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Chemico-Biological Interactions 223 (2014) 1–9

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Chemico-Biological Interactions
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Astin B, a cyclic pentapeptide from Aster tataricus, induces apoptosis
and autophagy in human hepatic L-02 cells
Li Wang, Ming-Dan Li, Pei-Pei Cao, Chao-Feng Zhang ⇑, Fang Huang, Xiang-Hong Xu,
Bao-Lin Liu, Mian Zhang ⇑
Research Department of Pharmacognosy, China Pharmaceutical University, Longmian Road 639, Nanjing 211198, PR China

a r t i c l e

i n f o

Article history:
Received 15 February 2014
Received in revised form 21 August 2014
Accepted 4 September 2014
Available online 16 September 2014
Astin B
Liver injury
Aster tataricus

a b s t r a c t
Astins (including astin B) are a class of halogenated cyclic pentapeptides isolated from the medicinal herb
of Aster tataricus. However, our previous works showed that the herbal medicine was hepatotoxic in vivo,
and a toxicity-guided isolation method led to the identification of a cyclopeptide astin B. Astin B is structurally similar to cyclochlorotine, a well-known hepatotoxic mycotoxin. Thus, the aim of this study was
to determine the potential cytotoxic effects and the underlying mechanism of astin B on human normal
liver L-02 cells. We found that astin B has hepatotoxic effects in vitro and in vivo and that hepatic injury
was primarily mediated by apoptosis in a mitochondria/caspase-dependent manner. Astin B provoked
oxidative stress-associated inflammation in hepatocytes as evidenced by increased levels of reactive oxygen species (ROS), reduced contents of intracellular glutathione (GSH), and enhanced phosphorylation of
c-Jun N-terminal kinase (JNK). Furthermore, the mitochondria-dependent apoptosis was evidenced by
the depolarization of the mitochondrial membrane potential, the release of cytochrome c into cytosol,
the increased ratio of Bax/Bcl-2, and the increased activities of caspases-9 and -3. Interestingly, astin B
treatment also induces autophagy in L-02 cells, characterized by acidic-vesicle fluorescence, increased
LC3-II and decreased p62 expression. Autophagy is a protective mechanism that is used to protect cells
from apoptosis. The presence of autophagy is further supported by the increased cytotoxicity and the
enhanced cleaved caspase-3 after co-treatment of cells with an autophagy inhibitor, also by increased
LC3-II and decreased p62 after co-treatment with a caspase inhibitor. Taken together, astin B, most likely
together with other members of astins, is the substance that is primarily responsible for the hepatotoxicity of A. tataricus.
Ó 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
The hepatocyte is especially vulnerable to injury due to its central role in xenobiotic metabolism including the metabolism of
drugs. Therefore, drug-induced liver injury is the most frequent
reason for post-marketing warnings and withdrawal [1]. The death
of hepatocytes and other types of hepatic cells is a characteristic
feature of drug-induced injury [2]. Based on morphological appearance, cell death has been classified into several modes such as
apoptosis, necrosis, necroptosis, autophagy, and cornification [3].
Of these, apoptosis is considered to be a common pathway for
execution of hepatocytes upon liver injury. In response to xenobiotic metabolism, hepatocytes often generate excess reactive
oxygen species (ROS), which evoke oxidative stress-associated
⇑ Corresponding authors. Tel./fax: +86 25 86185137.
E-mail addresses: (C.-F. Zhang),
(M. Zhang).
0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

inflammation, leading to mitochondrial dysfunction [4,5].
Mitochondria play a key role in the regulation of redox homeostasis and apoptosis in cells [6]. A loss of mitochondrial function
allows the release of a number of proapoptotic factors, such as
cytochrome c, which induces caspase activation to trigger
mitochondria-dependent apoptotic cell death [7]. Accumulating
evidence demonstrates that hepatocyte apoptosis is tightly associated with drug-induced liver injury [6,8]. However, autophagy is
understood to be a mechanism of protection against various forms
of human diseases, including drug-induced liver injury, with an
extremely complex interplay [9,10].
