PF2401-SF, standardized fraction of Salvia miltiorrhiza, induces apoptosis of activated hepatic stellate cells in vitro and in vivo.

During the course of our attempts to develop a potential herbal medicine, we had previously prepared PF2401-SF, a standardized fraction of S. miltiorrhiza, and reported its hepatoprotective activity in vitro as well as in vivo. Since apoptosis of activated hepatic stellate cells (HSCs) is a well-accepted anti-fibrotic strategy, in this study, we investigated the direct effect of PF2401-SF on t-HSC/Cl-6 cells in vitro and on CCl4-induced liver injury in vivo. We evaluated the activation and cleavage of hallmarkers of apoptosis, namely, caspase 3, 8, 9 and PARP. Upregulation of the pro-apoptotic Bax protein and downregulation of the anti-apoptotic Bcl2 protein were also analyzed. Furthermore, in the PF2401-SF treated rats, apoptosis induction of activated HSCs was demonstrated by reduced distribution of α-SMA-positive cells and the presence of high number of TUNEL-positive cells in vivo. Our data suggest that PF2401-SF can mediate HSCs apoptosis induction, and may be a potential herbal medicine for the treatment of liver fibrosis.

2',4',6'-Tris(methoxymethoxy) chalcone induces apoptosis by enhancing Fas-ligand in activated hepatic stellate cells.

Suppression of hepatic stellate cell (HSC) activation and proliferation, and induction of apoptosis in activated HSCs have been proposed as therapeutic strategies for the treatment and prevention of the hepatic fibrosis. We previously showed that 2',4',6'-tris(methoxymethoxy) chalcone (TMMC), a synthesized chalcone derivative, inhibits platelet-derived growth factor-induced HSC proliferation at 5-20 µM. Here, we showed that TMMC induces apoptosis in activated HSCs at higher concentrations (30-50 µM), but is not cytotoxic to primary hepatocytes. Moreover, TMMC induces hyperacetylation of histone by inhibiting histone deacetylase (HDAC) in activated HSCs. Interestingly, TMMC treatment remarkably increased Fas-ligand (FasL) mRNA expression in a dose-dependent manner. Cycloheximide treatment reversed the induction of TMMC on apoptosis, indicating that de novo protein synthesis was required for TMMC-induced apoptosis in activated HSCs. In addition, FasL synthesis by TMMC is closely associated with maximal procaspase-3 proteolytic processing. In vivo, TMMC reduced activated HSCs in CCl(4)-intoxicated rats during liver injury recovery, as demonstrated by α-smooth muscle actin expression in rat liver. TMMC treatment also resulted in apoptosis, as demonstrated by cleavage of poly(ADP-ribose) polymerase in rat liver. In conclusion, TMMC may have therapeutic potential by inducing HSC apoptosis for the treatment of hepatic fibrosis.

Tanshinone II A induces apoptosis and S phase cell cycle arrest in activated rat hepatic stellate cells.

Tanshinone IIA, a major component extracted from the traditional herbal medicine, Salvia miltiorrhiza Bunge, improves blood circulation and treats chronic hepatitis and hepatic fibrosis. Activation of hepatic stellate cells (HSCs) is the predominant event in liver fibrosis. The therapeutic goal in liver fibrosis is the reversal of fibrosis and selective clearance of activated HSCs. We used rat HSCs transformed by Simian virus 40 (t-HSC/Cl-6) to overcome the limitations inherent in studying subcultures of HSCs. Treatment of t-HSC/Cl-6 cells with tanshinone IIA inhibited cell viability in a dose- and time-dependent manner. Tanshinone IIA induced apoptosis as demonstrated by DNA fragmentation, poly(ADP-ribose) polymerase and caspase-3 cleavage, increased Bax/Bcl-2 protein ratio, and depolarization of mitochondrial membranes to facilitate cytochrome c release into the cytosol. Furthermore, this compound markedly induced S phase cell cycle arrest, and down-regulated cyclins A and E, and cdk2. Thus, tanshinone IIA induces apoptosis and S phase cell cycle arrest in rat HSCs in vitro.

Preventive effects of a purified extract isolated from Salvia miltiorrhiza enriched with tanshinone I, tanshinone IIA and cryptotanshinone on hepatocyte injury in vitro and in vivo.

