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.