Astins, mainly including astins A–I, are a class of natural halogenated cyclic pentapeptides isolated from the root of Aster tataricus
L. f. (RAT, Compositae) that has been used for over 2000 years in
traditional Chinese medicine for the relief of coughs and the
removal of phlegm. This class of compounds exhibits antitumor
and immunosuppressive activities [11–13]. However, our previous
studies discovered the potent hepatotoxicity of RAT in mice, and a

USA). 2. Haimen. Astin B is structurally similar to cyclochlorotine. a well-known hepatotoxic mycotoxin isolated from Penicillium islandicum [15]. were grown in RPMI-1640 medium supplemented with 10% FBS. 100 U/mL penicillin and 100 lg/mL streptomycin and incubated at 37 °C in a humidified atmosphere (5% CO2). Bax. Materials and methods 2. Astin B also induces autophagy in L-02 cells. Beyotime. 2. 1. China). 2. Antibodies for c-Jun N-terminal kinase (JNK). and measured at an .05 compared with the control group (0 lM). Effects of astin B on cell viability and lactate dehydrogenase (LDH) activity in L-02 cells. 1A) was given [14].8 0. Louis. China) in accordance with the manufacturer’s instructions. Finland) at a wavelength of 490 nm. Bcl-2. from which a halogenated cyclic pentapeptide astin B (Fig. toxicity-guided isolation method led to the identification of an astin-rich fraction (71. we evaluated the effects of astin B on the modulation of cell death. Measurement of intracellular ROS and GSH The generation of intracellular ROS was assessed using the ROS-specific fluorescent dye 2. a normal human liver cell line from the Cell Bank of the Chinese Academy of Sciences (Shanghai. Drugs and chemicals Astin B (Fig. 2. (B) Cells were treated with astin B for 12.2% of the relative content). USA. Nanjing.1 * 120 100 80 60 40 20 0 0 15 30 45 60 0 15 Astin B (µM) 30 45 60 Astin B (µM) Fig. and caspase-3 were purchased from Cell Signal Technology Inc. phosphorylated JNK (pJNK). the cells were washed with PBS.). MTT was added to each well to reach a final concentration of 0.2 L. and 48 h. but the hepatotoxic effects and the underlying mechanisms were unknown. For experiments in cells. Chlorogenic acid (CGA) was obtained from Nanjing Zelang Medical Technology Company with a purity of 98% (Nanjing. Cell culture L-02 cells. Cytotoxicity assay L-02 cells were seeded at an initial density of 1  105 cells/mL in 96-well plates for 24 h and were incubated with fresh medium containing different concentrations of astin B for 12.).6 * 0. USA). The supernatant was collected. Our experiments indicate that astin B has marked toxic effects in vitro and in vivo and induces hepatic cell death mainly by apoptosis through a mitochondria/caspasedependent pathway. / Chemico-Biological Interactions 223 (2014) 1–9 Cl O A N HN O Cl O NH HN OH N H O O OH B 180 * 1 * Relative viability 0.4.7 * * * * 140 * * 0.4 0. solutions of astin B and CGA were prepared in DMSO and diluted to the desired concentrations in FBS-free medium. Anti-b-actin antibody and secondary anti-mouse and anti-rabbit antibodies were obtained from Lianke Biotechnology (Hangzhou. (A) Chemical structure of astin B. 24.5 C 160 * 0. and LDH leakage was measured using commercially available kits.9 * 0. After incubation. Fetal bovine serum (FBS) was obtained from Hyclone (Thermo. the cells were incubated with astin B for 24 h. 1A) was obtained from previous experiments [14] with a purity of more than 98%. China). The medium was renewed every 2 days until the cells were grown to confluence. and cell viability was determined by the MTT assay. Japan).1. China).5-dimethylthiazol-2-yl)-2. The insoluble formazan was collected and dissolved in DMSO and then measured using a microplate reader (Thermo. ⁄p < 0. rinsed three times with serum-free culture media. (C) Cells were treated with astin B for 24 h.7-dichlorofluorescein diacetate (DCFH-DA. 24. L-02 cells were seeded at an initial density of 1  105 cells/mL in 96-well plates. In this study.5-diphenyl tetrazolium bromide (MTT) were obtained from Sigma–Aldrich (St.5 mg/mL. which appears to protect cells from apoptosis to some extent. For the assay of lactate dehydrogenase (LDH).2. 3-methyladenine (3-MA) and 3-(4. The DMSO concentrations in the experiments never exceeded 0. (Massachusetts. Wang et al. and LDH activity was determined with a commercial kit (Jiancheng.3 LDH release (%) 1. Data in B and C are expressed as the mean ± SD from three independent experiments with n = 6 for MTT and n = 4 for LDH. and 48 h. loaded with 10 lmol/L DCFH-DA at 37 °C for 30 min away from light.3. Earle’s balanced salts solution (EBSS).1%. After 12 h exposure to astin B. USA. South America). RPMI-1640 medium was obtained from Gibco BRL (NY. All of the other reagents were purchased from Amresco (Ohio. Anti-LC3 antibody was obtained from MBL (Nagano. apoptosis and autophagy in human normal liver L-02 cells. China).

