolytic genes (Cpt1a, Lcad, Acox1, and Ucp2) . Also, mitochondria is a primary site for fatty acid oxidation. LC8 overexpression also increased the hepatic expression of a key regulator of mitochondrial biogenesis, PGC-1β, and its target genes (Mt-co1, Mt-co3, Mt-nd4, and Mt-cyb). TNF-α induces hepatic steatosis by enhancing SREBP-1 expression  and by inhibiting both PPAR-α and PGC-1β expression .
Fat accumulation in the liver causes inflammation and mitochondrial dysfunction, leading to an increase in oxidative stress and consequently liver damage [44–46]. Increased ROS might exacerbate mitochondrial dysfunction by generation of mitochondrial DNA mutation and highly reactive aldehydes (4-HNE and MDA) produced through lipid peroxidation. LC8 overexpression attenuated MCD diet–induced oxidative stress and liver injury as indicated by the reductions in hepatic ROS, lipid peroxidation, Nrf2 activation, and serum ALT level observed in LC8 Tg mice fed the MCD diet. ROS mediate NF-κB activation, which in turn induces TNF-α expression, leading to inflammation and oxidative stress by increasing ROS [47, 48]. LC8 is a redox-sensitive molecule that is oxidized by TNF-α as well as ROS and forms an intermolecular disulfide bond . Such oxidation allows LC8 to dissociate from IκBα, leading to IκBα phosphorylation and degradation, followed by NF-κB activation . This implies that LC8 not only inhibits NF-κB, but also scavenges ROS.
Sustained liver inflammation leads to liver fibrosis. The pathogenesis of hepatic fibrosis is achieved through interplay of various hepatic cells, including hepatocytes, Kupffer cells, and hepatic stellate cells . Hepatic stellate cells are activated to myofibroblasts by periostin derived from hepatocytes. Immune cell–derived cytokines/chemokines such as TNF-α and MCP-1 promote the survival of hepatic stellate cells and accelerate liver fibrosis . TNF-α provokes hepatocytes to produce periostin, which can induce the expression of α-SMA, TGF-β, and collagen in hepatic stellate cells [51, 52].
The MCD diet is a widely adopted dietary model to study NASH. It can induce steatohepatitis with inflammation, oxidative stress, apoptosis, and fibrosis ; thus, it has been used as one of the useful models for studying the onset and progression of NASH, which is associated with inflammation, oxidative stress, and fibrosis [54–56]. However, it does not fully match all characteristics of human patients with NASH; notably, mice fed an MCD diet lose weight rather than become obese . To confirm our findings, a high-fat and high-fructose dietary model that can lead to an obese mouse with severe steatosis, inflammation, oxidative stress, and insulin resistance needs to be analyzed .
In conclusion, our observations and prior studies demonstrate that LC8 could alleviate MCD diet–induced steatohepatitis by attenuating hepatic fat accumulation, inflammation, oxidative stress, and liver fibrosis through downregulation of NF-κB, ROS, and TNF-α, which influence each other (Fig. 7). Thus, increasing intracellular LC8 could be a potential therapeutic strategy for patients with NASH and NAFLD.