Pharmacological Research 170 (2021) 105739

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Pharmacological Research
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Review

Hepatic stellate cells role in the course of metabolic disorders development

– A molecular overview
Nabila Bourebaba a, b, Krzysztof Marycz a, b, *
a
Department of Experimental Biology, Faculty of Biology and Animal Science, Wrocław University of Environmental and Life Sciences, Norwida 27B, 50-375 Wrocław,
Poland
b
International Institute of Translational Medicine, Jesionowa 11, 55-114, Malin, Wisznia Mała, Poland

A R T I C L E I N F O A B S T R A C T

Keywords: Fibrosis is characterized by an abnormal accumulation of extracellular matrix (ECM) constituents in the liver
Hepatic stellate cells parenchyma that lead to hepatic cirrhosis. After liver injury, the hepatic stellate cells (HSCs) undergo a response
Hepatic fibrosis called "activation", transforming the cells into proliferative, fibrogenic and contractile myofibroblasts, repre­
Metabolic disorders
senting the main collagen-producing cells in the injured tissue. Activated HSCs are considered as pro-
CGKI
inflammatory cells producing cytokines and several hepatomatogens; they are additionally involved in the
PTP1B
recruitment of Kupffer cells, circulating monocytes and macrophages through the production of chemokines.
Moreover, HSC have been proposed as being involved in the development of insulin resistance mainly mediated
by their inflammatory properties, which undeniably links their activation to the development of diabetes and
Non-alcoholic fatty liver disease. In addition, when the liver is injured, a complex interaction between hepa­
tocytes and HSCs occurs, inducing mitochondrial dysfunction, which contributes to the accumulation of fats in
hepatocytes that trigger to liver lipotoxicity. These mechanisms underlying the activation of HSC suggest their
major role in the development of metabolic disorders. It turns out that several molecules including MicroRNAs
and proteins have the ability to inhibit the activation and the proliferation of HSCs, which makes them inter­
esting therapeutic targets for the subsequent management of metabolic conditions. This review focuses on the
mechanisms and molecular pathways underlying the initiation and onset of metabolic disorders following HSCs
activation, as well as on molecular therapeutic targets, which could limit their fibrogenic transdifferentiation and
therefore improve the liver condition in the course of metabolic imbalance.

1. Introduction interactions, cytokine networks and sinusoidal blood flow modulation,

and participate in hepatic immune tolerance through their immuno­
The knowledge and the understanding of hepatic cell function and modulatory properties [4]. On the other hand, these cells are involved in
biology are essential in the development of appropriate therapeutic the initiation of hepatic fibrosis, which is a process that disrupts the
approaches for the management of liver-related diseases. Each different homeostasis of ECM and leads to a build-up of connective tissue in the
cell type plays a specific role in the liver. Parenchymal cells, mainly liver following their activation into fibrogenic myofibroblast-like cells
hepatocytes, constitute 80% of the total hepatic volume and are [5].
responsible for the majority of hepatic functions; among them, hepatic Non-alcoholic fatty liver disease (NAFLD) is one of the most
stellate cells (HSCs) formerly called Ito cells, lipocytes, or fat storing commonly responded to liver diseases and encompasses a wide range of
cells were recently recognized as being among the key players in liver downstream pathologies, ranging from simple steatosis with steatohe­
metabolic dysfunctions [1]. Normally, these cells appear as the site of patitis to advanced fibrosis and cirrhosis [6]. In NAFLD; insulin resis­
retinoids (vitamin A) storage [2,3]. HSCs also contribute in the main­ tance (IR), oxidative stress and subsequent lipid peroxidation,
tenance of healthy liver architecture by regulating the replacement of its proinflammatory cytokines, adipokines and mitochondrial dysfunction
extracellular matrix (ECM). Stellate cells exert a key role in cell-to-cell contributes to it appearance, directly by increasing de novo lipogenesis

* Corresponding author at: Department of Experimental Biology, Faculty of Biology and Animal Science, Wrocław University of Environmental and Life Sciences,
Norwida 27B, 50-375 Wrocław, Poland.
E-mail address: krzysztof.marycz@upwr.edu.pl (K. Marycz).

https://doi.org/10.1016/j.phrs.2021.105739
Received 11 April 2021; Received in revised form 7 June 2021; Accepted 19 June 2021
Available online 23 June 2021
1043-6618/© 2021 Elsevier Ltd. All rights reserved.
N. Bourebaba and K. Marycz Pharmacological Research 170 (2021) 105739

