Antifibrotic agents emerging from traditional herbal medicine

Introduction 

Hepatic fibrogenesis is a complex and dynamic pathological process that is controlled by the synthesis and degradation of extracellular matrix materials, including fibril-forming collagens. The proliferation and apoptosis of fibroblastic cell populations in the liver participate in the process as well. Stellate cells comprise the hepatic sinusoidal wall and play multiple roles in the fibrotic process. Transformation of stellate cells from the vitamin A-storing to the ‘myofibroblastic’ phenotype closely correlates with hepatic fibrogenesis during chronic liver trauma. Molecular analyses of this phenotypic change of stellate cells, the so-called ‘activation’, have made great progress, in particular, in the field of intracellular signal transduction of transforming growth factor-β. TGF-β and platelet-derived growth factor-BB (PDGF-BB), as well as collagen gene expression. Using the information on stellate cell activation, therapeutic strategies against fibrosis in human liver disease are being developed.

The stellate cell as a principal player in liver fibrosis 

Hepatic stellate cells, which reside in the space of Disse, in close contact with both sinusoidal endothelial cells and hepatocytes, play multiple roles in the pathophysiology of the liver [1]. Quiescent stellate cells represent a retinol-storing phenotype and metabolise a small amount of basement membrane-forming substrata, such as laminine and type-IV collagen. When the liver parenchyma suffer from chronic injury through various disease aetiologies, such as iron overload, alcohol consumption, infection of hepatitis B virus (HBV) or hepatitis C virus (HCV), non-alcoholic steatohepatitis, autoimmune hepatitis and bile duct obstruction, the elimination of damaged hepatocytes leads to stellate cells departing from the sinusoidal wall and becoming activated. This process is considered to be triggered by oxidative stress due to lipid hydroperoxide and reactive aldehyde generated in and released from damaged hepatocytes, via paracrine stimulation of PDGF-BB, insulin-like growth factor-1 (IGF-1) and TGF-β, and by early extracellular matrix material changes including the production of a splice variant of cellular fibronectin (EIIIA isoform) [2]. Activated stellate cells change their phenotype to ‘myofibroblasts’ that produce increased amounts of extracellular matrix materials, show augmented contractility accompanied by the expression of smooth muscle α-actin and the production of endothelin-1 (ET-1), secrete TGF-β and monocyte chemotactic protein-1 (MCP-1), lose retinoid and exhibit active apoptosis.

TGF-β is a key regulatory molecule for extracellular matrix material metabolism and functions as an autocrine and a paracrine mediator. Cellular sources of TGF-β1 are diverse, including hepatic stellate cells, Kupffer cells, hepatocytes, sinusoidal endothelial cells and platelets. Proteolytic cleavage of latent TGF-β-binding protein (LTBP) is believed to be a prerequisite for the release and generation of bioactive (mature) TGF-β, which is induced by urokinase plasminogen activator (uPA) or tissue PA (tPA) [3]. The impact of TGF-β1 on liver fibrosis has been well documented in a TGF-β1 knockout mouse model, via the marked attenuation of liver fibrosis development using the soluble type-II TGF-β receptor, and in adenoviral delivery of the dominant-negative TGF-β receptor. The role of the Smad cascade in TGF-β signaling has been characterised in stellate cells [4]. Furthermore, recent work has indicated that bone morphogenic protein-7 antagonises TGF-β signaling through Smad1/5/7 and Id-2, and thereby suppresses collagen gene expression [5].

The contraction of stellate cells, particularly induced by ET-1, causes the constriction of sinusoids, leading to a persistent disturbance of intrahepatic microcirculation and portal hypertension [6], [7]. ET-1 synthesis by stellate cells is regulated by endothelin-converting enzyme-1 (ECE-1) during hepatic wound healing. ET-1 release is increased in stellate cells, whereas it is markedly decreased in endothelial cells after liver injury, depending on the ECE-1 mRNA expression and its stability. Blockade of ET receptors using specific ET receptor antagonists markedly improves the development of liver fibrosis and portal hypertension in rat models.

Is hepatic fibrosis reversible? 

Several recent reports have indicated that hepatic fibrosis and even cirrhosis may regress [8], [9]. These observations have questioned the established theory that cirrhosis is an incurable liver disease, particularly from a pathological point of view, and increased efforts to develop antifibrogenic therapies. In experimentally induced liver fibrosis in rodents, the cessation of liver injury, for instance, by stopping hepatotoxin administration, results in fibrosis regression, usually mediated by the reduction of TIMP-1 and apoptosis of the stellate cell lineage. In humans, spontaneous resolution of liver fibrosis can occur after successful treatment of the underlying disease. In particular, chronic HCV infection has been studied most extensively, and interferon (IFN) therapy with viral eradication results in fibrosis improvement, although the precise cellular and molecular mechanisms remain unknown [10]. The marked regression of liver fibrosis is logically supported by experimental evidence showing that rodent stellate cells undergo apoptosis in culture. Recent studies, in most cases using rat cells, indicate that stellate cells in culture undergo apoptosis via pentapeptide GRGDS (Gly–Arg–Gly–Asp–Ser), recombinant matrix metalloproteinase-9, an antibody against focal adhesion kinase, Fas/Fas ligand, nerve growth factor (NGF) [11], tumor necrosis factor-α (TNF-α), IGF-1, interferon γ, selective ligands for peripheral benzodiazepin receptors, high-dose sphingosine-1-phosphate, gliotoxin, adenoviral overexpression of p53 or retinoblastoma protein, and so on.

