Studies on Antiproliferative Effects of Phthalides from Ligusticum chuanxiong in Hepatic Stellate Cells

Abstract

Suppression of hepatic stellate cell (HSC) growth and activation, and induction of apoptosis, have been proposed as therapeutic strategies for the treatment and prevention of liver fibrosis. Our previous study showed that the Chinese herb Ligusticum chuanxiong (LC) inhibits platelet-derived growth factor (PDGF-BB)-induced HSC proliferation. The present study was designed to investigate the active principles and their action mechanisms. With a bioactivity-directed fractionation approach, DNA synthesis (bromodeoxyuridine (BrdU) incorporation), cell cycle related proteins and apoptosis markers were determined to evaluate the inhibitory effects of active principles of LC. Two phthalides, Z,Z′-6,8′,7,3′-diligustilide (1) and levistolide A (2), from LC significantly abrogated PDGF-BB-induced proliferation in both rat and human HSC lines. These inhibitory effects of compounds 1 and 2 were associated with reduction of α-smooth muscle actin and collagen expressions. The cell cycle promoting proteins, cyclins D1, D2, E, A and B1, were downregulated while the inhibitory proteins p21 and 27 were up-regulated. JNK phosphorylation was up-regulated by compounds 1 and 2. In HSC-T6, the two compounds induced apoptosis through the activation of caspases 9 and 3, increase in cytosolic cytochrome c release, and downregulation of Bcl-2 and Akt phosphorylation. Moreover, neither phthalides caused direct cytotoxicity to either HSCs or rat primary hepatocytes under experimental concentrations. These results indicate that two phthalides from LC inhibited PDGF-BB-activated HSC proliferation possibly through cell cycle inhibition and apoptosis mechanisms. They might be potential anti-fibrotic drugs for the treatment and prevention of hepatic fibrosis.

Abbreviations

BrdU:bromodeoxyuridine

EtOAc:ethyl acetate

ECM:extracellular matrix

HSC:hepatic stellate cell

JNK:c-jun N-terminal kinase

LC:Ligusticum chuanxiong

NAC:N-acetylcysteine

PDGF:platelet-derived growth factor

α-SMA:α-smooth muscle actin

TNF:tumor necrosis factor

Key words

Hepatic stellate cells - Ligusticum chuanxiong - Umbelliferae - platelet-derived growth factor - phthalides - apoptosis

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