and indirectly by increasing free fatty acid (FFA) flux to the liver via also the tumor necrosis factor α (TNF-α). Several studies have demon­
decreased inhibition of lipolysis, leading to adipose tissue (AT) strated its link with the inflammatory state and the development of in­
dysfunction which, is characterized by decreased production of adipo­ sulin resistance; because the stimulation of TNF-α leads to the
nectin and greater release of leptin, conditioning a low grad phosphorylation of the serine of the substrate 1 of the insulin receptor
pro-inflammatory microenvironment, what supports an association be­ (IRS-1) which strongly decreases the signalling mediated by insulin. It
tween NAFLD and metabolic syndrome. An increase in circulating FFA therefore contributes to insulin resistance in chronic inflammation [13].
enhances their absorption by the liver, leading to an accumulation of Several studies on the mechanisms that can counteract the activation
lipids in the liver cells. This excessive accumulation of toxic lipids is of stellate cells refer to various molecules, most of which are MicroRNAs.
associated with dysfunction of organelles such as the endoplasmic re­ Indeed, the over expression of the latter seems to down-regulate the
ticulum (ER) and mitochondria, which increases the secretion of activation of HSCs. However, Micro RNAs are not the only antifibrotic
pro-inflammatory cytokines via the nuclear factor -β (NF-κβ) pathway. molecules, as some proteins including cGMP-dependent protein kinase I
At this stage, the repair capability of the liver tissue is exceeded, trig­ (cGKI) have been proposed as promising therapeutic targets for the
gering to the development of fibrosis [7]. This is consistent with the fact regulation of hepatic fibrosis. In this review, we will discuss the exact
that 80% of people with NAFLD have at least one risk factor for devel­ mechanism underlying stellate cells activation in the liver and its
oping metabolic syndrome and 33% have all of the features. However, crosstalk with lipotoxicity, inflammation and insulin resistance devel­
obesity is found in 30–100% of subjects with NAFLD; in 304 patients opment, in order to identify possible new therapeutic targets for meta­
with NAFLD without diabetes mellitus, the prevalence of metabolic bolic disorders management.
syndrome increased from 18% in people of normal weight to 67% in
obese people. Indeed, the presence of multiple metabolic disorders such 2. Stellate cells’ structure and function
as diabetes mellitus, obesity, dyslipidaemia and hypertension is associ­
ated with severe and potentially progressive liver disease such as fibrosis The liver parenchyma is made up of several types of cells which are:
[8]. Fibrosis, is characterized by the abnormally high accumulation of hepatic parenchymal cells (epithelial component); sinusoidal endothe­
ECM constituents in the hepatic parenchyma and can lead to hepatic lial cells (characterized by the presence of prominent pores); hepatic
cirrhosis. After liver injury, stellate cells undergo a response through stellate cells (perisinusoidal mesenchymal cells); Kupffer cells (tissue
their activation, which corresponds to the transformation of quiescent residing macrophages) and other cellular subtypes including pit and
cells into proliferative, fibrogenic, and contractile myofibroblasts dendritic cells. Hepatic parenchymal cells are relatively large cells with
(activated HSCs). Thus, the HSCs constitute the main a volume of about 5000 µm, its cellular elements are organized in and
collagen-producing cells in the injured tissue [9]. The HSCs activation around the sinusoids, and the perisinusoidal space of Disse separates the
process is complex; typical features include loss of vitamin A, the epithelium (parenchymal cells) from the complex of HSCs and sinusoidal
acquisition of stress bundles, the development of a prominent rough endothelium. HSCs are pericytes of liver and are localized in the space
endoplasmic reticulum, a striking increase in the production and between parenchymal cells (PC) and endothelial cells (EC) of the hepatic
secretion of extracellular matrix related proteins namely types I, III and lobule [14]. In normal liver, stellate cells are quiescent and make up
IV collagens, fibronectin, laminin and proteoglycans. Moreover, during about 1.4% of total liver volume and are present at a ratio of about 3.6–6
the activation of HSCs, the latter significantly proliferate, thus releasing cells per 100 hepatocytes (1:20). Stellate cells (from Latin stella, which
pro-inflammatory cytokines, matrix-degrading enzymes and their in­ means star) usually exhibit a star-like configuration due to their den­
hibitors, and exhibit paracrine signalling with a variety of other cells dritic cytoplasmic processes which coat the adjacent endothelial cells in
such as inflammatory cells [2]. In addition, activated stellate cells a partial manner, such as the astrocytes around the terminal cerebral
(AHSCs) are considered as being themselves pro-inflammatory cells, vessels [15]. The localization and the long cytoplasmic HSC favor their
since they produce a wide range of cytokines and chemokines and interactions with neighboring cell types. HSCs are in direct contact with
secrete several hepatomatogens such as hepatocyte growth factor endothelial cells, and interact with parenchymal cells via microspins
(HGF), interleukin 6 (IL-6), interleukin 8 (IL-8), monocyte chemo­ from cytoplasmic processes. The interaction of cells with ECM compo­
attractant protein-1 (MCP-1); they are furthermore involved in the nents affects various cellular functions, including cell differentiation,
recruitment of Kupffer cells, monocytes and macrophages from the cir­ migration, proliferation as well as survival [14] (Fig 1).
culation into the liver region affected by chemokine overproduction, Vitamin A has the role of regulating various cellular activities such as
these properties promote the proliferation of liver progenitor cells and proliferation, differentiation, morphogenesis and tumorigenesis. HSCs
hepatocytes, stimulate angiogenesis in the wounded area and assist in exhibit large perinuclear lipid droplets, which serve as the primary
the recruitment of hematopoietic stem cells [10,11]. storage site for vitamin A and are essential in the regulation of retinoic
Hepatic insulin resistance is significantly implicated in diseases such acid homeostasis; it stores up to 80% of the whole body’s vitamin A in
as type II diabetes and NAFLD related to metabolic syndrome. However, the form of retinyl palmitate in the cytoplasmic lipid droplets. In addi­
at the hepatocyte cells level, insulin resistance is caused by a combina­ tion, the concentration of circulating vitamin A in the blood is regulated
tion of pathological alterations including hyperglycaemia and hyper­ by HSCs and this, through the process of endocytosis mediated by spe­
insulinemia, the formation of end products of advanced glycation, the cific receptors; however, cells absorb circulating retinol in the form of a
increase in free fatty acids, oxidative stress and altered adipocytokine retinol complex by a specific binding protein called retinol binding
profiles. Hepatic insulin resistance causes changes in glucose meta­ protein (RBP); likewise, retinol circulates in the blood through its high
bolism, which interferes with cell survival and proliferation. On the affinity binding to RBP. The concentration of RBP – retinol (blood holo-
other hand, the production of fatty acids by hepatocytes remains stim­ complex) is regulated in the physiological range (1.4 mM) by HSCs,
ulated by compensatory hyperinsulinemia, resulting in steatosis. How­ which contain high levels of cellular proteins involved in the metabolism
ever, in the liver, the activation of sinusoidal endothelial cells, Kupffer of retinoid, like the cytosolic retinol binding protein (CRBP) and
cells and stellate cells will induce chemo-attraction with the inflam­ etherifying retinol bound to the membrane enzymes. These proteins
matory cells, and the activation of HSCs will lead to a fibrogenic mainly optimize the intracellular retinol transport and maintenance
response; upon liver injury, inflammatory cells are recruited into the retinol ester deposits. When distributed to cells, for it several functions,
liver; resulting in the amplification of intrahepatic inflammation and including the reform of the complex with RBP, its return to the blood­
hepatocyte damage [12]. Chronic inflammation resulting from obesity is stream. This is why the HSCs are important for the regulation of vitamin
closely related to hepatic insulin resistance. Indeed, the appearance of A homeostasis [15].
the latter coincides with the increase in the levels of interleukins 6 and 8 HSCs also produce small amounts of ECM components, such as
(IL-6 and IL-8), the C-reactive inflammatory marker protein (CRP) and laminin and type IV collagen to subsequently form the membrane

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N. Bourebaba and K. Marycz Pharmacological Research 170 (2021) 105739

