Suppressive Effects Of Leflunomide On Hepatic Stellate Cell Proliferation And Its Molecular Mechanism Of Action

Hepatic fibrosis is a wound-healing process that occurs when the liver is injured chronically. Hepatic stellate cells (HSC) are responsible for the excess production of extracellular matrix (ECM) components. The activation of HSC, a key issue in the pathogenesis of hepatic fibrosis, is mediated by various cytokines and reactive oxygen species (ROS) released from the damaged hepatocytes and activated Kupffer cells. Therefore, inhibition of HSC activation and its related subsequent events, such as increased production of ECM components and enhanced proliferation, are crucial goals for intervention in the hepatic fibrogenesis cascade. To explore the cellular and molecular mechanisms of leflunomide on liver fibrosis, we choose HSC as a target for the pharmacological, molecular, and other novel therapeutics for hepatic fibrosis. One focus of this review is the inhibition of two cytokines, leptin and platelet-derived growth factor, which are important in hepatic fibrogenesis. The main contents are divided into three sections, as follows: 1. Effects of leflunomide on CCl₄/leptin-induced liver fibrosisIn vivo, leflunomide (1,3,9mg·kg^-1, ig) treatment showed protective effects on CCl₄/leptin-induced liver fibrosis, as indicated by decreased plasma transaminase activities, HA production, and Hyp content in liver tissue. Reversibility of liver fibrosis after treatment with leflunomide was also showed by marked decreases in type I collagen and smooth muscle actin immunostaining, a marker of fibrogenic myofibroblasts. In vitro, we demonstrated that following a fibrogenic stimulus of leptin, hepatic stellate cells (HSCs) underwent a complex activation process characterized by increased proliferation and excessive deposition of type I collagen. Studies with special chemical inhibitors demonstrated that this process involved JAK (Janus protein tyrosine kinase)/STAT (signal transducer and activator of transcription), MAPK, and PI3K/AKT (Protein kinase B) signal pathways. Pretreatment with A771726, leflunomide's metabolite, significantly inhibited the deposition of type I collagen in HSCs and the proliferation of primary HSC by interrupting the three proliferative signal transduction pathways in vitro, which was indicated by [³H] thymidine incorporation and cell cycle analysis. Furthermore, leptin-induced cyclin D1 protein expression which correlates well with HSC proliferation was also significantly inhibited by A771726. On the other hand, A771726 also prevented leptin-induced Kupffer cell (KC) activation and hepatic stellate cell collagen synthesis induced by Kupffer cell-conditioned medium (KCCM). Collectively, these results provided a novel insight into the mechanisms by which leflunomide may exert in liver fibrosis.2. Suppressive effect of leflunomide on rat hepatic stellate cell proliferation in vitro involved on PDGF-BB-elicited activation of three mitogen-activated protein kinasesIn this section, we demonstrated that following a fibrogenic stimulus by KCCM, primary HSCs showed a significant increase in cell proliferation, enhanced expression of PDGFR-βprotein and increased phosphorylation of tyrosine in PDGFR-βas well as associated activation of three major members of the MAPK family. Studies with respective neutralizing antibodies against released cytokines demonstrated that PDGF-BB played an essential role in this complex activation process. Administration of A771726, leflunomide's metabolite, markedly blunted these effects mainly by interrupting the PDGF-BB-elicited MAPK signal transductions.3. Simultaneous suppression on proliferation and induction of apoptosis by leflunomide in hepatic stellate cells induced by Kupffer cell-conditioned mediumActivation and proliferation of hepatic stellate cells are central in the development and progression of hepatic fibrosis, whereas their removal by apoptosis may contribute to the termination of this response. The aim of this study is to examine the possible mechanism of A771726, leflunomide's metabolite, on the fate of HSCs, focusing on its growth regulation and apoptosis. We demonstrated that during the course of stimulation by KCCM, primary HSCs showed marked proliferation, which process was accompanied by increased expression of cytokine receptors, including PDGFR-βand DR5. Administration of A771726 significantly suppressed primary HSC proliferation and simultaneously induced apoptosis in HSCs. Briefly, KCCM treatment resulted in a increased phosphorylation of tyrosine in PDGFR-β, followed by the JAK/STAT, and the MAPK signal pathways. Studies with special chemical inhibitors demonstrated that JAK2 played an essential role in the tyrosine phosphorylation of both STAT3 and ERK1/2. Pretreatment of A771726 markedly blunted these effects. Furthermore, KCM-induced cell cycle-related proteins were also significantly inhibited by A771726. On the other hand, administration of A771726 substantially increased the expression of DR5 on HSC surface. This event was accompanied by marked increases in cleavage of Procaspase-8, Bid and Bcl-2, and release of Cytochrome c into the cytosolic fraction. Collectively, suppressing proliferation and priming apoptosis were newly identified mechanisms by which A771726 exerted in HSCs.