4. Discussion
Fibrosis is a complex disease, driven at the cellular level by activation of quiescent HSCs and characterized by the sustained induction of a fibrotic gene program (Ding et al., 2015). The importance of preventing HSC activation during liver fibrosis treatment is undisputed. Traditional Chinese medicine which is composed of variant monomeric compounds from herbs are potential therapeutics for liver fibrosis. The natural product PB isolated from a Chinese herb Physalis species, Solanaceae, exhibited promising suppressive effect on liver fibrosis in two classic animal models and HSCs. The mechanism of such effect may be associated with inhibiting of the LAP2α and HDAC1 interaction and regulating GLI1 acetylation. This conclusion is based on four major observations: i) BDL and CCl4-challenged mice treated with PB exhibited histological amelioration of liver damage and fibrosis. ii) The mRNA and protein levels of liver fibrogenic markers were downregulated by PB administration in vitro and in vivo . iii) We identify the role of PB in repressing GLI1 independent of the canonical Hh signaling pathway. PB increased the acetylation of GLI1 and blocked nuclear translocation of GLI1. iv) PB inhibited the LAP2α-HDAC1 complex formation, which increased acetylation of GLI1 and exerted a therapeutic effect on hepatic fibrosis.
Increasing evidence showed that GLI1 is indispensable in liver pathophysiology. Recently literature reported that smoothened-independent GLI1 signaling directs differentiation of HPCs to fibrogenic cholangiocytes, and that it is upregulated during this fibrosis progression, while inhibiting the GLI1 expression suppresses fibrosis progression (Chen et al., 2020). GLI1 also plays an important role in activation of HSCs (Zhang et al., 2020), and GLI1 targeted inhibitors have been reported to improve fibrosis in mice and humans (Guerrero-Juarez & Plikus, 2017). GLI1 is regulated by different cascades, including the hedgehog and TGF pathways (Nye et al., 2014). After TGFβ1 treatment, elevated expression of GLI1 and the nuclear localization of GLI1 in normal fibroblasts, keratinocytes, and various cell lines has been detected (Dennler et al., 2007; Katoh & Katoh, 2009), which was in accordance with our findings. In the present study, we found that PB inhibited expression of GLI1 and reduced the nuclear localization of GLI1, reversed the TGFβ1‐induced upregulation of myofibroblastic markers (α‐SMA and collagen I). On the contrary, overexpression of GLI1 increased the level of myofibroblastic markers in HSCs and reversed anti-fibrotic effect of PB. Based on these results, GLI1 can upregulate fibrogenic gene expression and promoted liver fibrosis, thus, blocking GLI1 is anticipated as a promising strategy. PB may inhibit liver fibrosis, as well as TGFβ1 induced HSC activation through inhibiting GLI activity.
Acetylation is a posttranslational modification that can either alters the biological activities of proteins or alter subcellular localization (Blanco-Garcia, Asensio-Juan, de la Cruz & Martinez-Balbas, 2009; Nye et al., 2014). Previous investigates have shown that HDACs are a group of enzymes capable of removing acetyl groups from histone and other proteins (Kim et al., 2010), and that activity and recruitment are required for transcriptional activation of genes including Gja1 , Irf1 , and Gli1 (Zupkovitz et al., 2006). Among HDACs, HDAC1 is positive modulators of the activator members of the GLI family (Canettieri et al., 2010). GLI1 physically interacts with HDAC1, and HDAC1-mediated deacetylation of GLI1 results in the transcriptionally active (Canettieri et al., 2010; Falkenberg et al., 2016; Geng et al., 2018; Gurung, Feng & Hua, 2013). GLI1 deacetylation leads to the growth of medulloblastoma (Canettieri et al., 2010) and is increased in resistant basal cell carcinomas (Mirza et al., 2019). Conversely, in medulloblastoma and glioblastoma cells, the reduction of GLI1 deacetylation leads to decreased proliferation and increased apoptosis (Mazza et al., 2013). In accordance with these studies, we showed that PB increased GLI1 acetylation indicating an inactive state, and decreased the expression of αSMA and COL1A1 in HSCs, thereafter preventing hepatic fibrosis. LAP2α acts as a scaffold to recruit HDAC1 to GLI1 (Gotic & Foisner, 2010), GLI1 deacetylation increased GLI1 association with LAP2α to form an activating complex that withstood stringent high-salt conditions (Mirza et al., 2019). Our study identified that PB had no effect on the total expression of HDAC1 and LAP2α, but it prevented LAP2α from recruiting HDAC1 and inhibited the formation of HDAC1-LAP2α complex, thus downregulates the expression of GLI1 and its downstream target genes. Whereas, PB did not directly affect the interaction between LAP2α-GLI1. In addition, it was reported that liver fibrosis can be blocked by induction of HDAC inhibitors(Liu et al., 2013). Loh et al. showed that potent and selective inhibitors of class I only HDAC enzymes profoundly inhibit hepatocyte death and type 2 inflammation to prevent TAA‐induced liver fibrosis in mice (Loh et al., 2019). Previous studies have demonstrated that LAP2α localizes in the nucleoplasm is highly influential on cell proliferation and differentiation in regenerated tissue (Vidak, Kubben, Dechat & Foisner, 2015). LAP2α overexpression resulted in GLI1 hyperactivation, whereas knockdown depressed GLI1 expression in basal cell carcinomas cells (Mirza et al., 2019). In our system, we found that LAP2α deficiency reduced the expression of myofibroblastic markers thereby attenuate HSC activation. Our investigation is the first revealing that PB inhibits liver fibrosis by impairing HDAC1-LAP2α-GLI1 complex.
In summary, PB, a monomeric component of traditional Chinese medicine, is proved to ameliorate liver damage and fibrosis in BDL and CCl4‐treated mice. We evaluated the antifibrotic effect of PB on both HSCs and liver tissues, and revealed that the underlying mechanism of PB involves HDAC1/LAP2α‐mediated GLI1 deacetylation. This study is the first showing that PB inhibited the formation of HADC1-LAP2α complex. Based upon these findings, PB exerts therapeutic effects on liver fibrosis, which qualifies it being a novel candidate for hepatic fibrosis treatment in the near future.