Protective mechanisms of medicinal plants targeting hepatic stellate cell activation and extracellular matrix deposition in liver fibrosis

During chronic liver injury, hepatic stellate cells (HSC) are activated and proliferate, which causes excessive extracellular matrix (ECM) deposition, leading to scar formation and fibrosis. Medicinal plants are gaining popularity as antifibrotic agents, and are often safe, cost-effective, and versatile. This review aims to describe the protective role and mechanisms of medicinal plants in the inhibition of HSC activation and ECM deposition during the pathogenesis of liver fibrosis. A systematic literature review on the anti-fibrotic mechanisms of hepatoprotective plants was performed in PubMed, which yielded articles about twelve relevant plants. Many of these plants act via disruption of the transforming growth factor beta 1 signaling pathway, possibly through reduction in oxidative stress. This reduction could explain the inhibition of HSC activation and reduction in ECM deposition. Medicinal plants could be a source of anti-liver fibrosis compounds. Electronic supplementary material The online version of this article (doi:10.1186/s13020-014-0027-4) contains supplementary material, which is available to authorized users.


Introduction
Liver fibrosis is caused by inappropriate tissue repair via connective tissue deposition, which results from chronic liver injuries, including those from alcohol, chronic viral hepatitis, autoimmune diseases, parasites, metabolic diseases, and toxins or other drugs [1]. When fibrosis is not controlled, it can progress into cirrhosis. Cirrhosis was previously considered to be irreversible, but some studies suggest that fibrosis and cirrhosis could be reversible [2].
Liver fibrosis is a public health problem that results in significant morbidity and mortality [3]. Hundreds of thousands of people worldwide suffer from cirrhosis, partially because of the obesity pandemic combined with the high incidence of alcohol abuse and viral hepatitis [4]. Chronic viral hepatitis (B and C), alcoholic liver disease, and nonalcoholic fatty liver disease are the three most common causes of liver cirrhosis [5]. The prevalence of chronic liver diseases is predicted to increase, partially owing to the rising prevalence of obesity and metabolic syndrome, especially in developed countries [6].
The pathogenesis of liver fibrosis is complex and varies among different kinds of hepatic injuries. Usually after acute liver damage, parenchymal cells are regenerated to replace the necrotic and apoptotic cells. This regenerative process is associated with an inflammatory response and a limited deposition of extracellular matrix (ECM). When the liver is subjected to chronic injury, the regenerative response fails and hepatocytes are replaced with abundant ECM, which is mainly composed of collagen types I, III, and IV; fibronectin; elastin; laminin; and proteoglycans [7]. Hepatic stellate cells (HSC) are the main source of ECM [8].
There is no standard treatment for liver fibrosis [7], but a reduction in liver injury events, such as cessation of alcohol intake or successful viral hepatitis treatment can control fibrosis. Nevertheless, these actions are often insufficient to avoid eventual progression to cirrhosis in the vast majority of patients [9]. Although important advances have been made in understanding the pathogenesis of hepatic fibrosis over the past 20 years, efficient antifibrotic drugs have yet to be developed. There are two ways by which medicinal plants and their bioactive compounds and extracts could reduce liver fibrosis: via inhibition of HSC activation and via reduction of ECM deposition ( Figure 1). Liver fibrosis treatment should take into account the versatility of its pathogenesis and should act upon all pathways involved, beginning with HSC activation and ECM deposition.
Medicinal plants are often safe, cost-effective, and versatile, and are therefore popular potential antifibrotic agents. This review aims to describe the role of some hepatoprotective plants in the inhibition of HSC activation and ECM deposition in the pathogenesis of liver fibrosis. These plants include: Curcuma longa, Silybum marianum, Ginkgo biloba, Salvia miltiorrhiza, Glycyrrhiza glabra, Scutellaria baicalensis, Bupleurum falcatum, Phyllanthus species, Berberis aristata, Ginseng species, Andrographis paniculata, and Coffea species.