Fig. 1. Representation of the localization of stellate cells in the healthy liver. A: diagram of the hepatic sinusoid demonstrating the relative orientation of stellate cells
(in blue, indicated with arrows) within the sinusoidal architecture. B: higher resolution drawing of stellate cells situated within the subendothelial space [16].

basement. Upon exposure of stellate cells to soluble factors (lipid per­ (ET-1) is the major contractile stimulus for HSCs and is activated by a
oxides, products of damaged hepatocytes and signals from Kupffer and converting enzyme; once activated, these cells produce nitric oxide (NO)
endothelial cells), HSCs lose their contained lipid (retinyl palmitate), which is the physiological opponent of ET-1. The contractile activity of
and undergo a transition to myofibroblast-like cells. This transition re­ HSCs reflects the relative balance between these two antagonistic mol­
fers to HSCs activation and trigger to the subsequent production of large ecules, when this balance is lost and is in favour of endothelin, it causes
amounts of ECM in an accelerated manner [17] (Fig 2). an increase in contraction as hepatic disease progresses [21]. Activation
consists of two major phases:
3. Role of hepatic stellate cells in fibrogenesis
• The initiation (also called the "pre-inflammatory stage"); during
Hepatic fibrosis is an increased but reversible accumulation of initiation; the gene expression of HSCs phenotype changes, making
extracellular matrix components in the liver parenchyma in response to them sensitive to other cytokines and stimuli that appear after injury.
chronic damage and injury. These fibrous changes appear with chronic Initial paracrine stimulation, results in early activation, and changes
liver disease and are caused by a loss of homeostatic mechanisms that, in the surrounding extracellular matrix. Platelet-derived growth
under normal physiological circumstances (healthy liver), control factor (PDGF) signalling is one of the key factors in HSCs activation;
fibrogenesis which is definite as increased synthesis and deposition of first of all, PDGF binds its receptors and their subunits dimerize and
extracellular matrix. In the chronically injured liver, the excessive cre­ this is accompanied by a subsequent phosphorylation of tyrosine
ation of matrix is continuously stimulated. When the liver is exposed to residues of the intracellular domain, activating the Ras-MAPK
acute injury, the architectural changes in the liver are transient and pathway, the signalling of PI3K-AKT/PKB pathway and the activa­
reversible; on the other hand, in the event of chronic injury, the hepatic tion of the protein kinase C (PKC) family by mobilizing intracellular
parenchyma is gradually replaced by scar. It is well known that one of calcium ions.
the liver characteristics lies in its remarkable capacity for regeneration, • The perpetuation; after the HSCs activation, perpetuation results
even during persistent lesions; this is why cirrhosis often appears slowly. from the effects of these stimuli on maintaining an activated
Stellate cells are the main effector cells during hepatic fibrogenesis, phenotype. Sustained activation involves at least seven changes in
orchestrating ECM deposition in normal and fibrous liver. Activation of cellular behaviour: proliferation, chemotaxis, fibrogenesis, contrac­
HSCs to myofibroblast may be caused by multiple injuries to the liver, tility, matrix degradation, loss of retinoids, and release of white
including viral hepatitis, toxins, (non) alcoholic steatohepatitis and blood cells (WBC) chemoattractant/cytokine. These changes result in
autoimmune diseases [19,20]. Furthermore, expression of the three increased accumulation of extracellular matrix. During this stage;
transformative growth factor-h (TGF-h) isoforms, the most potent pro-inflammatory, profibrogenic and promyogenic stimuli are
fibrogenic cytokine described for HSCs, Platelet Derived Growth Factor released and act in an autocrine and paracrine manner [20].
BB (PDGF-BB), the most potent mitogen for HSCs, and Platelet Derived
Growth Factor (PDGF) receptors and its receptors are increased after 4. Implication of stellate cells activations in the initiation of
activation of HSCs. Activation of hepatic stellate cells causes cell growth liver metabolic disorders
contractility, which causes portal pressure to increase by both con­
stricting individual sinusoids and contracting the liver. Endothelin-1 Non-alcoholic fatty liver disease (NAFLD) is now recognized as a
major global cause of liver disease increasing the risk of developing
obesity and type 2 diabetes. NALFD encompasses a spectrum of disease
ranging from simple steatosis to non-alcoholic steatohepatitis (NASH),
passing through fibrosis, cirrhosis and finally hepatocellular carcinoma
[22]. Metabolic factors are important in the development of fibrosis
associated with NASH. These factors are part of the "multiple side ef­
fects" responsible for liver damage during NASH. The main
pro-fibrogenic protagonists, such as hepatic stellate cells and Kupffer
cells are activated by insulin resistance, apoptosis and local inflamma­
tion. Studies showed that oxidative stress, cell death, inflammation as
well as liver fibrosis are mainly due to the accumulation of fatty acids
and changes in the composition of membrane phospholipids; however,
increased saturation of the membrane can profoundly disrupt cell ho­
meostasis by altering the function of receptors, channels and membrane
transporters, and related signalling pathways that strongly contribute to
the development of metabolic disorders as insulin resistance and
Fig. 2. Activated stellate cells [18].

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elevated blood pressure [23]. 4.2. Inflammation