10% SDS. The cell lysates were centrifuged at 18. The assay was performed with a JC-1 kit in accordance with the manufacturer’s instructions (Beyotime). Liver tissue was excised and fixed in 10% phosphatebuffered formalin and embedded in paraffin. New Jersey. 3 2. One hour after the last administration. approximately 1  105 L-02 cells were cultured in 6-well plates and separately treated with or without astin B for 24 h. 2. After further incubation at 37 °C for 4 h. the cells were analyzed using a flow cytometer (Becton Dickinson.5 mM MgCl2. the cells were cultured with astin B for 24 h and then harvested by centrifuging.6. The proteins were probed with primary antibodies and HRP-labeled secondary antibodies and visualized using super ECL detection reagent (Beyotime). 1 mM dithiothreitol. the extracted samples were loaded at 20 lL/per lane. Flow cytometric analysis of apoptotic cells 2. California. Briefly. blood was collected from the orbital sinus and centrifuged at 12. The intracellular GSH level was determined with commercially available kits (Jiancheng.8. the L-02 cells were gently trypsinized and washed once with PBS. centrifuged at 800 rpm for 5 min. USA) in accordance with the manufacturer’s instructions. the mice were killed by cervical dislocation after isoflurane inhalation.10. Finland) at a wavelength of 405 nm. 50% glycerine. Statistical analyses were performed using one-way analysis of variance (ANOVA) .8. China) in accordance with the manufacturer’s instructions. In brief. The apoptotic cells with hypodiploid DNA content were measured by quantifying the sub-G1 peak in the cell cycle pattern. approximately 1  105 cells were seeded in 24-well plates and incubated with astin B for 12 h. Sections were prepared and stained with hematoxylin and eosin (H&E) for histopathological analysis. 2 lg/mL aprotinin and 1 mM phenylmethylsulfonyl fluoride) for 10 min on ice followed by centrifugation at 10.2. Billerica. 2.1. Nanjing. Protein concentrations were measured with a bicinchoninic acid protein assay kit (Beyotime). For GSH assay. Measurement of cytochrome c (cyt c) content After treatment with astin B for 24 h. Next.000 events were recorded per sample. Analysis of caspase activity The activities of caspase-3 and caspase-9 were evaluated using caspase-3 and caspase-9 activity assay kits (Beyotime).9.7.1. 1 mM EGTA. 10 mM NaCl. The cells were then observed and photographed by fluorescence microscopy. 1 mM EDTA. Analysis of mitochondrial membrane potential (DWm) The membrane potential assay is based on the JC-1 dye: JC-1 emits green fluorescence when the DWm is relatively low. which consists of 1 M Tris–HCl. China) at 37 °C for 1 min in the dark. Wang et al. Animals Male ICR mice (6–8 weeks of age) were obtained from the Laboratory Animal Center of Nanjing Qinglongshan (Nanjing. After gentle pipetting. In brief.000 rpm for 5 min at 4 °C to obtain serum for the test of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities by commercial kits (Jiancheng. Nanjing. the absorbance was measured using a microplate reader (Thermo. Acridine orange (AO) staining of autophagic cells Autophagic cells were detected by AO staining in accordance with published procedures [18]. Sub-G1 peak The apoptosis induced by astin B