Literature inclusion criteria
The 12 plant species were selected because of their known hepatoprotective activities. A three-step progressive searching method was applied using PubMed. In each of the steps, only pertinent articles were selected. First, a global search on the liver activity of each plant species was undertaken using the keywords "liver" and "plant species name". The antifibrotic activities were classified under two different pathways: inhibition of HSC activation and suppression of ECM deposition. Bioactive compounds and extracts from every reviewed species were selected. Second, a more advanced search was performed using the terms "liver fibrosis" and "plant species name or bioactive extract name or bioactive compound name". Finally, a search on the antifibrotic mechanisms of each species was performed using the terms "hepatic stellate cells activation", "extracellular matrix", "collagen", and "plant species name or bioactive extract name or bioactive compound name".

Inhibition of hepatic stellate cell activation
Role of hepatic stellate cell activation in the pathogenesis of liver fibrosis HSC activation, which includes initiation and perpetuation, is an early event in liver fibrogenesis. The activation of HSCs converts normal, quiescent vitamin A-rich cells into myofibroblast-like cells characterized by proliferation, chemotaxis, fibrogenesis, contractility, matrix degradation, retinoid loss, and white blood cell chemoattractant/cytokine release [10][11][12][13][14].
HSCs are initiated when gene expression and phenotype changes render the quiescent cells responsive to other cytokines and stimuli [10,12]. Several paracrine stimuli from damaged hepatocytes and other neighboring cell types, such as Kupffer cells, platelets, sinusoidal endothelium, and immune cells initiate the activation of HSCs [15]. Transforming growth factor beta 1 (TGF-β1), plateletderived growth factor (PDGF), and epidermal growth factor (EGF) mediate platelet stimuli [15,16]. By producing fibronectin and activating latent TGF-β1, injured endothelial cells provoke HSC activation. Kupffer cells are important sources of paracrine stimuli for HSCs because they express TGF-β1, transforming growth factor alpha (TGF-α), reactive oxygen species, and lipid peroxides. These Kupffer cell paracrines lead to matrix synthesis, cell proliferation, and the release of retinoids [15] and matrix metalloproteinase (MMP)-9 [17] for collagen synthesis through the activation of latent TGF-β1 [18]. Kupffer cells also inhibit fibrogenesis via the production of anti-inflammatory interleukin-10 and nitric oxide (NO), which decrease collagen synthesis, increase collagenase production, and reduce cell proliferation and contractility [15]. All these stimuli trigger important changes in the composition of ECM, especially an increase in type I and III fibril-forming collagens and fibronectin [10]. This compositional transformation of the matrix induces new fibrogenic stimuli, which further exacerbate fibrosis [12].
Perpetuation of HSC activation results in maintenance of the activated phenotype and therefore the generation of fibrosis [15]. Scars are formed through changes in HSC behavior, like proliferation, fibrogenesis, contractility, chemotaxis, retinoid loss, white blood cell chemoattractant/cytokine release, and matrix degradation [12].
HSCs migrate towards injured areas in the liver, which increases the fibrogenic response at a specific site via mediation of several chemoattractants, including PDGF, monocyte chemoattractant protein-1 (MCP-1), and chemokine receptor CXCR3 [10,12].
Fibrogenesis is defined as increased fibrotic matrix production, especially that of collagen type I [12]. TGF-β1, produced by HSCs and other neighboring cell types like Kupffer cells, sinusoidal endothelial cells, bile duct epithelial cells, and hepatocytes [11], is a potent fibrogenic signal [20] as it increases the production of collagen I and other matrix constituents like fibronectin and proteoglycans [12,21]. These effects are induced by the interaction between TGF-β1 and the membrane receptor complex formed by TβRI and TβRII, leading to the phosphorylation of intracellular mediators, namely Smad proteins. Smad proteins are divided into three groups: receptoractivated Smad (R-Smad), common-Smad (Co-Smad) and inhibitory-Smad (I-Smad). Phosphoylated R-Smad (Smad1, Smad2, Smad3, Smad5 and Smad8) dissociate from anchoring proteins and associate with Co-Smad (Smad4). R-Smad-Co-Smad complexes are transported into the nucleus where they act as transcription factors. This cascade is inhibited by I-Smad (Smad6 and Smad7) [20]. CTGF (through TGF-β-dependent and -independent pathways), endothelin-1, leptin, and angiotensin II have also been reported as fibrogenic factors for HSCs [11,22].
Stellate cell contractility is associated with portal hypertension and may lead to the collapse and shrunken state of cirrhotic livers [23]. The increased expression of contractile proteins, smooth muscle α-actin, and smooth muscle myosins via calcium-dependent and calciumindependent pathways mediate the contractility of HSCs [11]. Endothelin-1, NO, angiotensinogen II, eicosanoids, atrial natriuretic peptide, somatostatin, and carbon monoxide are some of the signals that contribute to the contractile phenotype of HSCs [12]. Because HSCs are so important to fibrogenesis, the inhibition of HSC activation is a possible therapeutic approach to reversing liver fibrosis [1,4]. For instance, PPARγ ligands [24], interferon gamma (IFNγ) [25], and antioxidants [26] reduce liver fibrosis by inhibiting TGF-β1 expression and therefore HSC activation.