The liver plays a central role in the onset of metabolic disturbances.
Indeed, white adipose tissue (WAT) causes an increase in the flow of As seen previously, HSCs are the main effector cells in hepatic
fatty acids (FA) to the liver, promoting the deposition of fat in hepato­ fibrosis. However, recent studies have elucidated the fundamental role
cytes. Hepatic steatosis being linked to insulin resistance and the pro­ of hepatic stellate cells in hepatic immunology. Indeed, of their role in
gression of atherosclerosis, is a factor aggravating the pathogenesis and the storage of vitamin A, which is essential for the proper functioning of
progression of the metabolic syndrome and its associated disorders [24]. the immune system; these pericytes represent a versatile source of many
soluble immunologic active factors, including interleukin 17 (IL-17) and
4.1. Lipotoxicity chemokines and can either act as antigen presenting cells (APC), or
exhibit autophagy activity. In addition, they respond to numerous
Lipotoxicity refers to the cellular toxicity that occurs during an immunological triggers via toll-type receptors (TLR4 and TLR9) and
abnormal accumulation of fat. The aggregation of fat in the liver (fatty transmit signals through pathways and mediators initially found in
liver) is a morphological feature of a myriad of conditions. Adipose immune cells, including activation of the inflammasome (like the NOD-
tissue is a connective tissue containing fat cells, called "adipocytes" and like receptor family, pyrin domain containing 3 (NLRP3) which is an
serves as the primary depot of excess calories and undergoes many activator protein and interacts with the Caspase Recruitment Domain
functional changes that characterize the obese fat cell and insulin Family Member 8 (CARD8) gene and thus regulates caspase 1 which
resistance. These adipocyte changes include enhanced lipolysis with allows the activation of interleukin 1 beta (IL-1β)) [30]. It is also found
release of free fatty acids (non-esterified fatty acids (NEFA)) and the that HSCs express human leukocyte antigens (HLA-I and HLA-II), lipid
release of adipokines and inflammatory cytokines [25]. After activation presenting molecules (CD1b and CD1c) and the factors involved in the
of HSCs mediated by the release of inflammatory factors (cytokines) and activation of T lymphocytes (CD40 and CD80). Exposure of HSCs to
reactive oxygen species (ROS) synthesized by damaged hepatocytes; an pro-inflammatory cytokines regulates these molecules. According to
interaction between them and hepatic stellate cells occurs inducing previous findings, activated cells strongly express both HLA-II and CD40
progression of steatohepatitis, causing mitochondrial dysfunction, molecules, confirming that activated HSCs can be considered as
which contributes to the accumulation of fat in the hepatocytes [26]. On antigen-presenting cells (APCs) during human fibrogenesis. Moreover,
the other hand, NAFLD is associated with an alteration of the mito­ activated HSCs express antigen presenting molecules, internalize mac­
chondrial beta oxidation. Indeed, studies have shown that acute or romolecules, and modulate T cell proliferation [31]. HSCs can modulate
chronic fatty deposits in the liver, is associated to lipid peroxidation. inflammation in the liver through several mechanisms, including the
However, it turns out that the increase in lipid peroxidation is correlated expression of chemokines. The secretion of the monocyte chemo­
with the severity of the steatosis [27]. Several studies indicate that attractant protein 1 (MCP-1) by HSCs accounts for most of the chemo­
increased saturation of phospholipid membranes may trigger a disrup­ tactic activity of monocytes. Several researches reported that activated
tion of cellular mechanisms; thus, the activated signalling pathways hepatic stellate cells contribute to the secretion of MCP-1; in fact, in
which result from it generate hepatic cells apoptosis and insulin resis­ these cells, CD40 activates the main pro-inflammatory pathways
tance, which leads to complications linked to metabolic disorders and through TNF receptor-associated factor 2 (TRAF2) and inhibitor of the
hepatic lesions [23]. As previously described, the loss of retinol (vitamin beta subunit of nuclear factor kappa-B kinase (IKK2) which stimulates
A) is one of the key events in the activation of HSCs, moreover, the the secretion of IL-8 and MCP-1 chemokines; also, it has been shown that
retinol derivatives that are found in the body circulate in the form of MCP-1, mediates most of the chemotactic activity of HSC monocytes
palmityls or stearyls, making retinol metabolism inseparable from lipid [32,33]. The pro-inflammatory cytokines, IL-1, TNF-α as well as the
metabolism. Levels of triacylglycerol (TAG) containing long poly­ Gamma interferon (IFNγ) are the most powerful stimuli of the MCP-1
unsaturated fatty acids (PUFAs) are significantly increased in activated protein and therefore rapidly induce an increase in the expression of
HSCs [28]. Effectively, hepatic stellate cells are characterized from other its mRNA. Other studies have demonstrated the ability of HSCs to pro­
hepatic cell types by the presence of abundant and highly specialized duce other ELR-CXC-type chemokines as well as chemoattractant neu­
lipid droplets, which play crucial role in the storage of the majority of trophils. In addition, HSCs play a key role in the activation of nuclear
hepatic retinoid reserves. These lipid droplets are made up of other factor-kB. Indeed, when the HSCs are activated, significant over­
non-retinoid lipids (triglycerides, cholesterol and its esters, expression of NF-kB mediated by cytokines IL-1 and TNF-α occurs. The
non-esterified free fatty acids and various phospholipids). During their fact that hepatic stellate cells is responsible for the secretion of
activation, HSCs lose their characteristic lipid droplets in order to pro­ numerous chemokines demonstrates the role of activated HSCs in the
duce the components of EMC. The retinoids that the lipid droplets induction of an inflammatory reaction in the liver as well as the crosstalk
release during activation can be used for the formation of retinoic acid. between inflammation and hepatic fibrosis [32]. Inflammation is
previous investigation found that during HSC activation in mice, very generally associated with insulin resistance, and obesity which is asso­
potent bioactive non-retinoid lipid species are synthesized. Indeed, these ciated with a chronic low-grade inflammatory condition, suggesting that
lipids as well as the long-chain polyunsaturated fatty acids (PUFA), inflammation may be a potential mechanism by which obesity leads to
endocannabinoids, related N-acylethanolamides (NAEs) and ceramides, insulin resistance. Inflammation induced by obesity is a key component
are formed during the degradation and remodelling of the HSCs lipid in the pathogenesis of insulin resistance and metabolic syndrome. Since
droplets proving that the total concentrations of free fatty acids during the sources of cytokines in insulin resistance states are the insulin target
the activation stage of HSCs are significantly elevated, where palmitic, tissues themselves, i.e. adipose and liver, these pro-inflammatory cyto­
oleic, palmitoleic and stearic acids are among the most abundant kines indeed induce IR in liver by inhibiting insulin signalling trans­
circulating free fatty acids. These data indicate that the degradation of duction. Obesity causes hepatic inflammation activating the
lipid droplets associated with the activation of hepatic stellate cells is inflammatory pathway resulting from either steatosis and/or increased
not only a passive lipolysis, the sole purpose of which is to supply the cell hepatocyte stress pathway responses leading to hepatocytes inflamma­
with inert lipid content, but rather an active process aiming at the tion. Kupffer cells are associated to activated macrophages, they release
transformation and the redistribution of highly bioactive lipid species in locally acting cytokines which further aggravate inflammation and he­
abundance, suggesting that the activation of hepatic stellate cells in­ patic insulin resistance; thus, the inflammatory cells will be secreted in
duces an overproduction of fatty acids in the liver which, after accu­ the damaged liver and the profibrogenic cytokines (transforming growth
mulation, would lead to hepatic lipotoxicity [29]. factor (TGF-1), interleukin (IL-6 and IL-1), and TNF-α) will be secreted
[34]. Adipocytes, which are cells present in adipose tissue and specialize
in storing lipids, have the capacity to produce and secrete factors called