was determined using a cell cycle and apoptosis analysis kit (Beyotime) in accordance with the manufacturer’s instructions. After a 24-h exposure. the cells were stained by 3 lL of Annexin-V-FITC (AV) and 3 lL of propidium iodide (PI) (BD Pharmingen.2. and 10 lL of RNase A in the dark for 30 min at 37 °C. 25 lL of propidium iodide (PI). the cells were lysed in preparation buffer (250 mM sucrose. After boiling for 10 min.000 rpm for 20 min at 4 °C to separate the cytosolic and mitochondrial fractions [17]. 2. / Chemico-Biological Interactions 223 (2014) 1–9 excitation wavelength of 488 nm and an emission wavelength of 530 nm using a microplate fluorescence reader (MD. USA). 2. 1.11. washed with PBS. 20 mM HEPES-KOH (pH 7. 2. and resuspended in 200 lL of binding buffer. For each experiment. Statistical analysis The data are expressed as the means ± SD (standard deviation) from at least three independent experiments. and then transferred to a PVDF membrane (Millipore.4). However. 2. China).8. Western blot analysis The L-02 cells (approximately 6  105 in number) were extracted with 100 lL of lysate buffer. L-02 cells from 6-well plates were collected and rinsed with cold PBS and later lysed using lysis buffer for 1 h on ice. 10. 10. USA). 2. Minnesota. JC-1 aggregates and emits a red fluorescence when the DWm is high [16]. suspended in distilled water) or distilled water by gavage for 7 days. USA).L. Two groups of mice (10 per group) were administered daily with astin B (10 mg/kg. Hepatotoxicity in mice All of the mice were acclimatized for 3 days prior to experimental procedures.5. and colored with AO (1 lg/mL) (Generay.000 rpm for 10 min at 4 °C. Animal experiments 2.11. USA). The cells were rinsed twice with JC-1 staining buffer and were then observed and photographed using an Olympus fluorescence microscope. Shanghai. After treatment. the cells were collected and fixed with 70% ethanol at 4 °C overnight. The care and treatment of these mice were in accordance with the Provisions and General Recommendations of the Chinese Experimental Animals Administration Legislation.000 events were recorded per sample. 2. 2. USA) for 15 min at room temperature in the dark and analyzed using a flow cytometer (Becton Dickinson.11. This study was approved by the Animal Ethics Committee of the School of Chinese Materia Medica.0% bromophenol blue. The contents of cyt c in the cytosol and mitochondria were determined by ELISA kits (R&D Systems. and b-mercaptoethanol.12. China Pharmaceutical University. The assays were performed in 96-well plates by incubating 10 lL of the cell lysate supernatant in 80 lL reaction buffer containing 10 lL Ac-DEVD-pNA (for caspase-3) and 10 lL Ac-LEHD-pNA (for caspase-9). centrifuged at 800 rpm for 5 min and stained with 500 lL of buffer. New Jersey. spectramax M3. fractionated on 12–15% tris–glycine precast gels. China). They were maintained with free access to pellet food and water in plastic cages at 21 ± 2 °C and kept on a 12-h light/dark cycle. 1. Annexin-V/PI staining After 24 h treatment. For each experiment. L-02 cells (1  105 cells/mL) in 6-well plates were separately treated with or without astin B for 24 h and were incubated with JC-1 staining solution (5 lg/mL) for 20 min at 37 °C in the dark.

