Betulin, betulinic acid and butein are inhibitors of acetaldehyde-induced activation of liver stellate cells
Introduction
Liver fibrosis is caused by a variety of etiologic agents, including chronic viral hepatitis, alcohol toxicity, autoimmune disease, and inherited metabolic disorders. For all of these diseases, there is a common pathologic mechanism leading to fibrosis that includes the generation and proliferation of smooth muscle α-actin (α-SMA)-positive myofibroblasts of periportal and perisinusoidal origin. These cells are produced as a consequence of the activation of hepatic stellate cells (HSCs). HSCs exist in the normal liver as quiescent retinoid-storing cells that activate in response to injury; once activated, these cells become proliferative and profibrogenic [1, 29]. The activated HSCs are a rich source of type I and III fibrillar collagen and also secrete high levels of tissue inhibitors of metalloproteinase (TIMPs) [31]. The activation of HSCs is mediated by several soluble factors, including growth factors, cytokines, chemokines, and oxidative stress products. Activation of HSCs is associated with sequential expression of several key cytokines and their surface receptors, which include transforming growth factor β (TGF-β) and its receptors [5]. Endogenous expression of TGF-β in the liver induces liver fibrosis, and blockade of TGF-β signalling by multiple methods has been shown to prevent the progression of liver fibrosis in experimental animals [40].
The development of liver fibrosis in alcoholics has been linked to the oxidation reaction of ethanol to the highly reactive compound – acetaldehyde. At concentrations that have been detected in hepatic venous blood during alcohol consumption, acetaldehyde has been shown to stimulate type I collagen synthesis and gene transcription in cultured rat and human HSCs through protein kinase C (PKC) activation [34]. Acetaldehyde has also been shown to increase levels of NF-κB (p65) and its binding to the α2(I) collagen promoter [23], which has been shown to be enhanced by the accumulation of H2O2 [6, 24, 33]. CYP2E1 is an important source of reactive oxygen species (ROS) in alcohol-induced injury and fibrosis by generating su-peroxide (O2–) and hydrogen peroxide (H2O2). Furthermore, inhibition of CYP2E1 activity by diallyl sulfide (DAS) has been shown to prevent the induction of collagen I gene expression in rat stellate cells overexpressing CYP2E1 [21]. Oxidative stress also activates the c-Jun NH2-terminal kinase (JNK), which is a protein that regulates the secretion of proinflammatory cytokines by cultured HSCs [20, 22].
Matrix metalloproteinases (MMPs) are a family of zinc metallo-endopeptidases that are rapidly expressed by HSCs in response to a diverse number of hepatic toxins [18]. Numerous in vitro experiments have examined the role of MMPs in the activation of HSCs. In addition, the proliferation of HSCs has been shown to be promoted by pericellular collagen I proteolysis acting via αvβ3 integrins [43]. Conversely, MMPs can also contribute to the regression of liver fibrosis through the cleavage of the fibrillar extracellular matrix (ECM) and have been shown to promote the apoptosis of activated HSCs. Thus, MMPs appear to play a dual role in liver fibrosis, depending on the activation state of HSCs [7].
To prevent the progression of hepatic fibrosis, various types of compounds that interfere with HSC proliferation and activation have been developed as anti-fibrogenic agents. Among these agents, butein (3,4,2’,4’-tetrahydroxychalcone) is a polyphenolic compound extracted from the stem bark of cashews and Rhus verniciflua Stokes that has been shown to suppress liver fibrosis induced by carbon tetrachloride [11]. In addition, butein has been shown to inhibit the myofibroblastic differentiation of rat HSCs [38]. Furthermore, butein derivatives with an improved bioavailability have been shown to have a potent antiproliferative effect. This effect is mediated by the activation of ERK, which leads to the transcriptional activation of AP-1 and consequently to heme oxygenase 1 expression in hepatic stellate cells [12]. However, butein also exhibits anti-inflammatory and anti-tumor effects through activation of other pathways, including ERK 1/2 and NF-κB signaling [12, 25, 42].