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N. Bourebaba and K. Marycz Pharmacological Research 170 (2021) 105739

"adipokines", pro-inflammatory cytokines and chemokines, such as and not in other hepatic cells, which means that cGKI has a specific
MCP-1. Additionally, obesity tends to alter adipocyte function by function of stellate cells, thus agreeing with the hypothesis that the
increasing the secretion of pro-inflammatory factors, including MCP-1, absence of cGKI triggers the activation of HSCs leading to liver inflam­
resulting in the recruitment of monocytes/macrophages into adipose mation and insulin resistance. Furthermore, the expression of (cGKI) in
tissue [35]. smooth muscle correlates with the elevation of macrophages in the liver,
which was inducing hepatic inflammation, and consequently provokes
4.3. Insulin resistance insulin resistance; thus, the absence of cGKI triggers activation of HSCs
leading to liver inflammation and subsequently insulin resistance [11].
Chronic inflammation occurring as a result of obesity is an essential The role of cGKI in the regulation of glucose homeostasis has been
part of understanding the pathogenesis of insulin resistance and meta­ elucidated showing that the lack of cGKI severely affected the secretion
bolic syndrome. However, insulin resistance is closely linked to the in­ of glucagon and increased the level of fasting glucose. These results were
crease in the levels of synthesis of IL-6 and IL-8, the C-reactive subsequently supplemented and other researchers reported that genetic
inflammatory marker protein (CRP) as well as TNF-α which is a pro- suppression of cGKI in some cells resulted in a change in metabolic
inflammatory cytokine secreted by monocytes and macrophages and phenotype, which included liver inflammation and fasting hyperglyce­
which acts on coagulation, endothelial function and lipid metabolism. mia. Indeed, the suppression of cGKI in hepatic stellate cells affects
Activation of its receptor leads to stimulation of nuclear factor-kappa B hepatic metabolism via a paracrine mechanism which involves an in­
(NFκB) signalling via the Ikkb kinase. Several studies have indeed crease in macrophages and IL-6 [39]. Thus, shedding light on the link
demonstrated the link between the increase in TNF-α levels in the in­ between the activation of HSCs and the development of insulin resis­
flammatory state and the appearance of insulin resistance; indeed, tance. Indeed, it would seem that the activation of the stellate cells
stimulation of TNF-α leads to serine phosphorylation of Insulin receptor would induce hepatic insulin resistance indirectly through
substrate 1 (IRS-1) which attenuates its ability to transduce insulin- inflammation.
mediated cellular events. Furthermore, TNF-α succeeded in affecting
insulin signalling not only through IRS-1, but also by reducing the gene 5. Potential therapeutic targets for the modulation of stellate
expression of IRS1, the 4 ’glucose transporter (Glut4) and the nuclear cells activation
receptors of Peroxisome proliferator-activated receptors (PPARγ)
lowering insulin-stimulated glucose uptake capabilities. This therefore Liver fibrosis is a progressive liver disease, that is why it is important
contributes to insulin resistance in chronic inflammation [13,34]. Liver to target and reduce the disease at an early stage before it later develops
cells are not just passive witnesses in their changes; but they are also into cirrhosis or cancer of the liver. There is currently no direct drug
effector cells that can exacerbate IR in hepatocytes by increasing the aimed at alleviating or reversing liver fibrosis, so it would be wise to take
secretion of cytokines such as TNF-α and IL-6 [12]. The low-grade a serious look at this global health problem. There are effective antiviral
inflammation associated with metabolic disorders is responsible for agents that could target the underlying causes of the hepatitis B fibrotic
the decrease in insulin sensitivity. Indeed, the expression of certain in­ outcome and C, but not for other aetiologies of this liver disease (alco­
flammatory cytokines being increased, activates several signalling holic and non-alcoholic steatohepatitis, autoimmune diseases, etc.)
pathways involved in the development of insulin resistance [36]. Initial which remain badly resolved. However, a viable therapeutic approach
researches showed first that the pro-inflammatory cytokine TNF-α is emerges which includes HSCs as a target; nonetheless, there are
able to induce insulin resistance. Then, this concept quickly spread currently three approaches, namely apoptosis, senescence and rever­
beyond TNF-α and included, IL-6 and MCP-1 which are produced by sion, but all this remains hypothetical, several molecules of natural
activated hepatic stellate cells [13]. The progression of inflammation is origin (salvianolic acids, oxymatrine, curcumin, tetrandrine, etc.) from
explained by the activation of the nuclear factor-κB pathway (NF-κB) as medicinal plants have been studied based on the traditional remedies
well as the phosphorylation of c-Jun N-terminal kinases (JNK), which formerly used, however nothing is really conclusive to this day [40].
are considered as key factors in the development of metabolic diseases Many cellular changes are associated with the activation of HSCs,
(type 2 diabetes, insulin resistance.) associated with inflammation in the both at the hepatic level (increase in the synthesis of extracellular matrix
liver. In fact, cytokine signalling at the cell surface activate the NF-κB proteins making HSCs fibrogenic, and increase in their proliferation)
pathway regulating the expression of genes that stimulate the immune and at the whole organism level (appearance of complications linked to
response by inducing the expression of intracellular pro-inflammatory metabolic disorders). This is why an effective treatment aimed at
cytokines, which in addition to blood pro-inflammatory cytokines, reducing or inhibiting the activation of HSCs reducing their harmful
induce phosphorylation of JNK responsible for gluconeogenesis and thus effects [21]. The activation of HSCs, plays a crucial role in the patho­
induce insulin resistance. In summary, JNK phosphorylation produces physiology of the process of development of insulin resistance charac­
pro-inflammatory cytokines thus promoting insulin resistance directly; teristic of type II diabetes and metabolic disorders. A study in mice
on the other hand, the activation of NF-κB is also at the origin of the showed that cGMP -dependent protein kinase I (cGKI) deficiency
production of pro-inflammatory cytokines inhibiting insulin signalling (cGKI-KO) exhibited hepatic insulin resistance and impaired glucose
via the activation of the IKKβ/NF-κB and JNK pathways in hepatocytes metabolism, and since cGKI is detected in the HSCs and not in the rest of
and macrophages which promote increased expression of many poten­ the hepatocytes, the cGKI would prove to be a modulator of the acti­
tial markers and mediators of inflammation leading to insulin resistance. vation of the HSCs and of its metabolic consequences. In addition, it
The activation of JNK, promotes the phosphorylation of the insulin re­ appears that a nitric oxide deficiency in the liver could be the cause of
ceptor substrate 1 (IRS-1) (at the serine sites), which results in deregu­ the activation of HSCs and this is due to the alteration of nitric oxide
lation of normal insulin receptor signalling/IRS-1 [13,37]. This is (NO)/cGMP/cGKI signalling [11]. On the other hand, MiRNAs partici­
because IRS-1 phosphorylation at serine 307 (Ser307) occurs in response pate in the control of various biological processes, including prolifera­
to cytokines, inhibiting the association of its tyrosine protein binding tion, differentiation and apoptosis. However, it has been shown that
domain with the receptor subunit at insulin, thereby preventing IRS-1 these molecules are involved in the regulation of the activation of HSCs.
binding to the receptor and insulin-dependent activation of PI 3-kinase For example, the loss of expression of miR-33a inhibits activation of
(PI3K) which plays a key role in insulin’s stimulation of glucose trans­ hepatic stellate cells via activation of the PI3K/Akt and Peroxisome
port, inducing insulin resistance associated with an impairment of IRS-1 proliferator-activated receptors (PPAR-α) pathway; therefore it would
tyrosine phosphorylation and IRS-1-associated PI3K activation [38]. be judicious to explore this avenue as a therapeutic target in the
A study in mice showed that immunostaining carried out on the liver regression of the activation of hepatic stellate cells and of the disorders
for cGMP-dependent protein kinase I (cGKI) was positive in stellate cells which result therefrom [41].