Salvia miltiorrhiza is traditionally used to treat liver disease in Asia. In this study, we tested the ability of a purified extract of S. miltiorrhiza (PF2401-SF) and its constituents, tanshinone I, tanshinone IIA, and cryptotanshinone, to protect against acute and subacute liver damage induced by carbon tetrachloride by measuring serum transaminase levels, the reduced form of glutathione (GSH), antioxidant enzyme activities, and lipid peroxidation levels in the liver. We also evaluated their ability to protect primary cultured rat hepatocytes from tertiary-butylhydroperoxide (tBH) or d-galactosamine (GalN). PF2401-SF was protective at 50-200mg/kg per day in acute liver injury and 25-100mg/kg per day in subacute liver injury. Tanshinone I, tanshinone IIA, and cryptotanshinon (40 microM), inhibited lactate dehydrogenase leakage, GSH depletion, lipid peroxidation and free radical generation in vitro. PF2401-SF and its major constituents, tanshinone I, tanshinone IIA and cryptotanshinone, can protect against liver toxicity in vivo and in vitro due to its antioxidant effects.

Dibenzocyclooctadiene lignans and lanostane derivatives from the roots of Kadsura coccinea and their protective effects on primary rat hepatocyte injury induced by t-butyl hydroperoxide.

A new dibenzocyclooctadiene lignan, acetylepigomisin R ( 1), and a new 3,4-seco-lanostane-type triterpene, seco-coccinic acid F ( 2), along with three known dibenzocyclooctadiene lignans, isovaleroylbinankadsurin A ( 3), kadsuralignan J ( 4), and binankadsurin A ( 5), and one lanostane-type triterpene, 20( R),24( E)-3-oxo-9 beta-lanosta-7,24-dien-26-oic acid ( 6), were isolated from the methanol extract of the Kadsura coccinea roots. Their structures were elucidated on the basis of spectroscopic evidence including ESI-MS, HR-EI-MS, 1D and 2D NMR. The protective effects of these compounds were evaluated in primary cultured rat hepatocytes intoxicated with 1.2 mM T-butyl hydroperoxide. Compounds 1, 3, and 5 showed protective effects with ED (50) values of 135.7, 26.1, and 79.3 microM, respectively.

1,2,3,4,6-penta-O-galloyl-beta-D-glucose from Galla Rhois protects primary rat hepatocytes from necrosis and apoptosis.

In this study, we investigated the hepatoprotective effects of four compounds from Galla Rhois [gallic acid methyl ester, gallic acid, an equilibrium mixture of 3-galloyl-gallic acid and 4-galloyl-gallic acid isomers, and 1,2,3,4,6-penta- O-galloyl- beta- D-glucose (PGG)] in primary rat hepatocytes undergoing necrosis or apoptosis. Treatment with gallic acid methyl ester (12.5 and 50 microM) or PGG (3.125, 12.5 and 50 microM) reduced hepatocyte necrosis induced by tert-butyl hydroperoxide. PGG treatment (4 and 20 microM) also altered hepatocyte apoptosis induced by glycochenodeoxycholic acid. Based on these results, we propose that PGG warrants further evaluation as a hepatoprotective agent, because it protected primary rat hepatocytes from both necrosis and apoptosis.

Cytoprotective constituent of Hoveniae Lignum on both Hep G2 cells and rat primary hepatocytes.

A phytochemical investigation of the EtOH extract of Hoveniae Lignum yielded four phenolic compounds, phloretin (1), 5-(4'-hydroxyphenyl)-gamma-valerolactone (2), (-)-epiafzelechin (3), and maesopsin (4). Compound 1 was hepatoprotective against tacrine-induced cytotoxicity in human liver-derived Hep G2 cells with an EC50 value of 37.55 +/- 0.42 microM. Compound 1 (0.4-200 microM) also significantly reduced tert-butyl hydroperoxide-induced cytotoxicity in rat primary hepatocytes as measured by the cellular leakage of lactate dehydrogenase and the level of aspartate transaminase.

Butein suppresses bile acid-induced hepatocyte apoptosis through a JNK-dependent but ERK-independent pathway.

