Graphical
Abstract
Keywords: Liver fibrosis; picroside Ⅱ; M1 macrophage; hepatic
stellate cell; natural killer cell; neutrophil; CXCL16.
Abbreviation: ALT, alanine aminotransferase; AST, aspartate
aminotransferase; AKP, alkaline phosphatase;
CL, clodronate liposome; Col1a1,
collagen type I alpha I chain;
Csf3r, colony-stimulating
factor 3 receptor; CM, culture medium; CXCL16,
C-X-C motif chemokine ligand 16;
CXCR6, C-X-C motif chemokine receptor 6; ECM,
extracellular matrix; GZMB, granzyme
B; HSC, hepatic stellate cell;
IFN-γ, interferon-gamma; IL-2, interleukin-2; JAK1, Janus kinase 1;
Mdr2, multidrug resistance protein 2;
MMP2, matrix metallopeptidase 2;
MPO, myeloperoxidase; NETs, neutrophil extracellular traps; PSC, primary
sclerosing cholangitis; PIC Ⅱ,
picroside Ⅱ; STAT, signal transducer
and activator of transcription; TBA, total bile acid; TGF-β,
transforming growth factor-β; Timp1, tissue inhibitor of
metalloproteinase 1; TRAIL, tumor necrosis factor-related
apoptosis-inducing ligand; WGCNA, weighted gene co-expression network
analysis; α-SMA, α-smooth muscle actin.
Introduction
Hepatic fibrosis is a complex pathological process of recurrent
inflammation induced by various hepatic injuries and is characterized by
fibrous scar formation, due to the dysregulated overproduction and
gradual excessive accumulation of excessive extracellular matrix (ECM)
(Parola & Pinzani, 2019). If get out of control, this process may
continue to deteriorate severe liver diseases such as cirrhosis and
hepatocellular carcinoma. Throughout the entire process, inflammatory
reactions triggered by hepatocellular injury or necrosis of hepatic
parenchymal or non-parenchymal cells are the major risk factors for the
initiation and development of liver fibrosis, since the infiltration of
inflammatory immune cells and their released chemotactic peptides may
interact with each other and finally amplify the reaction of hepatic
stellate cell (HSC) activation (Ma et al., 2017). To break through the
bottleneck of anti-fibrosis drug development, ocaliva, an agonist of the
nuclear receptor farnesoid X receptor (FXR) that emerged as a vital
regulator in hepatic fibrosis, has been approved by the FDA for the
treatment of biliary fibrosis. However, its side effects including but
not limited to, pruritus, fatigue, abdominal pain, immunologic
derangement, dizziness, and even in severe cases, acute liver failure,
have restricted its popularization and application (Shah & Kowdley,
2020). Hence, there is an urgent need to explore the pathogenesis and
specific targets of novel drugs for alleviating liver fibrosis.
The deficiency of multidrug resistance protein 2 (Mdr2) protein, a
crucial transport protein closely associated with bile secretion, is
reported to result in the accumulation of toxic bile acids and
subsequent hepatocyte and cholangiocyte injuries, effectively mimicking
the clinical pathological characteristics in primary sclerosing
cholangitis (PSC) (Liu et al., 2020; Ma et al., 2017). Additionally, the
Mdr2 knockout (Mdr2-/-) mouse is a well-known fibrotic
mouse model and has been widely used for investigating the pathogenesis
of liver fibrosis and evaluating drug efficacy (Liu et al., 2019).
Targeting inflammatory pathways or influencing immune cells in
anti-fibrotic therapies is promising in recent years. Especially, M1-
and M2-like monocyte-derived macrophages have been demonstrated to be
tightly associated with sclerosing cholangitis in
Mdr2-/- mice by means of chemokines like
CCL2-CCR2/CCR5 axis and FXR-related pathways, reminding us that abnormal
changes of macrophages are not only features but also indispensable
initial participants of hepatic fibrosis (Guicciardi et al., 2018; Shi
et al., 2022). Gevitha et al. focused on how the absence of
interferon-gamma (IFN-γ)-mediated signaling shaped the production of
chemokines and immune cell recruitment, and ultimately influenced liver
fibrotic in Mdr2-/- mice model. They also confirmed
that the increase of IFN-γ promotes the expression of granzyme B (GZMB)
and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) from
CD8+ T and natural killer (NK) cells, further
resulting in the inhibition of activated hepatic stellate cells (aHSCs)
and the remission of liver fibrosis (Ravichandran et al., 2019).
Besides, low doses of interleukin-2 (IL-2) upregulated the expression of
CD39 in T regulatory cells to inhibit the hepatic CD8+lymphocyte proliferation, further leading to diminished fibrosis in
Mdr2-/- mice (Taylor et al., 2018). However, the
potential mechanisms of pathological transformation from hepatic
inflammation to fibrosis and complex crosstalk between multiple immune
cells especially for macrophages and NK cells that lead to hepatic
fibrosis in Mdr2−/− mice have not been fully
elucidated.
Picroside Ⅱ (PIC Ⅱ), one of the
representative active components of the Picrorhizae rhizoma , has
been demonstrated to exert a therapeutic potential on
ischemia/reperfusion injury, liver damage, and organic cancer metastasis
through inhibiting inflammation, oxidative stress and angiogenesis (Li
et al., 2020; Ma et al., 2020). In recent years, researchers have
gradually turned their attention on the hepatic protective role and
immunoregulative function of PIC Ⅱ. In the alpha-naphthyliso
thiocyanate-induced cholestasis mice model, PIC Ⅱ significantly
activated FXR to regulate the activities of bile acid efflux
transporters and bile acid metabolizing enzymes in livers (Li et al.,
2020). Meanwhile, bile acids were reported to have a potential effect in
regulating the whole-body immune system via influencing the
balance of gut microbiome or inducing signal transduction as messengers
(Duan et al., 2022), which reminds us PIC Ⅱ might possess a direct
influence on disparate immune cells in the liver. Meanwhile, although
there is no direct regulatory effect on immune cells, PIC Ⅱ could
alleviate apoptosis and inflammation in sepsis by decreasing the
expression of toll-like receptor 4 (TLR4) and tumor necrosis
factor-alpha (TNFα) in an ischemic injured rat model (Huang et al.,
2016). Of note, PIC Ⅱ also inhibited NF-κB activation and achieved the
alleviation of inflammatory response and apoptosis in endotheliocytes by
regulating the sirtuin 1 (SIRT1)/lectin-like oxLDL receptor-1 (LOX-1)
signaling pathway (Wang et al., 2019). However, whether PIC Ⅱ was able
to protect the liver from Mdr2 deficiency-induced liver fibrosis and the
potential interaction between the PIC Ⅱ and different immune cells in
fibrotic livers still needs to be clarified.
In the current study, we aimed to systematically investigate the
pathological role of multiple immune cells involved in Mdr2
deficiency-induced liver fibrosis and to explore whether PIC Ⅱ could
achieve its hepatoprotective effects by influencing the function of and
interaction between different immune cells in the fibrotic liver. Our
findings not only evaluate how different immune cells regulate the
immune microenvironment and HSCs activation during the development of
liver fibrosis, but also offered a kind of natural ingredients as a
novel therapeutic option for removing the bottleneck of antifibrosis.
Methods and materials