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N. Bourebaba and K. Marycz Pharmacological Research 170 (2021) 105739

5.1. cGMP-dependent protein kinase I (cGKI) fibrosis. Indeed, overexpression of cGKI or its pharmacological activa­
tion decreases the state of fibrosis and this, through guanylate cyclase,
Cyclic GMP regulates cellular mechanisms (proliferation, differen­ which increases cGMP levels activating cGKI. This therefore indicates
tiation and apoptosis) and is involved in several physio pathological that drugs which increase cGKI activity may be promising targets in the
processes (inflammation, angiogenesis, etc.). cGMP is synthesized in regulation of HSCs activation and the subsequent modulation of un­
response to nitric oxide (NO) and natriuretic peptides, which is gener­ derlying metabolic dysfunctions [11] (Fig 3).
ated by three different isozymes by increasing the concentration of
cGMP by activation of soluble guanylyl cyclase (sGC). Most cells contain 5.2. Nitric oxide pathway and Protein Tyrosine Phosphatase 1B (PTP1B)
a protein kinase (cGK) (cGKI α, cGKI β or cGKII), which are targeted by
their distinct amino termini involved in the regulation of different Nitric oxide NO is synthesized from arginine and O2 by nitric oxide
cellular functions [39,42]. In liver, studies reported that immunostain­ synthase (NOS). Arginine can be synthesized by two enzymes that are
ing of cGKI has been shown to be positive in hepatic stellate cells and involved in the urea cycle which is expressed only in the liver (argino­
negative in hepatocytes, suggesting that cGKI exerts a specific and succinate synthetase (ASS) and arginosuccinate lyase (ASL)), starting
crucial function for HSCs; this observation correlates with the fact that from citrulline, which is formed as a by-product of the NOS reaction
the absence of cGKI in liver tissue favours the activation of HSCs, which [43]. The best characterized upstream activator of cGKI is NO, and
causes inflammation and insulin resistance. It turns out that inflamma­ defective NO signalling has been found to be characteristic of a diseased
tory modulators (pro-inflammatory pathways (TNF-α, IL1 and IL6)) liver with hepatic insulin resistance, liver fibrosis and cirrhosis. Under
expressed by activated HSCs play a determining role in mediating the normal physiological conditions, endothelial cells (CE) keep HSCs
effects of cGKI in this cellular activation. However, studies have shown inactivated and quiescent via NO; this therefore, informs us about the
that absence of cGKI leads to the activation of stellate cells by increasing involvement of altered NO/cGMP/cGKI signalling in the activation of
the transcription of alpha smooth muscle actin (α-SMA), which is in fact HSCs [44]. However, endothelial dysfunction is characterized by a
a characteristic marker of activated HSCs; furthermore, cGKI suppres­ change in its phenotype and its ability to produce nitric oxide (NO).
sion has been associated with a loss of receptor activated by peroxisome Many molecular mechanisms are involved in this endothelial dysfunc­
proliferators (PPARγ) expression, which is key mediator in HSCs acti­ tion including phosphorylation of nitric oxide synthase (eNOS); how­
vation and phenotypic alteration, thus maintaining HSCs in a quiescent ever, it was shown that the inhibition of the protein tyrosine
phase and this by suppressing several markers of HSCs activation such as phosphatase 1B (PTP1B) restored vascular relaxation and phosphory­
expression of collagen and alpha smooth muscle actin (alpha-SMA), cell lation of eNOS suggesting its potential interest in the treatment of
proliferation and migration; therefore, the loss of cGKI can be consid­ endothelial dysfunction and thereby the restoration of hepatic NO levels
ered as a crucial event that induces a possible trans differentiation of the allowing the maintenance of HSCs at rest [45].
quiescent stellate cells into myofibroblasts. cGKI downregulation par­ The phosphorylation of the protein tyrosine is a key post-
ticipates also in ECM remodelling, through the overexpressing the ma­ translational mechanism of several cellular processes (proliferation,
trix metallopeptidase (MMP2) that is responsible for the ECM digestion migration, differentiation and apoptosis). In a normal physiological
and remodelling, and by decreasing the transcription of tissue inhibitor state, protein tyrosine kinases (PTK) catalyse the phosphorylation of
of metallopeptidase (TIMP2), which inhibits MMP and activates MMP2 tyrosine; unlike protein tyrosine phosphatises (PTP), which are
and is overexpressed during fibrosis and HSCs activation. Likewise, the responsible for dephosphorylation and the deregulation of phosphory­
lack of cGKI tends also to increase the transcription levels of IL-8 and lation of tyrosine proteins which is associated with pathological states.
TLR-4 which are expressed by activated HSCs and strongly participate in PTP is emerging as next generation drug targets. Furthermore, protein
the increase of chemokine secretion, inducing macrophage chemotaxis tyrosine phosphatase 1B (PTP1B) which is the founding member of the
and subsequent mobilization. These data indicate that altered PTP superfamily, represents the non-transmembrane or cytosolic form of
cGMP/cGKI signalling in HSCs contributes to the onset of lipotoxicity, the enzymes; this is why PTP1B represents the main target of pharma­
liver inflammation, insulin resistance, and related metabolic disorders. ceutical industry. It is well known that PTP1B participates in the
In this sense, in vivo studies conducted on the human hepatic stellate cell appearance of metabolic disorders; however, more recent studies have
line LX2 in order to investigate the key metabolic pathways disturbed by shed light on the involvement of PTP1B in the pathological processes of
the ablation of cGKI, has suggested protective effects of cGKI against hepatic lesions [46]. Indeed, PTP1B seems to be a pivotal modulator in