Astin B induced oxidative stress in L-02 cells. 100 lM) for 12 h. A * * As apoptotic proteins. Astin B induces oxidative stress DCF fluorescence intensity 3. This may be evidenced from the reverse side that astin B can cause oxidative stress and liver injury. ⁄p < 0. Astin B regulates Bcl-2/Bax expression and increases caspase-3 and -9 activity To determine whether astin B induces oxidative stress. In contrast. 4B) in a concentration-dependent manner. B. 3B). The results showed that astin B induced concentration-dependent DWm collapse in L-02 cells after incubation with astin B for 24 h (Fig. 24. the phosphorylation of JNK was obviously enhanced after treatment with astin B for 3. we observed the effects of astin B on ROS production and GSH levels. 2B). Data in (A. (A) Cells were treated with astin B for 12 h. As shown in Fig. and phosphorylated JNK was determined using Western blot analysis. Astin B promotes ROS production and decreases GSH levels. Because JNK activity and oxidative stress are tightly associated. 3. the release of cyt c from the mitochondria into the cytosol in the L-02 cells was also detected. and 6.4. Astin B induces mitochondrial dysfunction To assess whether astin B affects the functioning of mitochondria.01).24% (p < 0. the intracellular ROS levels increased significantly when the cells were exposed to astin B for 12 h (Fig. and 48 h. The results of the MTT assay showed that astin B decreased cell viability in a concentration-dependent and timedependent manner (Fig. Values of p < 0. the cell viability decreased to 39. Increased LDH leakage was observed only in cells exposed to high concentrations of 45 and 60 lM. 30. Thus. 1C. there was no significant leakage of LDH in cells after treatment with astin B at 15 and 30 lM for 24 h. and D) are expressed as the mean ± SD from three independent experiments with n = 4–6. (C) Cells were treated with astin B (30 lM) at regular intervals. Caspases are most likely the most important effector molecules for the execution of apoptosis [21]. 2D). The ratio of Bcl-2 to Bax determines the survival or death of the cells following an apoptotic stimulus [20].1. Compared with untreated cells.05 were considered to be statistically significant. 6. and intracellular ROS was assessed by the ROS-specific fluorescent dye DCFHDA.76% (p < 0. After pretreatment with CGA for 12 h. As shown in Fig. #p < 0.4 250 200 D 1. Exposure of cells to astin B for 24 h significantly increased the cyt c content of the cytosol fraction (Fig.0 0.4 L. and 60 lM) groups increased by 7.05 compared with the control group (0 lM). we determined the changes in the mitochondrial membrane potential (DWm).2 0. the GSH levels in the cells were reduced in a concentration-dependent manner after treatment with astin B for 24 h (Fig.05).8 0. 24. Furthermore. / Chemico-Biological Interactions 223 (2014) 1–9 followed by Student’s two-tailed t-test. (D) Cells were pretreated with chlorogenic acid (CGA+. (B) Cells were treated with astin B for 24 h. 3A). 6. 1B). Chlorogenic acid (CGA) is a well-known antioxidant and potential 5000 hepatoprotective [19].2. 2A). and 48 h. compared with the corresponding groups without pretreated by CGA (Fig. leading to a state associated with apoptosis. After 48 h treatment. 9. Results 3. we further observed the effects of astin B on JNK phosphorylation in cells treated with astin B at 30 lM for 0. 12.03% (p < 0. following treated with astin B for 24 h.6 0. Leakage of LDH from cells is an indicator of cell necrosis. and cell viability was determined by the MTT assay. whereas Bax accelerates apoptotic death and counters the death repressor activity of Bcl-2. and 12 h. 3.3. 3. Treatment of L-02 cells with astin B significantly induced the expression of pro-apoptotic Bax and decreased the expression of anti-apoptotic Bcl-2 in a concentrationdependent fashion (Fig. 2. and the intracellular GSH level was determined using commercially available kits. Wang et al.0 0 0 15 30 Astin B (µM) 45 60 0 15 30 Astin B (µM) 60 Fig.4 0. Astin B inhabits proliferation of L-02 cells The potential cytotoxic effect of astin B was investigated by incubating L-02 cells with astin B at different concentrations for 12.05 compared with the corresponding CGA group. 4A).22% at 60 lM.2 Relative viability GSH (nmol/mg protein) 300 B * 150 100 * 50 * * 1.001). The results shown in C are representative of three independent experiments. When the L-02 cells were treated with astin B for C * 4000 3000 2000 1000 0 0 15 30 60 Astin B (µM) 1. . Bcl-2 is a powerful antagonist of apoptotic death programs. 3. the cell viability of astin B-treated (15. astin B significantly increased the Bax/Bcl-2 ratio (Fig. 2C.