We investigated the protective effect of butein on glycochenodeoxycholic acid (GCDC)-induced apoptosis in primary cultured rat hepatocytes. Treatment with GCDC at a concentration of 100 microM for 4 h induced apoptosis, and treatment with butein at concentrations of 30 microM inhibited the GCDC-induced apoptosis as shown by the reduced cleavage of poly(ADP-ribose) polymerase, DNA fragmentation, and activation of caspases-3, -8, and -9. c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) play fundamental roles in cell survival, proliferation, and apoptosis. GCDC alone induced ERK and JNK phosphorylation. Butein alone induced ERK activation, and ERK activation was greater in hepatocytes treated with butein and GCDC than in hepatocytes exposed to GCDC alone. Butein treatment reduced JNK activation induced by GCDC. Addition of U0126, an inhibitor of ERK, did not alter the proapoptotic effect of GCDC or the antiapoptotic effect of butein. Addition of SP600125, a specific JNK inhibitor, protected hepatocytes against GCDC-induced apoptosis. These data suggest that butein has a protective effect against GCDC-induced hepatocyte apoptosis and that the protective effect of butein is JNK dependent but ERK independent.

PF2401-SF, standardized fraction of Salvia miltiorrhiza and its constituents, tanshinone I, tanshinone IIA, and cryptotanshinone, protect primary cultured rat hepatocytes from bile acid-induced apoptosis by inhibiting JNK phosphorylation.

Bile acid-induced hepatocyte apoptosis plays an important role in cholestatic liver disease, and the role of apoptosis may be of therapeutic interest in preventing liver disease. The dried root of Salvia miltiorrhiza Bunge (Labiatae) has been used traditionally to treat liver diseases. We investigated the antiapoptotic effects of a standardized fraction of S. miltiorrhiza (PF2401-SF) and its components, tanshinone I, tanshinone IIA, and cryptotanshinone, in primary cultured rat hepatocytes. PF2401-SF was enriched with tanshinone I (11.5%), tanshinone IIA (41.0%), and cryptotanshinone (19.1%). Glycochenodeoxycholic acid (GCDC)-induced apoptosis, as shown by DNA fragmentation, poly(ADP-ribose) polymerase cleavage, and activation of caspases-8, -9, and -3. PF2401-SF and its components, tanshinone I, tanshinone IIA, and cryptotanshinone showed antiapoptotic activity. Treatment with PF2401-SF or with its components significantly inhibited the generation of intracellular reactive oxygen species. Hydrophobic bile acids activate c-Jun-NH(2)-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPK), and extracellular signal-regulated kinase 1/2, and PF2401-SF inhibited the phosphorylation of JNK and p38. All three components of PF2401-SF inhibited JNK phosphorylation. Addition of inhibitors of MAPK showed that inhibition of JNK decreased apoptosis. These data indicate that PF2401-SF and its components protect hepatocytes from GCDC-induced apoptosis in vitro by inhibiting JNK.

Bakuchiol-induced caspase-3-dependent apoptosis occurs through c-Jun NH2-terminal kinase-mediated mitochondrial translocation of Bax in rat liver myofibroblasts.

Liver fibrosis and cirrhosis may be reversible, possibly through the selective clearance of activated hepatic stellate cells/myofibroblasts by apoptosis. Hepatic stellate cells transdifferentiate into myofibroblast-phenotype cells in culture, a process that recapitulates hepatic stellate cell activation in vivo. Bakuchiol, a prenylated phenolic terpene isolated from the seed of Psoralea corylifolia L. (Leguminosae), reduced activated hepatic stellate cells when treated to rats during liver injury recovery period as demonstrated by alpha-smooth muscle actin immunostaining in rat liver and induced apoptosis in activated hepatic stellate cells/myofibroblasts as demonstrated by DNA fragmentation, activation of caspase-3, release of cytochrome c into the cytoplasm, translocation of Bax into mitochondria, and the proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) in vitro. Bakuchiol-induced apoptosis was prevented by z-DEVD-fmk, a specific inhibitor of caspase-3, and z-VAD-fmk, a general caspase inhibitor, suggesting that bakuchiol-induced apoptosis occurs through a caspase-3-dependent pathway in vitro. Bakuchiol treatment stimulated the activation of extracellular signal-regulated kinase 1/2 (ERK), c-Jun NH2-terminal protein kinase (JNK), and p38 mitogen-activated protein kinases (MAPK) in vitro. Pretreatment with SP600125 attenuated the bakuchiol-induced translocation of Bax into mitochondria, cytochrome c release into the cytosol, caspase-3 activation, and PARP cleavage. In contrast, preincubation with SB203580, a p38 MAPK inhibitor, and U0126, an ERK inhibitor, had no effect on bakuchiol-induced cell death and caspase-3 activity. Taken together, these findings indicate that bakuchiol induces caspase-3-dependent apoptosis through the activation of JNK, followed by Bax translocation into mitochondria in rat liver myofibroblasts.

