Induction of Apoptosis by Ligusticum chuanxiong in HSC-T6 Stellate

Chapter 21

Induction of Apoptosis by Ligusticum chuanxiong in HSC-T6 Stellate Cells Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on June 24, 2016 | http://pubs.acs.org Publication Date: January 12, 2006 | doi: 10.1021/bk-2006-0925.ch021

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Yun-Lian Lin , Ting-Fang Lee , Young-Ji Shiao , and Yi-Tsau Huang 2

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National Research Institute of Chinese Medicine and Institute of Traditional Medicine, National Yang-Ming University, Taipei, Taiwan

Activation of hepatic stellate cells (HSCs) is a key feature of liver fibrosis. During the development of hepatic fibrosis, activated HSCs are the primary source of extracellular matrix. Induction of H S C apoptosis has been proposed as one of therapeutic strategies for this desease. Ligusticum chuanxiong (LC) is a traditional Chinese herb that has been used in the treatment of cardiovascular diseases and to facilitate blood circulation. The present study showed that L C attenuated HSC-T6 growth. B y M T T cell viability and lactate dehydrogenase release assay, L C reduced the cell viability in a dosedependent manner and without significant L D H release. The apoptotic features were observed by cell arrested in S phase, the appearance of a sub-G1 peak and apoptotic cells. The induction of apoptosis by L C was through the activation of caspase-3 and inducing expression of the cell cycle inhibitory proteins, p21 and p27, with no direct cytotoxicity on primary rat hepatocytes. However, tetramethylpyrazine, an active principle of L C had no effect in this studies. The results indicate that L C had a selective effect on HSCs and induce H S C apoptosis, thereby minimizing fibrogenesis. These provide the theoretical basis for L C used to treat and prevent hepatic fibrogenesis, but the active components of L C need to be explored further.

© 2006 American Chemical Society

Wang et al.; Herbs: Challenges in Chemistry and Biology ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on June 24, 2016 | http://pubs.acs.org Publication Date: January 12, 2006 | doi: 10.1021/bk-2006-0925.ch021

282 Hepatic fibrosis is a wound-healing response to various chronic liver injuries (7,2). Stellate cells play a crucial role in the development of liver fibrosis (3,4). A s a result of this process, hepatic stellate cells undergo transformation from vitamin A storing quiescent cells to myofibroblasMike activated cells (5). Activated HSCs exhibit the expression of highly proliferative activity and the accumulation of extracellular matrix (ECM), including the appearance of α-smooth muscle actin (aS M A ) and elevation in type I collagen, the main pathologic feature of hepatic fibrosis (6,7). Suppression of HSC activation and proliferation, and inducing HSC apoptosis have been proposed as one of the therapeutic strategies for the treatment and prevention of hepatic fibrosis (8). Plant-derived antioxidants may emerge as potential anti-fibrotic agents by either protecting hepatocytes against ROS or inhibiting the activation of hepatic stellate cells (9), such as resveratrol (10), curcumin (77), salvianolic acid Β (72), epigallocatechin-3-gallate (EGCG) (75-75). Chinese herbal medicine by facilitating blood circulation and dispersing blood stasis has recently attracted much attention for preventing oxidative stress-related diseases including cancers, cardiovascular diseases and degenerative diseases and opened a new route for anti-hepatic fibrosis drug development (16,17). Ligusticum chuanxiong Hort (LC), a traditional Chinese herb, has been widely used to treat irregular menstrual cycles, cardiovascular diseases and facilitate blood circulation (18) and is claimed to induce vasodilation. Several reports indicated the beneficial effects of LC. Tetramethylpyrazine, an active principle of L C , has been shown as a vasodilator (19), a potassium channel opener to lower calcium influx into cultured aortic smooth muscle cells (20), attenuator of iron-induced oxidative damage and apoptosis in cerebellar granule cells (27), scavenger of superoxide anion and decrease nitric oxide production in human poly-morphonuclear leukocytes (22), antiplatelet aggregation (23), portal hypotensive effect (24) and hepatoprotective and therapeutic effect on econazole-induced liver injury (25). However, the effect of L C on liver fibrosis is still not mentioned. The aim of this study is to elucidate whether L C has any effect on stellate cells. HSC-T6 cells which are immortalized rat liver stellate cells (26) were used to investigate the mechanism involved. The ethanolic extract of the slices of Rhizoma L chuanxiong (LC) was prepared for this study. Cell cycle was analyzed by flow cytometry and apoptotic cells was investigated by TUNEL-staining. Caspase-3 activity was determined by DEVD-p-nitroanilide (pNA) substrate. Western blotting was used for detecting cell cycle proteins. In the present study, we demonstrated, for the first time, that L C induced apoptosis in activated stellate cells, cell cycle arrest at S phase by inducing the expression of cell cycle-dependent kinase inhibitor, p21 and p27, and the activation of caspase-3 in vitro.