Hepatic stellate cell activation as a target of antifibrotic medicinal plants
All the reviewed medicinal plants were shown to suppress HSC activation. Since the activation of HSCs occurs via a complex network of signaling pathways, different targets have been investigated to explain the antifibrotic activity ( Table 1).
VEGF and its receptors were suppressed by curcumin [45] and coffee [122], which could also explain their amelioration of angiogenesis in the fibrotic liver. Additionally, curcumin reduced TGF-α levels [43].
Cyclins, and cyclin inhibitors, are essential proteins for the control of the cell cycle, and potential therapeutic targets for inhibiting HSC proliferation. The cyclin D1 gene was down-regulated by curcumin through PPARγ activation [30]. Inhibition of HSC proliferation by salvianolic acid A was mediated by the induction of cell cycle inhibitory proteins p21 and p27, down-regulation of cyclins D1 and E, and suppression of protein kinase B phosphorylation [63].
Medicinal plants also regulate the contraction of HSCs. For example, salvianolic acid B lowered portal pressure and attenuated the contraction of HSC by inhibiting the Ras homolog family member A signaling pathway [97] and decreasing the HSC free calcium ion concentration [93].

Reduction in extracellular matrix deposition
Role of extracellular matrix deposition in the pathogenesis of liver fibrosis Liver fibrosis is a dynamic process in which the equilibrium between ECM synthesis and degradation is impaired [11]. HSCs, neutrophils, and macrophages are the main cellular contributors to ECM degradation [1], while HSCs, portal myofibroblasts, bone-marrowderived cells, and the epithelial-mesenchymal transition are responsible for ECM synthesis [14]. All these cells involve several molecular effectors such as matrix proteins, MMPs and TIMPs. Transforming growth factor beta 1, b connective tissue growth factor, c platelet derived growth factor, d transforming growth factor alpha, e vascular endothelial growth factor, f advance glycation end-product, g low-density lipoprotein, h hypoxia-inducible factor-1α, i placental growth factor, j platelet activate factor, k monocyte chemotactic protein-1.
During fibrogenesis, several ECM proteins, especially collagens type I, III, and IV; proteoglycans; laminin; and fibronectin are over-expressed by activated HSCs and other cells, which results in pathological scar formation [131]. Accumulation of ECM not only depends on upregulation of ECM production, but also on MMP-TIMP expression [132,133]. In the early phases of liver injury, HSCs transiently express MMP-3 and MMP-13, which is a matrix-degrading phenotype. In the later stages of liver injury, the inhibition of fibrillar liver collagen degradation prevails over ECM synthesis, as evidenced by increased TIMP-1 expression, which leads to a decrease in collagen degradation by MMP-1 and MMP-13 [133]. TIMP-1 was found to protect HSCs from apoptosis [134]. Regulation of the plasminogen activation system, including enhanced production of the urokinase-plasminogen activator (uPA) and uPA receptor and modulation of plasminogen activator inhibitor type 1 (PAI-1), is another method by which HSCs regulate the ECM [135][136][137]. Macrophages also take part in matrix breakdown [9,138], since macrophages produce MMPs, like MMP-13 [139,140] and MMP-9 [141,142] through the mitogenactivated protein kinase and NF-κB pathways [143].
The dynamic process of matrix degradation and synthesis could be a therapeutic target to reversing liver fibrosis and restoring normal liver architecture [4,11,144]. Indeed, MMP-8 cloned into an adenoviral vector reversed fibrosis in cirrhotic animal livers [145]. In vivo reduction of liver fibrosis has also been observed with similar therapeutic approaches involving MMP-1 [146], TIMP-1 scavengers [147], and uPA [148].
Since ECM proteins, MMPs, and TIMPS are overexpressed when HSCs are activated, the inhibition of HSC activation and proliferation is the main method by which plants can suppress ECM deposition. The antifibrotic mechanism is mainly the result of down-regulating the TGF-β1/Smad signaling pathway.

