Sheng-Lan Zeng, Rong-Zhen Zhang, Na Wang, Ting-Shuai Wang, Cong Wu, Xiao-Bin Qin, De-Wen Mao?

1. The First Clinical Faculty of Guangxi University of Chinese Medicine,Nanning 530022,China

2. The First Affiliated Hospital of Guangxi University of Chinese Medicine,Nanning 530022,China

Keywords:Hepatic fibrosis Hepatic stellate cell Cell activation Therapy

ABSTRACT The incidence of liver fibrosis is increasing worldwide, and if left untreated, it will later develop into cirrhosis with a high mortality rate. In this paper, the activation pathway and related mechanism of stellate cells in liver fibrosis are introduced, and some current therapeutic methods are summarized. These results suggest that stellate cells play an important role in liver fibrosis, and targeted therapy for the purpose of inhibiting the activation of stellate cells and inducing their apoptosis is expected to be an effective regimen to reverse liver fibrosis.However, there are some problems such as insufficient in-depth study of related mechanisms and imperfect experiments. In future animal experiments and clinical trials, more studies can be carried out to provide high-quality protocols for the treatment of liver fibrosis.

Liver fibrosis is a complex fibrotic and inflammatory process resulting from chronic liver injury and is an early step in the development of cirrhosis [1]. Progressive liver fibrosis may be caused by chronic infection with hepatitis B or C virus, alcoholism,nonalcoholic fatty liver disease, nonalcoholic steatohepatitis,and other relatively rare diseases, such as autoimmune hepatitis,hemochromatosis, and cholangitis. Cirrhosis is the end stage of progressive liver fibrosis, and according to statistics, about 1% to 2% of people worldwide suffer from liver fibrosis, and more than 1 million people die of liver fibrosis each year [2, 3]. At present,effective strategies to prevent and treat liver fibrosis are still lacking.Liver fibrosis occurs through an integrated signaling network that regulates extracellular matrix deposition. Hepatic stellate cells are activated in this process and are induced into a myofibroblastlike phenotype with contractility, proliferation, and fibrogenesis,resulting in the accumulation of collagen and other extracellular matrix components, and the continuous stimulation and accumulation of these substances leads to the destruction of liver structure and hepatic nerve function, resulting in decreased liver function [4]. Recent data suggest that termination of the fibrotic process and restoration of the defective pathway can reverse advanced fibrosis or even cirrhosis [5]. Therefore, understanding the pathogenesis of liver fibrosis will help to develop better treatment options, and hepatic stellate cells play an important role in this mechanism. This article mainly introduces the activation pathway of hepatic stellate cells in liver fibrosis and targeted therapy with the goal of inhibiting their activation or inducing apoptosis of activated hepatic stellate cells, providing a reference for the treatment of liver fibrosis with stellate cells as a target in the future.

1. Hepatic stellate cells: myofibroblasts

Since molecular markers of different germ layers coexist in hepatic stellate cells, their origin and identity remain unclear.Under physiological conditions, hepatic stellate cells reside in the Disse space and exhibit a dormant phenotype, the main function of which is to store vitamin a in lipid droplets [6]. Hepatic stellate cells are closely associated with endothelial cells, and they function as sinusoidal pericytes. In the setting of liver injury, hepatic stellate cells of the dormant phenotype are activated to become type I collagen-producing myofibroblasts. In chronic fibroproliferative diseases affecting multiple organs such as the lung, kidney and liver, the presence of myofibroblasts, which are fibroblast-like cells with contractile properties, is a key common feature. Proliferating myofibroblasts are a major source of extracellular matrix molecules,such as type I and III collagen, as well as other proteins that constitute pathological fibrous tissue [7]. After chronic injury induced by CCl4 treatment, hepatic stellate-cell-derived myofibroblasts proliferated rapidly and accumulated around the central vein, and the myocardial fibroblast population in the resulting lobules accounted for 14% of the total number of hepatocytes [8]. Activated hepatic stellate cells secrete endothelin-1, an effective vasoconstrictor that promotes cell proliferation, fibrogenesis and contraction [9].

2. Mechanism of hepatic stellate cell activation

The activation of hepatic stellate cells is associated with a variety of factors, such as epithelial cell injury, changes in extracellular matrix, transforming growth factor β (TGF-β) and SMAD signal transduction, and chronic infection with hepatitis virus. This article mainly describes the following.