Therapeutic strategy for preventing liver fibrosis 

An anti-oxidative agent, N-acetyl-l-cysteine, triggers the degradation of the PDGF receptor β mediated by cathepsin B and inhibits PDGF signaling and PDGF-dependent DNA synthesis [11]. The agent additionally affects the expression of TGF-β receptor type-II in stellate cells. N-acetyl-l-cysteine markedly attenuates liver fibrosis development in rats [17]. Hepatocyte growth factor (HGF) gene therapy is another candidate for the treatment of liver fibrosis. Repeated transfections of the human HGF gene into skeletal muscle suppress the increase of TGF-β1, inhibit fibrogenesis and hepatocyte apoptosis, and completely resolve fibrosis in the cirrhotic rat liver [12]. The continuous administration of recombinant human HGF (rhHGF) is also effective for liver fibrosis. Although rhHGF reduces mRNA levels of pro-collagen α2(I), α1(IV), and TGF-β1, the detailed molecular mechanism of these anti-fibrotic effects of HGF remains to be elucidated.

In Japan, we have used herbal compounds to reduce the hepatic fibrotic process. Glycyrrhizin is an extract of the root of the plant Glycyrrhiza glabra (liquorice), and has been used for the treatment of chronic hepatitis for more than 30years in Japan. The first double-blind, controlled clinical study on the effect of glycyrrhizin was performed in 1977. Intravenous injection of 80mg glycyrrhizin/day significantly reduced aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels of chronic hepatitis caused by HBV and non-Anon-B (NANB) hepatitis. Ikeda reported that carcinogenesis rates in patients with high mean values of ALT were significantly lower in the glycyrrhizin-treated group [13]. Although the anti-fibrotic action of glycyrrhizin has not been fully elucidated because the development of hepatocellular carcinoma is considered to be closely related to liver fibrosis, these reports support the usefulness of glycyrrhizin as an anti-fibrotic agent in human. The suppression of rat stellate cell activation was evidenced by using carbenoxolone, a disodium salt of the 3-O-hydrogen succinate of glycyrrhizic acid. The carbenoxolone-mediated inhibition of PDGF-BB-stimulated stellate cell DNA synthesis is caused by the reduction of cyclins D1 and D2 and by the prevention of the phosphorylation of Raf-1 [14].

Sho-saiko-to (TJ-9) is composed of seven kinds of herbs and is the most popular herb used for chronic liver disease in East Asia. For hepatocytes, TJ-9 shows cytoprotection, inhibition of oxidative stress, and inhibition of malondialdehyde production. In stellate cells, TJ-9 inhibits cell proliferation, collagen generation, and oxidative stress development. In addition, TJ-9 was reported to suppress the occurrence of hepatocellular carcinoma in type-B liver cirrhosis [15]. In 1999, Shimizu, in collaboration with us, reported direct evidence showing that TJ-9 inhibited liver fibrosis in rats induced by either dimethylnitrosamine or porcine serum. TJ-9 treatment reduced smooth muscle α-actin expression in both a model of liver fibrosis and hepatic levels of collagen and malondialdehyde, indicating that TJ-9 has an anti-fibrotic and anti-oxidative action [16]. Sakaida reported that TJ-9 was effective for collagen deposition and GST-positive nodule formation in a choline-and l-amino acid-deficient dietary model. Thus, both in the clinical field and experimental models, TJ-9 evidently attenuates the progression of liver fibrosis, resulting in the suppression of hepatocellular carcinoma in chronic hepatitis [17].

Inchinko-to (TJ-135) is an herbal preparation long used for liver cirrhosis and jaundice [18]. Inchinko-to consists of three herbs, Artemisiae Capillaries spica, Gardenia fructus and Rhei rhizoma. TJ-135 decreased the serum level of ASL and ALT and the expression of stellate cell activation markers, such as PDGFRβ and smooth muscle α-actin, in a thioacetamide-induced liver injury model. In the culture of stellate cells, TJ-135 inhibited the phosphorylation of PDGF receptors, Raf-1/MEK/MAPK activation, and Akt phosphorylation under PDGF-BB stimulation. Among the components in TJ-135, we identified emodin included in R. rhizoma as a representative for the anti-proliferative action of stellate cells [19].