. 3.8 0. / Chemico-Biological Interactions 223 (2014) 1–9 A B 400 Cyt c (ng/mg protein) 350 300 Mitochondria Cytosol 250 * * 200 * * 150 * * 100 50 0 0 15 30 0 45 60 15 30 45 60 Astin B (µM) Fig. 50 lM). and (B) the ratio of Bax/Bcl-2 was calculated by densitometry.4 0. was determined by the MTT assay. ⁄p < 0.4 E zVAD - 1. Cells were treated with astin B for 24 h: (A) Bax and Bcl-2 expressions were determined by Western blot. The results shown in (A) are representative of three independent experiments. 4. the reader is referred to the web version of this article. The proportion of green fluorescence emission represents the DWm collapse degree. (A) L-02 cells were treated with astin B for 24 h and subsequently incubated with JC-1 dye for 20 min. and the content of cytochrome c was measured with ELISA kits. Wang et al.05 compared with the control group (0 lM) in (B–D) or with the corresponding z-VAD-group in E. then observed and photographed by an Olympus fluorescence microscope. a pan-caspase inhibitor.) 5 A B * Ratio of Bax/Bcl-2 4 * 3 2 * 1 0 0 15 30 45 60 Astin B (µM) 4 9 C Caspase-9 Fold of control Fold of control * * * Caspase-3 7 3 2 D 8 * * 1 6 * 5 * 4 * 3 2 1 0 0 0 15 30 45 60 0 15 1. Astin B decreased the mitochondrial membrane potential (DWm) and promoted cytochrome c release.05 compared with the control group (0 lM). (B) Cells were treated with astin B for 24 h. The results shown in A are representative of three independent experiments. (For interpretation of the references to colour in this figure legend. Astin B regulated Bcl-1/Bax expression and increased activities of caspases-9 and -3 in L-02 cells.2 Relative viability 30 45 60 Astin B (µM) Astin B (µM) * 1 * zVAD + * 0. ⁄p < 0. (E) Cell viability in the presence of z-VAD-fmk (z-VAD+.5 L.2 0 0 15 30 60 Astin B (µM) Fig. The data in B-E are expressed as the mean ± SD from three independent experiments with n = 3. Data in (B) are expressed as the mean ± SD from three independent experiments with n = 4. Enzymatic activities of (C) caspase-9 and (D) caspase-3 were measured using commercial kits.6 0.

30. the activities of caspases-3 and -9 were increased in concentration-dependent manners (Fig. the expressions of LC3-I/LC3-II and p62. the population of apoptotic cells. As shown in Fig. The results showed that the treatment of L-02 cells with astin B significantly enhanced LC3-II and reduced p62 expression (Fig. and (C and D) apoptotic and necrotic cells were clarified by Annexin V-FITC (AV) and propidium iodide (PI) staining. Cells were treated with astin B for 24 h. 3. 15. and apoptotic cells were determined by flow cytometry. both key indicators for autophagosome formation [23. after exposure to astin B for 12 h. 6A.24]. Wang et al. which can clearly discriminate among the four types of cells. i. 6E). The data in (B) and (D) are expressed as the mean ± SD from three independent experiments. autophagic vacuoles were visible in the cells. 3. In addition. along with lower increases of necrotic and late apoptotic cells from 4.6. 5A and B). These results indicated that astin B-induced LC3-II enhancement and p62 redaction were the A C * B * 12 * 10 60 Cell number (%) Cells of sub-G1 peak (%) 16 14 * 8 6 4 40 10 0 30 Astin B (µM) 45 60 AV+PI- 20 0 15 * * AV+PI+ 30 2 0 D 50 * 0 * 15 ** 30 * 45 * 60 Astin B (µM) Fig. These results indicate that apoptosis is one of the major ways for astin B to induce L-02 cell death. indicating apoptotic cell death induced by astin B in L-02 cells. 4E).03% to 47. 5.22% (Fig.e. On the other hand. and 60 lM for 24 h. After treatment of the cells with astin B at 0. were examined by Western blot analysis. Therefore. 4E). compared with the groups that were only treated by astin B with corresponding concentrations (Fig. of the L-02 cells.6 L. (A and B) The population of sub-G1 peaks (indicated by an arrow) was measured by propidium iodide staining.5. early apoptotic cells (AV+/PI). .. and significantly reduced cell viability in L-02 cells (Fig. 6B). The results shown in (A) and (C) are representative of three independent experiments. a method of AO staining was employed to detect autophagic effects in L-02 cells. Compared with treatment with astin B alone. 