2.1 TGF-β and SMAD Signaling

The TGF-β family consists of 33 members, including TGF-βs,activins, and BMPs [10]. TGF-β protein is present in three isoforms,TGF-β1, TGF-β2, and TGF-β3, which are the most extensively and intensively studied isoforms in liver fibrosis [11]. In the case of liver injury, macrophages can produce TGF-β and activate hepatic stellate cells, which secrete potential TGF-β and form an autocrine positive feedback loop through SMAD2 and SMAD3 to drive the formation of fibrosis, and SMAD7 acts as a negative regulator in an autocrine regulatory feedback loop [12], for example, binding TGFβRI to inhibit the interaction of SMAD2, inducing TGFβRI degradation as well as regulating the Wnt/β-catenin pathway to inhibit TGF-β-induced apoptosis [13]. Ligation of TGF-β1 with its receptors TGFβRI and TGFβR2 induces phosphorylation of SMAD2, SMAD3 and interaction with SMAD4. SMAD2, SMAD3,and SMAD4 complexes can transport to the nucleus and induce the expression of fibrotic genes, that is, type I collagen [14].

CD147 is a glycosylated protein expressed in the membrane of hepatic stellate cells [15], and there is an interaction between CD147 and TGF-β1. On the one hand, TGF-β1 increased the expression of CD147 and promoted the migration and contraction of LX-2 cells through SMAD2, SMAD3, and SMAD4-dependent mechanisms.On the other hand, overexpression of CD147 triggers the expression of TGF-β1, α-SMA and COL1α1 by upregulating ERK1/2 and Sp1[16].

2.2 Platelet-derived growth factors (PDGF)

PDGF is a key mitotic source in the liver and a chemoattractant that drives hepatic stellate cell proliferation and migration. The expression of PDGFβ is induced during the initiation of hepatic stellate cell activation and enhances the inflammatory and fibrotic response to chemical injury through the ERK, AKT and NF-κB pathways [17]. After CCl4-induced liver injury in rats, PDGFβ and PDGFRβ are dramatically up-regulated, in part through downstream activation of ERK, and also induce stellate cells to secrete macrophage colony-stimulating factor, so PDGF signaling may underlie certain immunomodulatory functions of stellate cells.Loss of PDGFRβ inhibits the fibrotic response during liver injury,but PDGFRβ in stellate cells is essential for tissue regeneration after partial hepatectomy [18].

2.3 Chronic Infection with Hepatitis Virus

Chronic infection caused by HBV and HCV has become one of the major triggers of fibrotic liver disease throughout the world.Viral genes and proteins can directly or indirectly promote hepatic stellate cell activation. HBVe antigen directly induces the activation and proliferation of rat hepatic stellate cells in vitro through the TGF-β pathway, and viral core and X proteins similarly activate human LX-2 cells through PDGFβ signaling [19]. Viral core and nonstructural proteins directly induce inflammatory and profibrotic pathways in hepatic stellate cells, and HCV core protein may promote epithelial-mesenchymal transition of hepatic parenchymal cells through TGFβ signaling [20].

2.4 Intestinal Dysfunction and Dietary Structure

Increased intestinal permeability can be observed in advanced liver disease, intestinal bacteria and related metabolites can be translocated to the liver, and bacterial molecules can signal through TLR on stellate cells to induce their activation and subsequent fibroinflammatory response. TLR4 is a ligand for lipopolysaccharide,a bacterial membrane component, which can promote stellate cell activation and fibrosis in vivo [21]. Dietary cholesterol exacerbates liver fibrosis because free cholesterol accumulates in hepatic stellate cells, which leads to increased TLR4 signaling and downregulation of bone morphogenetic proteins and activin membrane-bound inhibitors. Hepatic stellate cells are sensitive to TLR4, and this pathway can be used as a target for anti-fibrotic therapy [22].

2.5 Hedgehog Pathway

The Hedgehog pathway is an important system in the regulation of progenitor cell fate during liver fibrosis. Smooth homologs drive epithelial regeneration by promoting myofibroblast mesenchymalepithelial transition derived from hepatic stellate cells by upregulating hedgehog ligand release and activation [23]. The absence of smooth homologs in hepatic stellate cells significantly attenuates fibrosis during liver injury, suggesting that the Hedgehog protein pathway is involved in stellate cell activation. Interestingly, blocking signaling in activated hepatic stellate cells during liver injury can also prevent the accumulation of hepatic progenitor cells, which may imply that signaling pathways in stellate cells are involved in the regulation of epithelial cell regeneration during injury repair [24]. The Hedgehog pathway is a potential target for fibrosis therapy.

In the development of liver fibrosis, the activation of hepatic stellate cells plays an important role, and the activation pathway involves a complex mechanism of action between a series of cytokines and cell signaling pathways. Because of the criticality of hepatic stellate cells, drug studies targeting the signaling pathways involved in the mechanism of hepatic stellate cell activation have emerged in endlessly and have also been widely used in clinical practice.