Fig. 3. Representative diagram of the proposed mechanism of action of cGMP-dependent protein kinase I (cGKI) in inhibiting the activation of hepatic stellate cells.

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N. Bourebaba and K. Marycz Pharmacological Research 170 (2021) 105739

fibrogenesis. However, the precise role of PTP1B in hepatic fibrosis and Table 1
HSCs activation is still unclear. Previous investigations highlighted that Summary of MicroRNA/lncRNA innate changes during progression of hepatic
expression of PTP1B was significantly high in fibrotic liver but reduced fibrosis (activation of HSCs).
after spontaneous recovery; in addition, data suggested that PTP1B is MicroRNA/lncRNA type The innate changing during the References
upregulated upon HSCs activation. The activation of HSCs correlates pathology
with several phenomena, whose characterization could help to the miR-150 • Its expression is significantly reduced [48–50]
better understanding of HSCs trans-differentiation pathways and thus during liver fibrosis and in liver cells
anticipate their therapeutic inhibition for hepatic fibrosis and associated which express TGF-β1.
miR-194 • Its expression is lacking in human [48–50]
metabolic defects management. The most likely mechanism put in place
livers with advanced fibrosis, and
for the resolution of fibrosis lies in HSCs apoptosis induction HSCs. downregulated during hepatic stellate
However, this cannot solve the problem entirely since about 50% of cells activation.
activated HSCs escape apoptosis and revert to an inactivated phenotype Hedgehog (Hh) • It is normally inactive, but it is [39]
(iHSC). Several studies have demonstrated the potential role of PTP1B in reactivated during liver injuries such
as fibrosis to promote liver
the development of fibrosis; it was therefore suggested that deletion of reconstruction;
the PTP1B gene could significantly decrease fibrosis and inhibit collagen • Hepatocytes in apoptosis process
deposition by suppressing the expression of α-SMA and Col1α1, indi­ secrete Hh ligands that promote the
cating that the loss of PTP1B could prevent activation of HSCs and proliferation of hepatic progenitor
cells and trigger the activation of
attenuate excess collagen deposition; in summary, the inhibition of
HSCs.
PTP1B can reduce the activation of hepatic stellate cells via 2 possible Long intergenic non- • Down-regulation of lincRNA-p21 [51]
ways; either by restoring NO levels produced by endothelial cells and coding RNA-p21 expression in animal liver fibrosis,
thereby in the liver; or, by inducing an under expression of collagen (lincRNA-p21) activated HSCs and liver cirrhosis.
(α-SMA and Col1α1) produced by activated HSCs; this confirms that
pharmaceutical inhibition of PTP1B is a promising remedy [45,47] (Fig
than 1000 miRNAs are known to be encoded by the human genome, half
4).
of which have been validated experimentally. Mammalian miRNAs play
a critical role in many biological processes (differentiation, prolifera­
5.3. Targeting microRNA regulation tion, resistance to oxidative stress and suppression of tumors such as
those of the miR-34 family, which are the transcriptional targets of p53
MicroRNAs (miRNAs) are small non-coding RNAs about 19–25 bases and mediate its functions tumor suppressors). However, miRNAs are
in length, they are present in all living species and control gene deregulated in several diseases, particularly in cancers, for example
expression by degradation messenger RNA or inhibiting its translation; miR-21 and miR-221 are highly expressed and function as oncogenes
in fact, they recognize their target genes thanks to the base pairing be­ (oncomiR). Thanks to their capacity of gene regulators, miRNAs are
tween a sequence which is called "starting sequence" of a precise length considered to be interesting therapeutic targets for the modulation of
(between 6 and 8 nucleotides) at the 5 ’end and the sequence comple­ several pathological pathways. A number of recent studies have shown
mentary to the messenger RNA level of target genes. MiRNAs can repress that miRNA deregulation is involved in the activation of HSCs; the
gene expression by two possible mechanisms: either by degradation of reduced expressions of miR150 and miR194 correlate with the activa­
the targeted mRNAs and this by almost perfect pairing; or either by tion of HSCs, and it appears that their overexpression efficiently inhibits
translational repression caused by imperfect matching. To date, more the activation and proliferation of HSCs [48–50]. Hedgehog (Hh)

Fig. 4. Expected Role of Nitric Oxide (NO) and Protein Tyrosine Phosphatase 1B (PTP1B) in maintaining HSCs quiescent state.

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Table 2
Overview of the therapeutic targets and their mechanisms involved in the modulation of hepatic stellate cells activation.
Therapeutic Targets Localisation Mechanism References

