Autoimmune hepatitis (AIH) is a complex immune-mediated inflammatory liver disease of unknown cause that can affect people of any age, gender, and ethnicity (Mack et al., 2020). AIH is diagnosed histologically by interface hepatitis and high serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and immunoglobulin G and the presence of autoantibodies (Floreani et al., 2018). Patients can be asymptomatic, chronic, or present in various forms, that progress further to hepatic fibrosis, cirrhosis, liver failure, or others overlapping syndromes between AIH and primary biliary cirrhosis or primary sclerosing cholangitis (Boberg et al., 2011). Immunosuppressive agents are frequently used to treat AIH. However, the effectiveness varies greatly among patients and long-term use of immunosuppressants can be associated with serious side effects (Wang et al., 2020). Therefore, there is a need for alternative therapeutic agents to treat AIH. As a traditional Chinese medicine, Tripterygium wilfordii Hook F. has significant anti-inflammatory and immunosuppressive properties and is widely used for treating autoimmune and inflammatory diseases such as rheumatoid arthritis, Systemic Lupus Erythematosus, and kidney disease (Chen, G. et al., 2022; Zhang et al., 2022). However, the effect of TWT on Con A-induced liver injury thus far has not been investigated.

Celastrol is the main active ingredient of TWT. It has been shown to produce a variety of beneficial effects, including anti-inflammatory, anti-obesity, anti-cancer, bacteriostatic and immunomodulatory (Chen et al., 2020; Liu et al., 2015; Yu et al., 2018). Celastrol has been shown to alleviate thioacetamide (TAA) and α-naphthyl isothiocyanate (ANIT) -induced cholestatic liver injury by modulating the SIRT1-FXR signaling pathway (Zhao et al., 2019). It remains to be determined whether celastrol could protect against AIH. In addition to its hepato-protective effect, celastrol has also been shown to enhance heat shock factor 1 (HSF1) and promote energy expenditure through activation of a PGC1α-dependent metabolic program, which improves mitochondrial function and protects against high-fat diet-induced obesity (Ma et al., 2015). A more recent study has revealed a protective role of celastrol against Alzheimer's disease through an up-regulation of the transcription factor EB (TFEB)-mediated autophagy and lysosomal biogenesis in vitro and in mouse brain (Yang, C. et al., 2022).

TFEB is a master regulator of the autophagy–lysosomal pathway. It has been widely shown to play a protective role against the development and progression of many diseases (Martini-Stoica et al., 2016). For example, up-regulation of TFEB through activation of PGC-1α can drastically reduce huntingtin protein aggregation and alleviate Huntington's disease neurodegeneration partly by decreasing oxidative stress (Tsunemi et al., 2012). Up-regulation of TFEB expression using a genetic or pharmacological approach can effectively block the development of α-syn–induced toxicity and ameliorate Parkinson's disease (Decressac et al., 2013). TFEB overexpression also reduces weight gain and improves metabolic syndrome in high-fat diet-induced obesity by increasing lysosomal biogenesis and autophagy (Settembre et al., 2013). Given the mounting evidence that TFEB plays a critical role in neurodegenerative and metabolic diseases, it remains to be determined whether dysregulation of TFEB-mediated autophagy is involved in the pathogenesis of liver diseases and whether pharmacological approaches to increase TFEB (e.g., with 4-OI) could protect against Con A-induced liver injury.

Intravenous injection of Concanavalin A (Con A) can induce acute liver inflammation in mice by specific activation and recruitment of T cells (Heymann et al., 2015; Zhao et al., 2021). The induction time is relatively short and the pathological features are highly reproducible. Therefore, Con A-induced acute liver inflammation model has become a popular experimental model in hepatitis research. The nuclear receptor PXR has been long recognized and widely studied for its role in xenobiotics metabolism and drug clearance (Xie et al., 2019). Emerging evidence also suggested a role of PXR in the regulation of immune or inflammatory responses (Zhou et al., 2009). For example, pregnenolone 16α-carbonitrile (PCN), a specific PXR activator, improves Con A-induced liver injury (Kodama et al., 2017) and dextran sulfate sodium (DSS) -induced colitis in mice (Liu et al., 2018). These studies have demonstrated the potential advantages of PXR activation in the treatment of inflammatory diseases, but the underlying mechanism of PXR in an animal model of Concanavalin A-induced AIH is still unclear.

Herein, the effect of TWT and celastrol on Con A-induced hepatitis and the underlying mechanism were studied using a combination of mass spectrometry-based metabolomics, molecular biology and cell-based approaches. Using 4-octyl itaconate (4-OI) as an itaconate mimicker, we found 4-OI improved Con A-induced liver injury by enhancing TFEB-mediated autophagy by activating PXR in primary hepatocytes. These results indicate that PXR and TFEB-mediated lysosome autophagy play a pivotal role in Con A-induced liver injury. Importantly, our study is the first to provide evidence for the dysregulation of TFEB in Con A-induced liver injury and that targeting the TFEB-mediated autophagy might provide therapeutic benefits for the treatment of Con A-induced liver injury. Collectively, the present study suggested that PXR and TFEB are potential therapeutic targets for the treatment of AIH, and TWT might be a promising candidate for the treatment of autoimmune liver diseases.