Highlights
Inhibition of HSC activation and subsequent ECM deposition via medicinal plants is the result of TGF-β1/ Smad signaling disruption. The down-regulation of TβRI and II and modulation of Smads suggests a common mechanism upstream to the pathway affected by the plants. Since oxidative stress is involved in the activation of HSCs, antioxidative properties could be the common mechanism by which the plants inhibit HSC activation and ECM deposition.
The antifibrotic properties of medicinal plants have mainly been observed in liver fibrosis models in vitro and in vivo. Clinical studies are sparse and mainly recruited chronic hepatitis B and C patients to assess the hepatoprotective effects of medicinal plants. Silymarin, glycyrrhizin, and S. miltiorrhiza have been somewhat successfully tested. Glycyrrhizin treatment induced a decrease in alanine transaminase and maintenance and improvement of necroinflammation in chronic hepatitis C nonresponders, and those patients unlikely to respond to interferon therapy when they receive at least three injections weekly over the course of 4, 22, or 52 weeks [190][191][192][193]. Since glycyrrhizin had no effect on hepatitis C RNA [190], investigation of the hepatoprotective mechanisms, such as inhibition of HSC activation and ECM deposition, is necessary.
Salvia injection and salvianolic acid B, have been tested in patients infected with hepatitis B. Salvianolic acid B, administrated over 6 months, reversed liver fibrosis and inflammation more effectively than IFNγ in patients with hepatitis B, as suggested by significant improvement in serum hyaluronic acid, laminin, type IV collagen, and procollagen III peptide as compared with the IFNγ group [194]. The same results, as well as decreased levels of alanine transaminase and aspartate transaminase were observed in fibrotic and cirrhotic patients infected with hepatitis B virus who were treated with Salvia injections over 45 [195] or 60 days [196]. However, another study on chronic hepatitis B patients treated with S. miltiorrhiza injection showed improvements only in symptoms, physical signs, and liver functions, not antifibrotic effects. Therefore, additional clinical studies are required to understand the effect of S. miltiorrhiza injection on liver fibrosis [197].
Clinical studies on silymarin administration for the treatment of hepatitis C were inconclusive. Silymarin has antiviral activity and has been associated with reduced progression from fibrosis to cirrhosis in advanced hepatitis C patients [198]. However, it has low oral bioavailability at 0.73% in rat plasma [199][200][201] and has not always been associated with hepatic improvement. For example, it was not effective in patients with chronic viral hepatitis C infection that who also unsuccessfully treated with interferon-based therapy [202].
Besides HSC activation and ECM deposition, other events like oxidative stress, inflammation, and immune responses are involved in the fibrogenic response [10,11,13,14]. These pathways are potential targets that could help reduce hepatic fibrosis. Nevertheless, several medicinal plants have been shown to inhibit HSC activation and ECM deposition during liver fibrosis.

Competing interests
The authors declare that they have no competing interests.
Authors' contributions FD and DEC conceived and designed the study. FD and DEC analyzed the data. FD, DEC, JEM, MTG, and CR wrote the manuscript. All authors read and approved the final manuscript.