A ginsenoside metabolite, 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol, triggers apoptosis in activated rat hepatic stellate cells via caspase-3 activation.

We investigated the apoptotic effects of the protopanaxadiol ginsenosides, Rb (1) and Rb (2), and their intestinal bacterial metabolite, 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol (M1), and of the protopanaxatriol ginsenoside, Rg (1), and its intestinal bacterial metabolite, 20(S)-protopanaxatriol, in activated rat hepatic stellate cells (HSCs) transformed by Simian virus 40 (T-HSC/Cl-6). As HSCs play a central role in liver fibrosis, agents that selectively induce apoptosis of HSCs could be used to treat this disease. Apoptosis was measured using cell viability tests, DNA fragmentation analysis, and immunoblot analysis of poly(ADP-ribose) polymerase cleavage. M1 (40 microM for 24 h) significantly induced apoptosis in activated rat HSCs. M1 induced apoptosis in a dose-dependent manner as shown by DNA fragmentation, an increased population of cells in the sub-G1 phase, and reduced mitochondrial transmembrane potential. M1 induced caspase-3 activity in a dose- and time-dependent manner. A specific inhibitor of caspase-3 prevented induction of apoptosis by M1 as shown by DNA fragmentation analysis. It is concluded that M1 induces apoptosis in T-HSC/Cl-6 cells via caspase-3 activation.

Polyozellin inhibits nitric oxide production by down-regulating LPS-induced activity of NF-kappaB and SAPK/JNK in RAW 264.7 cells.

Polyozellin, isolated from Polyozellus multiplex (Thelephoraceae), was investigated for its anti-inflammatory activity in the murine macrophage cell line RAW 264.7. Polyozellin inhibited both lipopolysaccharide (LPS)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression in a dose-dependent manner. The effects of polyozellin on the activation of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein (MAP) kinases in these cells were studied in order to elucidate the underlying mechanism. Polyozellin suppressed the activation of both LPS-induced NF-kappaB and the stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), but had no effect on the extracellular signal-regulated kinase (ERK) or p38. These data suggest that polyozellin suppresses iNOS expression by inhibiting the activation of NF-kappaB and SAPK/JNK, leading to the inhibition of NO production.

Honokiol reduces oxidative stress, c-jun-NH2-terminal kinase phosphorylation and protects against glycochenodeoxycholic acid-induced apoptosis in primary cultured rat hepatocytes.

Hydrophobic bile acid-induced apoptosis plays an important role in cholestatic liver disease, and its prevention may be of therapeutic interest. The aim of this study was to investigate the protective effect of honokiol on glycochenodeoxycholic acid-induced apoptosis in primary cultured rat hepatocytes. Glycochenodeoxycholic acid is a hydrophobic bile salt that accumulates intrahepatically during cholestasis and induces hepatocyte apoptosis at pathophysiological concentrations. Primary rat hepatocytes were pretreated with honokiol at concentrations of 40, 20 and 10 microM 5 min before glycochenodeoxycholic acid treatment. Incubation of hepatocytes with glycochenodeoxycholic acid at a concentration of 100 microM for 4 h induced apoptosis as shown by DNA fragmentation, chromatin condensation and cleavage of poly(ADP-ribose) polymerase. Pretreatment with honokiol at concentrations of 40, 20 and 10 microM significantly inhibited the generation of intracellular reactive oxygen species and reduced activation of caspases-8, -9, and -3 and cleavage of poly-(ADP-ribose) polymerase. Glycochenodeoxycholic acid treatment up-regulated phosphorylation of stress-activated protein kinase/c-jun-NH2-terminal kinase which was inhibited by honokiol treatment. Inhibition of stress-activated protein kinase/c-jun-NH2-terminal kinase phosphorylation by SP600125 protected hepatocytes from apoptosis induced by glycochenodeoxycholic acid. These data indicate that honokiol protects hepatocytes from apoptosis induced by glycochenodeoxycholic acid in vitro and this protection may be due to reduced oxidative stress and inhibition of stress-activated protein kinase/c-jun-NH2-terminal kinase phosphorylation.