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Effect of L C on HSC-T6 Cell Growth The doubling time of HSC-T6 cells was about 24 h in FBS-containing medium. To evaluate the effect of L C on HSC-T6 growth, preconfluent cells were treated with different concentrations of L C extract in serum free medium for 24 h and 48 h. Cell viability was determined by M T T reduction analysis. As shown in Figure 1, treatment of HSC-T6 with L C decreased cell viability in a dose-dependent manner with an I C of 233± 7 μg/mL and 257 ± 5 μg/mL for 24 h and 48 h., respectively. The appearance of cell morphology (not shown) exhibits shrinkage with few extracellular matrix ( E C M ) production and lasted throughout the whole period of treatment. L C cytotoxicity to HSC-T6 cells was also measured as a percentage of lactate dehydrogenase release (LDH) after 24 h treatment (Table I). Compared with control, no significant difference of L D H leakage was found up to a


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Figure 1. The dosage response ofLC in HSC-T6 by MTT assay. 1 χ 10 HSC-T6 cells were treated with or without various concentrations of LC for 24 and 48 h. MTT reduction assay was performedfor cell viability. Each point represents the mean of three independent experiments.


284 Table I. Lactate Dehydrogenase Release in Cultured HSC-T6 Treated with L C

LC fag/mL) 0


Total L D H (%)

50 31 ± 3

100 35 ± 3

200 32 ± 1

33 ± 2

Values are means ± SEM; n=3. Lactate dehydrogenase was calculated as medium LDH/(medium LDH+cellular LDH)

concentration of 200 μg/mL. On the basis of these observations, L C at 200 μg/mL and 24 h treatment was chosen to conduct the following experiments.

Induction of HSC-T6 Apoptosis and the Expression of p21 and p27 by L C Cytoplasmic and nuclear shrinkage, chromatin condensation, membrane blebbing, internucleosomal degradation of D N A and apoptotic body formation, are characteristic morphological changes of apoptosis (27). Morphological assessment by microscope as well as T U N E L staining and flow cytometric sub-Gl cell analysis were further used to evaluate the effect of L C on HSC-T6 survival. As shown in Figure 2, compared with control, L C significantly increased the number of positive TUNEL-staining of nuclei with fluorescein dye, suggesting an increase in apoptotic cells at 24 h after L C treatment. N o significant changes of L D H leakage were previously found in a dose- and time-course studies, suggesting that L C induced D N A damage. LC-induced apoptosis in HSC-T6 cells was also evaluated by sub-Gl cell analysis with propidium iodide (PI) by flow cytometry. The profile of D N A content was obtained by measuring the fluorescence of PI binding to D N A (Figure 2). Cells with lower D N A staining than that of diploid cells were considered as apoptoic. There was an accumulation of subploid population or so-called "sub-Gi peak after LC-treated cells, when compared with the untreated groups, up to 10% and 22% of total cell counts at 24 h and 48 h, respectively. In addition, L C enhanced the expression of cell cycle inhibitory protein, including p21 and p27 (Figure 3) by about 3.0 and 2.2 fold, respectively. The above results suggest that the reduction of viability after L C treatment might result from apoptosis in HSC-T6 cells.



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Figure 2. LC induces HSC-T6 apoptosis. Preconfluent cells were treated with or without 200 pg/ml of LC for 24 hCells were stained as the protocol provided by the TUNEL kit manufacturer.

Figure 3. LC alters the expression of cell cycle related proteins in HSC-T6 cells. Cells were treated with or without 200 pg/mlofLC for the indicated time. P21 andp27 were detected by Western blot with antibodies. Each result represented three independent western blotting analyses.


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Effects of L C on the Activation of Caspase-3 in HSC-T6 Cells Caspase-3 is an executive enzyme for cell apoptosis that functions at the late stages of protease casecade. Caspase-3 activity assay was measured by colorimetric assay using acetyl-Asp-Glu-Val-Asp-/?NA (Ac-DEVD-/?NA) as the specific substrate. Inhibitor of caspase-3 was employed to investigate whether apoptosis was involved in LC-mediated cell death. A c - D E V D - C H O is the cell-permeable inhibitor for caspase-3. The inhibitor block the activity of caspase-3 but did not interfere with its activation. It was used to rule out non-specific protease activity. Treatment of L C for 24 h, the specific activity of caspase-3 was increased by 2.2 fold of control. Compared with control, the inhibitor of caspase-3 completely abolished the induction of caspase-3 activity (Figure 3). Curcumin was used as a positive control, has been demonstrated as an inducer of H S C apoptosis (//), increased caspase-3 activity by about 3.0 fold at 30 μΜ.

Figure 3. LC induced the activation of Caspase-3 in HSC-T6 cells. Cells were treated with 200 pg/mL of LC for 24 h. Activity of caspase -3 was determined by the cleavage ability of Ac-DEVD-pNA in cell extracts. Results are means ± SEM from three independent experiments. Significant differences between control and drug treated group. * p

Induction of Apoptosis by Ligusticum chuanxiong in HSC-T6 Stellate

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