Ralf Weiskirchen
Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and
Clinical Chemistry (IFMPEGKC), RWTH University Hospital Aachen, Aachen,
Germany; Email:
rweiskirchen@ukaachen.de
Word count: 1193
Liver fibrosis is an excessive scarring process resulting in progressive
disruption of the normal tissue architecture and impaired organ
function. It comprises many different etiologies and its consequences
present a substantial medical and economic burden worldwide (Weiskirchen
et al., 2018). Mechanistically, fibrosis is initiated by parenchymal
cell destruction resulting in tissue damage that is associated with an
inflammatory response. This in turn provokes the local activation of
mesenchymal cells which have the capacity to produce extracellular
matrix compounds such as collagens. Most important in this process are
hepatic stellate cells (HSCs) that are pericytes in the perisinusoidal
space of the liver. Upon activation, these cells lose their quiescent
phenotype and transit in a process called transdifferentiation into
proliferative fibrogenic α-smooth muscle positive (α-SMA)
myofibroblasts. These are the central cellular drivers of hepatic
fibrogenesis in experimental and human liver injury. Although the
cellular and molecular mechanisms of hepatic fibrosis including
important pro-inflammatory and pro-fibrogenic soluble mediators
(chemokines, cytokines) and signaling pathways are identified, there
exist no specific and effective antifibrotic pharmacological therapies.
Beside synthetic drugs, complementary and alternative medicine is
presently in the focus of basic and clinical research. In particular, a
number of herbal remedies and active ingredients thereof show promising
effects against hepatic fibrosis either experimentally in cell culture,
in pre-clinical animal models, or even in the first clinical trials
(Weiskirchen 2015). These compounds counteract the intercellular
production or activity of reactive oxygen species, prevent hepatic
infiltration of circulating inflammatory acting blood cells, or
interfere with relevant signaling pathways or mediators involved in the
production or turnover of extracellular matrix.
The present study by Zhu and colleagues investigated the antifibrotic
activities of Physalin B (PB) in two models of hepatic fibrosis, in
primary cultures of mouse HSC, and in the well-established immortalized
human HSC cell line LX-2.
PB possesses a unique 13,14-seco -16,24-cyclo -steroidal
skeleton and an H-ring with a C14-O-C27bond forming an intriguing cage-shaped structure (Fang et al., 2003). It
is a main active physalin of Physalis angulata L . belonging to
the nightshade family (Solanaceae ). When the authors applied PB
in mice that were either simultaneously subjected to repeated
applications of carbon tetrachloride or to a bile duct surgery that are
both established experimental models of hepatic fibrosis, the
seco-steroid ameliorated hepatic fibrosis as assessed by overall reduced
hepatic collagen deposition, lowered blood markers of liver damage
(alanine aminotransferase, aspartate aminotransferase), and diminished
expression of fibrogenic markers (collagen type I, tissue inhibitor of
metalloproteinase 1 (TIMP1), α-SMA, and transforming growth factor-β
(TGF-β)). In cultured HSCs, PB decreased α-SMA expression during
spontaneous activation and transdifferentiation. Moreover, PB was
effective to blunt TGF-β-induced activity of a luciferase-based COL1A1
reporter in human HSC line LX-2. Similarly, PB repressed
glioma-associated oncogene 1 (GLI1) mRNA expression and activity of a
GLI1-dependent reporter construct in primary mouse HSCs and in the
immortalized human HSC line LX-2, while the expression of GLI2 and GLI3
were unaffected. Interestingly, the drug effectively blocked
GLI1-induced expression of collagen and α-SMA in cultured HSCs, most
likely by hindering GLI1 nuclear localization that in turn resulted in
reduced mRNA expression of typical GLI1 target genes such asHedgehog Interacting Protein , Cyclin D , Cyclin E ,
and c-myc . Moreover, PB induced GLI1 acetylation by blocking
complex formation between Lamina-associated polypeptide 2α (LAP2α) and
histone deacetylase 1 (HDAC1) which normally promotes GLI1
deacetylation. The same stimulatory effect on GLI1 acetylation was
induced by the HDAC inhibitor vorinostat confirming the protective
effect of the LAP2α/HDAC1 complex on GLI1 deacetylation.