Skullcapflavone I from Scutellaria baicalensis induces apoptosis in activated rat hepatic stellate cells.

The therapeutic goal in liver fibrosis is the reversal of fibrosis and the selective clearance by apoptosis of hepatic stellate cells (HSCs), which play a central role in liver fibrogenesis. In this study, the apoptotic effect of wogonin, oroxylin A, 2',5,6',7-tetrahydroxyflavone, skullcapflavone I, and baicalein, isolated from the dried root of Scutellaria baicalensis, was investigated in activated rat HSCs, T-HSC/Cl-6 cells transformed with the Simian virus 40. Among the isolated compounds, skullcapflavone I (20 microM for 24 h) significantly induced apoptosis in activated rat HSCs while there was no change in the cell viability of hepatocytes. Skullcapflavone I increased caspase-3 and -9 activities accompanied by the proteolytic cleavage of poly(ADP-ribose) polymerase. Specific inhibitors of caspase-3 and caspase-9 prevented the apoptotic process induced by skullcapflavone I. From these results, skullcapflavone I from S. baicalensis selectively induced apoptosis in T-HSC/Cl-6 cells via caspase-3 and caspase-9 activation.

Protective effect of (S)-bakuchiol from Psoralea corylifolia on rat liver injury in vitro and in vivo.

The aim of this study was to investigate the protective effect of (S)-bakuchiol isolated from the seed of Psoralea corylifolia, on liver injury. Primary rat hepatocyte intoxication was induced by tert-butyl hydroperoxide (tBH), carbon tetrachloride (CCl4) or D-galactosamine (D-GalN). Liver injury was induced by either CCl4 or D-GalN in rats. In vitro, the cellular leakage of lactate dehydrogenase and cell viability following treatment with hepatotoxicants were significantly improved by bakuchiol treatment at a concentration range of 25-200 microM for tBH, 100-200 microM for CCl4 and 100-200 microM for D-GalN-induced hepatocyte injury. Treatment with bakuchiol significantly inhibited lipid peroxidation and intracellular glutathione depletion in hepatocytes induced by tBH, CCl4 or D-GalN. Treatment with bakuchiol (25 or 50 mg/kg, p.o.) at 1, 24 and 48 h after subcutaneous injection of CCl4 significantly reduced the levels of aspartate transaminase and alanine transaminase in serum. Histological observations revealed that fatty acid changes, hepatocyte necrosis and inflammatory cell infiltration in CCl4-injured liver was improved when treated with bakuchiol. Bakuchiol treatment (25 and 50 mg/kg, p.o.) also significantly reduced the levels of aspartate transaminase and alanine transaminase in an acute liver injury model induced by D-GalN. From these results, bakuchiol has a protective effect against tBH, CCl4 or D-GalN-induced hepatotoxicity in vitro or in vivo.

Aloe emodin-induced apoptosis in t-HSC/Cl-6 cells involves a mitochondria-mediated pathway.

The aim of our study was to clarify the apoptosis pathway induced by aloe emodin, an hydroxyanthraquinone present in aloe vera leaves, in rat hepatic stellate cells transformed by simian virus 40 (t-HSC/Cl-6), which retain the features of activated rat stellate cells. Apoptosis was determined by DNA fragmentation, caspase activity assay and western blotting analysis. Treatment of t-HSC/Cl-6 cells with 12.5, 25, or 50 microM aloe emodin inhibited t-HSC/Cl-6 cell viability in a dose- and time-dependent manner. The induction of apoptosis by aloe emodin was confirmed by typical DNA ladder formation and annexin v-propidium iodide flow-cytometric analysis. Aloe emodin treatment of t-HSC/Cl-6 cells caused activation of caspase-3 and caspase-9, detected with a caspase activity assay, although no change was observed in caspase-8 activity. Western blotting showed caspase-3 and caspase-9 active forms and the subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. Aloe emodin induced mitochondrial membrane depolarization. Our data also show that cytochrome c increased in the cytosol but decreased in the mitochondria in a time-dependent manner. Increased Bax and unchanged Bcl-2 levels resulted in an increased Bax/Bcl-2 ratio. Thus, our research provides evidence that aloe emodin-induced apoptosis involves a mitochondria-associated apoptosis pathway.