3. Stellate cell-targeted therapy

The development of liver fibrosis may lead to cirrhosis and a series of complications, such as portal hypertension and hepatic encephalopathy. Although there is a lack of therapeutic means to directly target and reverse liver fibrosis. However, termination of chronic liver injury was observed to result in regression of liver fibrosis and a decrease in activated hepatic stellate cells accompanied by regression of inflammatory tissue. These results suggest that targeting hepatic stellate cells may be an anti-fibrotic therapeutic strategy regardless of the cause of liver injury.

3.1 IL-30

IL-30 attenuates liver fibrosis and is an ideal therapy for liver fibrosis. IL-30 allows NKT cells to accumulate in the liver, promotes NKG2D expression on the surface of hepatic NKT cells, and enhances their toxicity to activated hepatic stellate cells, thereby inhibiting liver fibrosis [25].

3.2 Ursolic acid

Ursolic acid is a pentacyclic triterpenoid with a wide range of pharmacological activities in various edible fruits and medicinal plants. It has been shown that ursolic acid induces apoptosis in activated hepatic stellate cells and not in isolated hepatocytes and static hepatic stellate cells. Ursolic acid inhibits TGF-β1-induced static hepatic stellate cell activation and transformation by inhibiting NADPH oxidase expression and Hedgehog pathway [26]. In rats pretreated with TAA for 6 weeks, ursolic acid injection significantly resolved liver fibrosis within 48 hours. Moreover, ursolic acid improved liver fibrosis caused by chronic administration of TAA and BDL [27]. Zhang [28] et al found that the use of ursolic acid in rats with CCl4-induced liver fibrosis reduced liver and intestinal pathological damage, decreased serum lipopolysaccharide and procalcitonin levels, improved intestinal malnutrition and the expression of tight junction proteins claudin1 and occludin in the ileum of rats, inhibited intestinal NOX-mediated oxidative stress response, and had a protective effect on the intestinal mucosal barrier in rats with CCl4-induced liver fibrosis.

3.3 Resveratrol

Resveratrol is found mainly in red grape skin and is sometimes found in peanuts and berries. There are beneficial effects in different models of hepatic steatosis. Resveratrol can activate superoxide dismutase, superoxide dismutase activity is necessary to reduce oxygen free radicals, can protect it from lipid peroxidation, and restore the levels of liver function biomarkers of oxidative damage(MDA, SOD, protein carbonyl). It also inhibits the oxidative effect of down-regulating α-SMA and hepatic stellate cell activation and limits the progression of liver fibrosis [18, 29].

3.4 Celecoxib Derivative OSU-03012

OSU-03012 is a potential antifibrotic drug that is a noncyclooxygenase-inhibiting seloxifene derivative. OSU-03012 inhibited the proliferation of LX2 cells and prevented the secretion of fibrotic factors in a dose-dependent manner. In addition, it also inhibits liver fibrosis by inducing hepatic stellate cell senescence in G1 phase [29].

3.5 Curcumin

The antioxidant curcumin is a phytochemical present in turmeric,and curcumin inhibits its activation by inducing HSC senescence,thereby achieving the effect of inhibiting liver fibrosis. Curcumin promotes the expression of Hmga1, a marker of aging in the fibrotic liver of rats. Furthermore, curcumin increased the number of senescence-associated β-galactosidase in vitro. Meanwhile,curcumin induced hepatic stellate cell senescence by elevating the expression of hepatic stellate cell senescence markers P16,P21, accompanied by decreased abundance of hepatic stellate cell activation markers α-smooth muscle actin and α1-procollagen.Moreover, curcumin can affect the cell cycle and telomerase activity[30].

Drugs targeting stellate cells for the treatment of liver fibrosis contain both active components extracted from natural plants and synthetic compounds with different mechanisms of action, but all aim to inhibit stellate cell activation or promote apoptosis of activated cells. Although some achievements have been made, most of the studies focus on animal models of liver fibrosis induced by CCl4, and more in-depth mechanism and clinical research are needed for the treatment of liver fibrosis caused by viral or other diseases.

4. Outlook

In recent years, the treatment of liver fibrosis is becoming a huge medical burden, if not intervened, fibrosis can develop into cirrhosis,ultimately leading to organ failure or even death, the development of targeted therapy to inhibit the occurrence of fibrosis is very important. Inhibition of hepatic fibrosis by inhibiting hepatic stellate cell activation has gained much attention, and the molecular mechanisms of fibrosis and its relationship with hepatic stellate cells are essential for the discovery of new therapeutic targets. In this paper, we outline the activation mechanism of hepatic stellate cells in liver fibrosis and introduce strategies to inhibit hepatic stellate cell activity, providing new insight into potential therapeutic approaches for liver fibrosis. However, the activation mechanism of stellate cells is complex, and there are still problems such as insufficient in-depth study and many uncertainties in treatment options, which require further study of related animal models and clinical treatment in the future.