Anthocyanins isolated from the purple-fleshed sweet potato attenuate the proliferation of hepatic stellate cells by blocking the PDGF receptor

Abstract

During the process of liver fibrosis, hepatic stellate cells (HSCs) play a critical role in the increased formation and reduced degradation of extracellular matrix in the liver. We investigated the anti-proliferative effects of an anthocyanin fraction (AF), isolated from the purple-fleshed sweet potato, on platelet-derived growth factor (PDGF)-BB-dependent signaling pathways in HSC-T6 cells. HSC proliferation plays a pivotal role in liver fibrogenesis. The AF suppressed HSC activation, including PDGF-induced proliferation and α-smooth muscle actin (α-SMA) expression. Additionally, AF inhibited PDGF-BB-induced Akt and ERK1/2 phosphorylation. AF inhibited the phosphorylation level of PDGF receptor-β (PDGFR-β) following PDGF-BB stimulation, providing a mechanism for the inhibition of AF-mediated kinase. These results suggest that AF suppresses HSC proliferation by blocking PDGFR-β signaling, inhibiting Akt and ERK1/2 activation and α-SMA expression.

Introduction

Hepatic fibrosis, a response to chronic liver injury, results from the excessive deposition and reduced degradation of extracellular matrix (ECM) proteins and can ultimately lead to cirrhosis of the liver (Hsiang et al., 2005). During hepatic fibrosis, hepatic stellate cells (HSCs) undergo phenotypic transformation from vitamin A-storing quiescent cells to myofibroblast-like activated cells. Activated HSCs are proliferative and fibrogenic, and accumulate ECM, including α-smooth muscle actin (α-SMA).

During hepatic fibrogenesis, HSCs are activated by reactive oxygen species, growth factors, and pro-fibrogenic cytokines released from damaged hepatocytes and Kupffer cells (Kisseleva and Brenner, 2007), and their cognate receptors are associated with this transition. Among these factors, autocrine and paracrine signaling via platelet-derived growth factor (PDGF), a potent ligand for PDGF receptors (PDGFRs), stimulates HSC growth and proliferation (Lotersztajn et al., 2005, Pinzani and Marra, 2001). PDGF is composed of two chains (A and B) that form three isoforms (AA, AB, BB). Two types of PDGFRs have been identified: α (PDGFR-α) and β (PDGFR-β). A- and B-chains of PDGF bind to PDGFR-α, whereas only the B-chain binds to PDGFR-β. These isoforms promote myofibroblast proliferation and chemotaxis, but also stimulate collagen production and promote cell adhesion (Bonner, 2004, Apte et al., 1999, Luttenberger et al., 2000).

Upon the binding of PDGF-BB to PDGFR-β, the receptor undergoes dimerization, resulting in the autophosphorylation of tyrosines in the receptor's intracellular domain. This phosphorylation results in the recruitment of numerous proteins and ultimately leads to the activation of specific signaling pathways (Claesson-Welsh, 1994). PDGF activates the mitogen-activated protein kinase (MAPK) pathways responsible for mediating a broad array of cellular responses, including Akt, extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and p38 MAPK. The activation of these signaling cascades leads to PDGF-induced cell proliferation and differentiation, transcription factor activation, and cytokine expression and production. PDGF activity is also regulated by a variety of ECM proteins and glycoproteins that are up-regulated in the fibrogenic process (Bonner and Brody, 1995). MAPK pathways also play an important roles in the regulation of HSC activation by fibrogenic mediators (Fan et al., 2006). PDGF is a potent stimulator of HSCs. When activated, HSCs become both proliferative and fibrogenic, and accumulate ECM proteins, including α-SMA (Kisseleva and Brenner, 2008, Henderson and Iredale, 2007, Wallace et al., 2008). PDGF regulates several different phenotypic modifications in HSCs, including cellular proliferation and chemotaxis (Pinzani, 2002). Hepatic fibrosis is associated with increased PDGFR expression (Pinzani et al., 1998).

Anthocyanins, a class of natural polyphenol compounds, are widely distributed in fruits, beans, cereals, and vegetables. In animal models, the biological activity of anthocyanins includes powerful antioxidant effects (Shih et al., 2007), anti-inflammatory effects (Karlsen et al., 2007), and anti-tumor properties, through the stalling of pre-malignant cell growth (Shih et al., 2005). Anthocyanins may also help in preventing obesity, hyperglycemia (Tsuda et al., 2003), and asthma (Park et al., 2007). The consumption of vegetables and fruits containing abundant plant polyphenols is associated with a lower risk of lifestyle-related diseases, such as cardiovascular disease (Keli et al., 1996, Aviram and Fuhrman, 1998, Arts and Hollman, 2005). Recently, the purple sweet potato (Ipomoea batatas) has received much attention because of its unique color, nutritional value, and its role in health (Lila, 2004, Gülçin et al., 2005). There is a high anthocyanin pigment content in the tuber of some purple sweet potato cultivars. The anthocyanins from purple sweet potato are more stable than the pigments of strawberries, red cabbage, perilla and other plants. Thus, purple sweet potatoes have been regarded as a good source of stable anthocyanins as a food colorant and the purple sweet potato color may be recognized as a physiologically functional food factor. Indeed, purple sweet potato anthocyanins possess biological properties such as free radical scavenging, anti-mutagenicity, anti-carcinogenic activity, and anti-hypertensive effects (Ahmed et al., 2010). We previously reported that the anthocyanin fraction (AF) obtained from the purple-fleshed sweet potato has a potent hepatoprotective effect in acetaminophen (APAP)-induced hepatic damage in a mouse model (Choi et al., 2009). AF up-regulated the antioxidant activities of glutathione and glutathione S-transferase, and acted as a free radical scavenger. AF also inhibited APAP-induced hepatotoxicity through blockade of CYP2E1-mediated APAP bioactivation. However, anti-proliferative effects of AF have not been reported. In the current study, we investigated the anti-proliferative effects of AF obtained from the purple-fleshed sweet potato on PDGFR-β-dependent signaling pathways in HSCs.