L-02細(xì)胞系特性

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Pyrrolizidine alkaloid clivorine inhibits human normal liver L-02 cells growth and activates p38 mitogen-activated protein kinase in L-02 cells

Abstract

Clivorine, a pyrrolizidine alkaloid extracted from Chinese medicinal plant Ligularia hodgsonii Hook significantly inhibited human normal liver L-02 cells proliferation and decreased L-02 cells viability. The results of western blot showed that clivorine strongly evoked phosphorylation of p38 mitogen-activated protein (MAP) Kinase in L-02 cells, but had no effect on extracellular signal-related kinases MAP Kinase phosphorylation. Moreover, another pyrrolizidine alkaloid monocrotaline had no effect on phosphorylation of p38 MAP Kinase in L-02 cells. These studies document the effects of pyrrolizidine alkaloid clivorine on the MAPK cascade and on the growth of human normal liver L-02 cells for the first time, which may be a possible reason for the toxic effects observed in those plants containing pyrrolizidine alkaloids.

Keywords: Clivorine; Pyrrolizidine alkaloid; L-02 cell; p38 Mitogen-activated protein kinase

1.Introduction

Pyrrolizidine alkaloids (PAs) occur naturally in a wide variety of plant species such as Leguminosae, Boraginaceae and Asteraceae. Many PAs are hepatotoxic, genotoxic, cytotoxic and pneumotoxic (Mattocks, 1986; Huxtable,1989). Ligularia hodgsonii belonging to Family Asteraceae has been used for cough, hepatitis and inflammation in traditional Chinese medicine (TCM). L. hodgsonii contains many PAs of which clivorine is the most abundant component (0.151%) and has been reported to be a carcinogenic agent (Kuhara et al., 1980). Our early study has found that oral administration of clivorine to rat leads to death and that the major hurt is happened in the liver (unpublished data). However, there is no study of clivorine about its toxicity on human hepatocytes.

Many evidences have demonstrated that signal transduction pathways actively modulate the hepatocyte’s response to toxin-induced liver injury (Jones and Czaja, 1998).Mitogen-activated protein kinases (MAPKs) are evolutionary conserved enzymes in the intracellular signal transduction pathways connecting cell surface receptors to critical regulatory targets, and control many aspects of mammalian cellular physiology including cell growth, differentiation and apoptosis (Lewis et al., 1998; Gutkind, 2000; Chang and Karin, 2001). So far, at least four distinctly regulated groups of MAPKs: extracellular signal-related kinases (ERK1/2),jun amino-terminal kinases (JNK1/2/3), p38 proteins (p38a/b/g/d) and ERK5 have been found to be expressed in mammals (Gutkind, 2000; Chang and Karin, 2001).

This study is conducted to investigate the effects of clivorine on the growth of human normal liver L-02 cells and the potential effects on two major MAPKs: p38 and ERK1/2 kinases.

2.Materials and methods

2.1.Chemicals and reagents

Roswell Park Memorial Institute 1640 (RPMI 1640),modified Eagle’s medium (MEM), fetal bovine serum (FBS), trypan blue solution and horseradish peroxidaseconjugated goat anti-rabbit secondary antibody were purchased from GIBCO-BRL. Phosphospecific rabbit polyclonal antibodies against 180Thr and 182Tyr dual-phosphorylated p38, against total p38 (phosphorylation state independent), against 204Tyr-phosphorylated ERK1/2 and against total ERK1/2 (phosphorylation state independent) were purchased from New England Biolabs Inc. Nitrocellulose membranes (Hybond) and enhanced chemiluminescence detection system were obtained from Amersham Pharmacia. All other reagents unless indicated were from Sigma Chemical Co.

2.2.Treatment solutions

Clivorine isolated from L. hodgsonii was structurally elucidated by IR, NMR, MS spectra and its purity determined by HPLC was 99.5%. The structure of clivorine is shown in Fig. 1. The roots and rhizomes of L. hodgsonii were collected on September 19, 1993 in Emei County,Sichuan Province of People’s Republic of China. The material was authenticated and stored dry until extraction. A voucher specimen is deposited in the Department of Pharmacognosy, China Pharmaceutical University. Monocrotaline gifted from Prof. Zhi-Ben Tu (Wuhan Institute of Botany, Chinese Academy of Sciences) was isolated from Crotalaria sessiliflora (Roder et al., 1992).

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