cGMP-dependent protein kinase I • Synthesized in hepatic stellate cells in response to nitric • An overexpression of cGKI decreases the state of fibrosis [11,42]
(cGKI) oxide (NO) and natriuretic peptides through guanylate cyclase; and the suppression of
several markers of HSCs activation (collagen and
α-SMA), cell proliferation and migration;
• High cGKI activity may be promising in decreasing and
inhibiting the activation of HSCs and the subsequent
modulation of underlying metabolic dysfunctions.
Nitric Oxide Pathway and Protein • NO is synthesized from arginine (obtained from the • Endothelial cells (CE) keep HSCs inactivated and [43–45,
Tyrosine Phosphatase 1B (PTP1B) enzymes arginosuccinate synthetase (ASS) and quiescent via NO; through the phosphorylation of nitric 47]
arginosuccinate lyase (ASL) (involved in the urea cycle oxide synthase (eNOS). The inhibition of PTP1B restores
which takes place in the liver) vascular relaxation and phosphorylation of eNOS, used
in endothelial dysfunction treatment and thereby the
• Or synthesized from O2 by nitric oxide synthase (NOS) restoration of hepatic NO levels allowing the
maintenance of HSCs at the quiescent state;
• PTP1B participate in the onset of the liver fibrosis and
metabolic disorders; that’s why, the deletion of the
PTP1B gene could decrease fibrosis and inhibit collagen
deposition by suppressing the expression of α-SMA and
Col1α1, indicating that the loss of PTP1B could prevent
activation of HSCs.
MicroRNA • Present in all living species • The overexpression of miR150 and miR194 could inhibit [48–50,
the production of ECM proteins and the activation of 39]
HSCs and proliferation;
• Hedgehog (Hh) is activated when the liver is subjected
to injuries such as fibrosis, and it helps for its
reconstruction. Some Hh proteins, bind to the Hh
Patched (Ptc) receptor and release the Smoothened
(Smo) in the cytoplasm, which translocate proteins of
the glioblastoma family (Gli) in the nucleus, that will
play the role of transcriptional activators of Hh
signalling;
• An overexpression of one of the miR-378 family mem­
bers, could reduce the synthesis of Gli and inactivate
subsequently the Hh signalling pathway and would
reduce the activation of stellate cells;
• miR-29b attenuates the expression of Col1a1 and Col1a2
transcripts as well as the increased expression of α-SMA,
DDR2, FN1, ITGB1 and PDGFR-β, which are master
regulators involved in the activation of hepatic stellate
cells;
• miR-150 plays an antifibrotic microRNA role, because
its expression reduces the protein expression of type I
collagen and α-SMA, slowing and inhibiting the
proliferation of hepatic stellate cells.
lincRNAs (long intergenic non-coding • Present in all living species • lincRNA-p21 inhibits HSCs activated via p21, because it [41,51]
RNAs (lincRNAs) and heterogeneous resides upstream of the gene encoding the critical cell
group of long non-coding RNAs cycle regulator;
(lncRNA)) • LncRNA-p21 is a downstream transcriptional repressor
in the p53 pathway promoting apoptosis;
• LncRNA-p21 could increase the expression of the PTEN
gene through the competitive binding of miR-181b as
cRNA, thereby limiting the activation of HSCs via the
PTEN/Akt path;
• LncRNA-p21 sponges miR-17–5p to inhibit WIF1
through the Wnt/β-catenin pathway resulting in
suppression of HSC activation.

signalling is a morphogenic signalling pathway that plays an important that by raising the expression of one of the miR-378 family members,
role in embryonic development. In healthy adult liver, Hh signalling is significant reduction in the synthesis of Gli maybe be achieved and
considered inactive because maturing astrocytes barely express Hh li­ therefore inactivate the Hh signalling pathway and would reduce the
gands; however, it is reactivated when the liver is subjected to various activation of stellate cells [39]. Different investigations reported that
types of injuries such as fibrosis. Thus, the activation of the Hh pathway HSCs activity control maybe driven by different miRNAs, especially
promotes reconstruction of adult livers after injury. The Hh, Sonic Hh, during the synthesis of collagen that makes up ECM, such as miR-29b,
Indian Hh and Desert Hh ligands, bind to the Hh Patched (Ptc) receptor which is an mRNA regulator responsible for synthesis of type I
and release the Smoothened (Smo) in the cytoplasm, which translocate collagen, and could suppress the activation of HSCs since it markedly
proteins of the glioblastoma family (Gli) in the nucleus and will play the attenuates the expression of Collagen Type I Alpha 1 Chain (Col1a1) and
role of transcriptional activators of Hh signalling. In the damaged liver, collagen type I alpha 2 chain (Col1a2) transcripts as well as the
hepatocytes that are in the process of apoptosis secrete Hh ligands increased expression of α-SMA, Double Data Rate 2 Synchronous Dy­
triggering the proliferation of hepatic progenitor cells and also causes namic Random Access Memory (DDR2), Fibronectin 1 (FN1), Integrin
activation of HSCs. Therefore, Glis are good therapeutic targets for HSCs Subunit Beta 1 (ITGB1) and Platelet-derived growth factor receptor beta
activation control during hepatic fibrogenesis. Indeed, it would seem (PDGFR-β), which are key genes involved in the activation of hepatic

8
N. Bourebaba and K. Marycz Pharmacological Research 170 (2021) 105739

stellate cells. On the other hand, in an in-vitro study, the researchers Science Centre in Poland over the course of the realization of the pro­
noticed that the expression of miR-150 was downregulated in cells jects: ‘Inhibition of tyrosine phosphatase as a strategy to enhance insulin
which expressed TGF-β1; they therefore suggested that miR-150 could sensitivity through activation of chaperone mediated autophagy and
play the role of an antifibrotic microRNA. Thus, a restoration of miR-150 amelioration of inflammation and cellular stress in the liver of equine
clearly reduces the protein expression of type I collagen and α-SMA, metabolic syndrome (EMS) horses.’ (2018/29/B/NZ7/02662); and
slowing and inhibiting the proliferation of hepatic stellate cells [50]. "Exploring the role and therapeutic potential of sex hormone binding
Other ncRNAs, such as long intergenic non-coding RNAs (lincRNAs) globulin (SHBG) in the course of insulin resistance, inflammation, lip­
and heterogeneous group of long non-coding RNAs (lncRNA), have been otoxicity in adipose stem progenitor cells and adipocytes in equine
shown to be deregulated in several human pathologies, including he­ metabolic syndrome (EMS) mares” (No 2019/35/B/NZ7/03651).
patic. LncRNAs is structurally similar to mRNAs, however, they do not
encode proteins and are characterized by more than 200 nucleotides; Competing Interests
therefore, the regulation of the expression of these lncRNAs would be a
promising alternative in limiting the activation of hepatic stellate cells. Not Applicable.
As an example, the long intergenic non-coding RNA-p21 (lincRNA-p21)
inhibits HSCs activated via p21 [41]. lncRNA-p21, resides upstream of Acknowledgments
the gene encoding the critical cell cycle regulator Cdkn1a (also called
p21) and contains two exons. LncRNA-p21 functions as a downstream Not Applicable.
transcriptional repressor in the p53 pathway by activating the latter
promoting apoptosis. A study has also shown that lncRNA-p21 increased References
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