The finding that PB treatment mitigated liver fibrosis viainhibiting the interaction between LAP2α and HDAC1 that provokes
increased acetylation and nuclear translocation of GLI1 is novel. In a
related study, the same authors have recently shown that PB ameliorates
experimental non-alcoholic steatohepatitis (NASH) in mice by stimulating
autophagy and p62-Keap1-Nrf2 anti-oxidative signaling, suggesting that
this drug has also some general anti-inflammatory and hepatoprotective
activities (Zhang et al., 2021). In line, in a more previous report PB
was considered to inhibit NO production by lipopolysaccharide- or
interferon-γ-activated macrophages and to protect mice against a lethal
lipopolysaccharide challenge (Soares et al., 2003). Thus, PB has the
potential to counteract a broad range of biological processes that
contribute to the pathogenesis of hepatic fibrosis. In particular, the
inhibition of collagen, α-SMA, TGF-β, and TIMP1 provides evidence that
PB exerts anti-fibrogenic properties in the liver by inhibiting HSC
activation. During hepatic fibrosis, the upregulation of these
pro-fibrogenic marker genes is a hallmark indicating activation and
transdifferentiation of quiescent HSCs into proliferative, profibrogenic
and extracellular matrix-producing myofibroblasts.
The study provides novel molecular insights into the biological activity
of PB and in principal opens new avenues for the implementation of novel
anti-fibrotic therapies. However, the study has still some limitations.
In particular, the authors investigated the anti-fibrotic activities
only in in vitro and in vivo models of ongoing hepatic
fibrogenesis showing that the drug efficiently impacts disease
initiation and progression. It will be essential in future studies to
prove the curative effectiveness in suitable models. Moreover, previous
biological evaluation of different physalins in a panel of ten human and
murine cancer cell lines demonstrated that PB has a broad cytotoxic
activity towards most of the cell lines tested with a half-maximal
inhibitory concentration (IC50) in the range of 0.6
µM-2.7 µM (Fang et al., 2003). In the present study, Zhu and colleagues
determined an IC50 of 5 µM for the immortalized human
HSC line LX-2 and used in their animal experiments concentrations of 1-5
mg/kg body weight that were applied intraperitoneally for 14 (bile duct
ligation model) or 28 consecutive days (carbon tetrachloride model). The
concentrations used in luciferase reporter assays and cell apoptosis
testing were in the range of 0.25-1.0 µM, concentrations that might be
already toxic to other cell types. Moreover, inhibition of GLI1 by PB
might have several unwanted side effects. GLI1 is a transcriptional
effector of the Hedgehog (Hh) signaling pathway playing key roles in the
development and homeostasis of many organs and tissues. Transcriptional
suppression, aberrant posttranslational modification such as
hyper-acetylation, or untargeted trafficking of this zinc finger protein
will ultimately influence gene transcription and transcriptional output
of the Hh pathway. Moreover, the Hh-Gli pathway is relevant for
stimulating cell divisions in undifferentiated progenitor cells and for
committing cells to a specific fate.
Effective anti-fibrotic therapies should include the attenuation of
excessive matrix synthesis and deposition, the replacement of
dysfunctional liver tissue, and the restoration of the original tissue
architecture. Liver regeneration and remodeling is a well-coordinated
process requiring increased cell proliferation and differentiation.
Genetic cell lineage-tracing approaches have shown that damaged
hepatocytes and biliary epithelial cells are renewed from common liver
progenitor cells and liver repair requires the activity of immune cells.
Therefore, it will be of fundamental importance to test if the
beneficial effects of PD in preventing extracellular matrix synthesis
interfere with the dynamic and adaptive changes necessary in during the
process of reconstitution of functional liver tissue.
In sum, the current work has identified a new drug candidate effective
in experimental hepatic fibrosis. It will now be necessary to perform
well-designed safety and efficacy studies. Future work will show if the
observed experimental anti-fibrotic activities of thisseco -steroid can be factually translated to the clinic.
Funding : The author is supported by grants from the German
Research Foundation (projects WE2554/13-1 and W2554/15-1). The funder
had no role in the design of this comment or in the decision to publish
it.
Conflict of interest statement : The author has nothing to
declare.