Abstract
Background and Purpose: Liver fibrosis is one of the leading causes of morbidity and mortality worldwide of which no acceptable therapy exists. Accumulating evidence supports that glioma-associated oncogene homologue 1(GLI1) is a potentially important therapeutic target for liver fibrosis. This study investigates the antifibrotic activities and potential mechanisms of Physalin B (PB), a natural Solanaceae compound.
Experimental Approach: Mice subjected to CCl4challenge and bile duct ligation were used to study the antifibrotic effects of PB in vivo . Mouse primary hepatic stellate cells (pHSCs) and human HSC line LX‐2 also served as an in vitro liver fibrosis model. Liver fibrogenic genes, GLI1 downstream genes were examined using western blot and real-time PCR analyses. GLI1 acetylation and LAP2α-HDAC1 interaction were analyzed by coimmunoprecipitation.
Key Results: In animal models, PB administration attenuated hepatic histopathological injury, collagen accumulation, and reduced the expression of fibrogenic genes. PB dose‐dependently suppressed fibrotic marker expression in LX‐2 cells and mouse pHSCs. Mechanistic studies showed PB inhibited GLI activity in a non-canonical Hedgehog signaling. PB blocked lamina-associated polypeptide 2 α (LAP2α)/ histone deacetylase 1 (HDAC1) complex formation thereby inhibited HDAC1mediated GLI1 deacetylation. PB downregulated the acetylation and expression of GLI1, and subsequently inhibiting HSC activation.
Conclusions and Implications: PB exerted potent antifibrotic effects in vitro and in vivo by disrupting the LAP2α/HDAC1 complex, increasing GLI1 acetylation and inactivating GLI1. This indicates that PB may be a potential therapeutic candidate for the treatment of liver fibrosis.
Introduction
Liver fibrosis, a wound-healing response to chronic liver injury, is characterized by excessive deposition of extracellular matrix (ECM) in the liver. During chronic liver damage, HSCs are continuously activated and converted into myofibroblasts, which are the major source of ECM and the principal cell type in liver fibrogenesis(Friedman, 2008). This transient response must be tightly controlled, otherwise, it would become persistent and lead to excessive matrix accumulation and fibrosis(Lee, Wallace & Friedman, 2015). Thus, potential therapies for these pathologies should ideally possess pro regenerative and antifibrotic properties, for example, the ability of controlling hepatocyte proliferation and inactivating activated HSC(Hernandez-Gea & Friedman, 2011). Currently, there are no approved drugs that can effectively reverse liver fibrosis, further highlighting the urgent clinical need for novel antifibrotic therapies.
GLI (glioma-associated oncogene homologue) was identified as a gene that is amplified in gliomas(McMillin et al., 2014), and it is one of the strongest transcriptional activators of Hh signaling pathway, can also be regulated independent of the canonical Hh signaling pathway. The TGFβ signaling pathway activates GLI1 even in the presence of SMO antagonist (Dennler et al., 2007). In injured organs, resident GLI1+ cells were committed to the myofibroblast lineage, which directly regulates HSC fate (Machado & Diehl, 2018). Genetic ablation of GLI1+ cells ameliorated fibrosis and improved organ function, providing a proof of principle for therapeutic targeting of GLI1+ cells in fibrotic disease (Kramann et al., 2015). The aforementioned reports emphasize the importance of the mechanisms that terminate GLI1 activity and that may be impaired in disease. Hence, GLI1 is a potential biomarker and therapeutic target aiming at controlling activation of HSCs during liver fibrogenesis.
GLI1 acetylation, a modification that limits the activity of this transcription factor (Miele et al., 2017). Acetylation represents a key transcriptional event, finely tuned by histone acetyltransferases (HATs) and histone deacetylases (HDACs). It has been observed that loss of class I HDAC disrupts the transcriptional response to Hh activation, HDAC1 is shown to deacetylate the transcription factor GLI1 (Canettieri, Di Marcotullio, Coni, Greco & Gulino, 2010), which allows GLI1 to associate with chromatin and initiate transcription (Canettieri et al., 2010; Coni et al., 2013). Such deacetylation mechanisms are involved in GLI1-dependent cell proliferation, migration, differentiation, and survival.
Currently, few antifibrotic treatment strategies are available. Thus, there is an urgent clinical need for the development of antifibrotic candidates specifically targeting HSCs. Our study is engaged in evaluating the antifibrotic activities of natural compounds. We have utilized a high-throughput drug screening model based on the COL1A1 promoter to filtrate potential anti-hepatic fibrosis agents from natural substances. Physalin B (PB), a natural component derived from one of the best-known traditional Chinese medicinal plants, Physalis species, Solanaceae, was speculated having potential therapeutic effect for liver fibrosis by us. Previous studies have described the biological functions of PB, including anti‐inflammatory (Hsu et al., 2012; Yang, Yi, Wang, Xie, Sha & Dong, 2018), antimalarial(Magalhaes et al., 2006), proapoptotic(Soares et al., 2006), antinociceptive (Vandenberghe et al., 2008), and antitumor activities(Hsu et al., 2012; Ma, Han, Li, Hu & Zhou, 2015), but the effects of PB on liver fibrosis have not been reported thus far. We investigated the anti-fibrotic effects of PB on human stellate cell line LX-2, primary HSCs, and experimental hepatic fibrosis mice induced by CCl4 and bile duct ligation (BDL), finally elucidated the potential mechanisms underlying its function in vitro and in vivo .