Natural triterpenoids such as ursolic and oleanolic acids have been investigated for their hepatoprotective effects. The mechanisms underlying these protective effect are complex and include the suppression of enzymes that play a role in liver damage such as cyto-chrome P450, cytochrome b5, CYP1A, and CYP2A. These protective effects are also mediated by an increase in antioxidant substances like glutathione, metallothioneins, and glutathione-S-transferase that simultaneously provide protective effects for liver mitochondria. Oleanolic acid protects the mouse liver from hepatotoxic tetrachloromethane, acetaminophen, phalloidin, and cadmium, whereas it has no effect on the toxicity of α-amanitine and allyl alcohol [13., 14., 15., 16., 17., 28, 37]. The protective effect of betulin, betulinic acid, and oleanolic acid against ethanol-induced cytotoxicity in HepG2 cells has also been noted [35]. Ursolic acid has been observed to have a similar protective effect in in vitro models of ethanol-induced hepatic damage in rats [28]. However, the antifibrotic activity of betulin and betulinic acid and the mechanisms underlying their antifibrotic effects have not been examined.
Therefore, the aim of this study was to examine the protective effects of butein, betulin, and betulinic acid (Fig. 1 ) on acetaldehyde-induced cytotoxicity in rat liver stellate cells (HSC). Several markers of HSC activation were examined, including α-SMA, procollagen I, TNF-α, TGF-β1, ROS, MMP-2, TIMP-1, and TIMP-2.
Section snippets
Cell cultures
A rat liver stellate cell line, CFSC-2G, was kindly provided by Dr. Marcos Rojkind (Department of Clinical Investigation, Walter Reed Army Medical Center, Washington, D.C., USA). CFSC-2G cells were cultured in Eagle’s Medium (MEM) supplemented with 5% heat-inactivated fetal calf serum (FCS), 1% nonessential amino acids (NEAA), and 1% antibiotic and antimycotic, pH 7.4. The cells were seeded in tissue culture plates (Falcon, Bedford, MA, USA) and incubated at 37°C in a humidified atmosphere of
Acetaldehyde-induced HSC cytotoxicity is attenuated by betulin but not butein and betulinic acid
When HSCs were cultured on plastic plates and inhibited by a low (0.1%) level of FCS in the medium, they were very resistant to acetaldehyde toxicity. Acetalde-hyde at concentrations between 75–175 µM exhibited a low toxicity towards HSCs in vitro, as measured by the MTT method. Therefore, we tested higher concentrations of acetaldehyde in these experiments (up to 3 mM). As can be seen from Figure 2 , preincubation of HSCs with 10 µM betulin (a non-toxic concentration) inhibited the toxicity of
Discussion
Our study explored the multiple effects of butein, betulin, and betulinic acid on acetaldehyde-activated rat stellate cells. Pretreatment of HSCs with the chalcone compound butein or the triterpenes betulin and betulinic acid influenced several of the cytotoxic effects of acetaldehyde-induced HSC activation, caused a decrease in α-SMA and procollagen type I production, and inhibited HSC motility. To assess the mechanisms by which butein, betulin and betulinic acid inhibited HSC activation, we
Acknowledgments
This work was supported by grant No. 2 P05A 169 29 from the Polish Ministry of Science and Higher Education. We gratefully acknowledge Dr. Marcos Rojkind for the generous donation of the rat HSC line CFSC-2G.
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2012, Tzu Chi Medical JournalCitation Excerpt :Acetaldehyde, the first metabolite of ethanol, can affect collagen I production by upregulating the synthesis of TGF-β1 [18]. Acetaldehyde also induces rat hepatic stellate cells to increase α-SMA and TGF-β1 production [19]. Ethanol elevates the activities of VEGF and TGF-β1 [20].