Honokiol induces apoptosis via cytochrome c release and caspase activation in activated rat hepatic stellate cells in vitro.

The therapeutic goal in liver fibrosis is the reversal of fibrosis and the selective clearance of activated hepatic stellate cells (HSCs) by inducing apoptosis. Over the past several years, we have screened for natural products that mediate apoptosis in activated HSCs. Among the candidate compounds, honokiol, isolated from Magnoliae cortex, was found to induce apoptotic death in activated rat HSCs, while there was no cell viability change in hepatocytes, at concentrations of 12.5-50 microM. Apoptosis was identified by DNA fragmentation, activation of caspase-3 and -9, and the proteolytic cleavage of poly(ADP-ribose) polymerase, down-regulation of bcl-2 and the release of mitochondrial cytochrome c into the cytoplasm.

An isocoumarin with hepatoprotective activity in Hep G2 and primary hepatocytes from Agrimonia pilosa.

Phytochemical investigation of the aqueous extract of the roots of Agrimonia pilosa Ledeb. (Rosaceae), as guided by hepatoprotective activity in vitro, furnished two isocoumarins, agrimonolide (1) and agrimonolide 6-O-beta-D-glucoside (3), and (+)-catechin (2). Compound 1 showed hepatoprotective effects on both tacrine-induced cytotoxicity in human liver-derived Hep G2 cells and tert-butyl hydroperoxide-induced cytotoxicity in rat primary hepatocytes with EC50 values of 88.2 +/- 2.8 and 37.7 +/- 1.6 microM, respectively.

Water-soluble polysaccharide from Eleutherococcus senticosus stems attenuates fulminant hepatic failure induced by D-galactosamine and lipopolysaccharide in mice.

The aim of this study was to investigate whether Eleutherococcus senticosus stems could attenuate D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure in mice. E. senticosus, known as Siberian ginseng, is a popular folk medicine used as a tonic in Asia. Preparations of E. senticosus used in this study were as follows; (i) 70% ethanol extract (ii) water extract (iii) ethanol-soluble part of the water extract (iv) polysaccharide obtained as an 80% ethanol insoluble of the water extract. Preparations were given by intraperitoneal (300 mg/kg and 50 mg/kg) or oral (300 mg/kg) injection at 12 hr and 1 hr before a D-galactosamine/lipopolysaccharide injection. The intraperitoneal injection of water extract and polysaccharide significantly lowered serum levels of tumour necrosis factor-alpha, aspartate transaminase and alanine transaminase, improved the histologic changes in liver, inhibited hepatocyte apoptosis confirmed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method and DNA fragmentation assay, and suppressed the lethality induced by D-galactosamine/lipopolysaccharide. The oral administration of water extract and polysaccharide also reduced serum aspartate transaminase, alanine transaminase and tumour necrosis factor-alpha levels. In contrast 70% ethanol extract and ethanol-soluble part of the water extract had no protective effect when treated intraperitoneally or orally. These results indicate E. senticosus stems attenuate fulminant hepatic failure induced by D-galactosamine/lipopolysaccharide in mice and the protective effect is due to water-soluble polysaccharides in E. senticosus stems.

Tetrandrine induces apoptosis in hepatic stellate cells.

We have previously reported that tetrandrine reduced hepatic stellate cell activation and collagen accumulation in liver fibrosis induced by biliary obstruction. In the present study, we examined the apoptosis-inducing effect of tetrandrine on activated hepatic stellate cells, as the therapeutic goal in hepatic fibrosis is to eliminate the activated hepatic stellate cells by apoptosis. We used rat hepatic stellate cells transformed by Simian virus 40 (T-HSC/Cl-6) to overcome the limitations inherent in studying primary cultures of hepatic stellate cells. Tetrandrine treatment at doses of 25 and 50 microg/ml for 12 h induced apoptosis as confirmed by DNA fragmentation and increased sub-G1 DNA content as detected by flow cytometric analysis. Tetrandrine also induced the activation of caspase-3 protease and subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. In conclusion, our results demonstrate that tetrandrine induces apoptosis of T-HSC/Cl-6 cells, and these results should contribute to the development of new agents for the treatment of hepatic fibrosis.