5C and D). Astin B increased the apoptotic L-02 cells.05 compared with the control group (0 lM). co-treatment with 3-MA obviously attenuated LC3-II expression. we analyzed the sub-G1 peaks. early necrotic and late apoptotic cells (AV+/PI+). and late necrotic cells (died without apoptosis) (AV/PI+).. the early apoptotic L-02 cells increased obviously in a concentration-dependent fashion from 8. Co-treatment of astin B with z-VAD-fmk (a pan-caspase inhibitor) could increase the viability of the L-02 cells to some extent. i. After treatment with astin B at same concentrations for 24 h. These data indicate that the stimulus of astin B induces both autophagy and apoptosis in hepatocytes. 6C and D). co-treatment with z-VAD-fmk resulted in an accrual of autophagic pathway evidenced by increased LC3-II and decreased p62 (Fig. which may be one of the main hepatotoxic components in RAT. This finding was further evidenced by the Annexin-V/PI staining method. which may partially explain why the cell viability was only increased slightly on blocking apoptosis (Fig.09% to 14. rapidly enhanced caspase-3 expression. 4C and D). ⁄p < 0.12%. unaffected cells (AV/ PI). Astin B induces apoptotic cell death To confirm whether astin B induced cell death through apoptosis. / Chemico-Biological Interactions 223 (2014) 1–9 24 h.64% to 16. the connection between autophagy and apoptosis was investigated with astin B-induced L-02 cells by co-treatment with 3-MA (an autophagy inhibitor) or z-VAD-fmk (a caspase inhibitor). Furthermore.33% (Fig. the percentage of the sub-G1 peak gradually increased in a concentration-dependent manner from 4. These results suggest that astin B most likely mediates apoptotic cell death and that they do so mainly in a caspase-dependent manner. Astin B induces autophagy against apoptosis Autophagy is important for cell death decisions and can protect cells by preventing them from undergoing apoptosis [22].e. 45.

/ Chemico-Biological Interactions 223 (2014) 1–9 A C 1. the results shown in (B) are representative of five independent experiments at a magnification of 200. The results shown in (B. autophagy in the L-02 cells was detected by acridine orange (AO) staining. Earle’s balanced salt solution (EBSS) as positive control. Astin B induced autophagy in L-02 cells to protect from apoptosis. (A) ALT and AST levels in serum were determined by commercial kits. The data in (D) are expressed as the mean ± SD from three independent experiments. Hepatotoxicity of astin B in mice. and orange-colored vacuoles indicated positive results. and E) are representative of three independent experiments.01 compared with the control group. Wang et al. The results shown in A are representative of three independent experiments.4 0. C.8 0.05 0. (E) L-02 cells were treated with astin B (60 lM) with or without the presence of the pan-caspase inhibitor z-VADfmk (50 lM) for 12 h.6 0. (A) After treatment with astin B (30 lM) for 12 h. .7 L.2 Relative viability 1 D P ˘ 0. the levels of p62 and LC3-I/LC3-II were determined by Western blot analysis. and cell viability was measured by the MTT assay. (C and D) L-02 cells were exposed to astin B (60 lM) with or without the presence of the autophagy inhibitor 3-MA (2 mM) for 12 h. the levels of LC3-I/LC3-II and procaspase-3/cleaved caspase-3 were determined by Western blot analysis. A B 160 Enzyme activity (U/L) 140 ALT ** 120 AST 100 ** 80 60 40 20 0 Control Astin B Fig. 6. The data in (A) are expressed as the mean ± SD. 30. 7. Ten mice were orally administered with vehicle (control group) or astin B at 10 mg/kg once daily for 7 consecutive days.2 0 E B Fig. (B) After treatment with astin B (15. 60 lM) for 12 h. (B) Slices of liver were stained with H&E for histopathological analysis. ⁄⁄p < 0. the levels of p62 and LC3-I/LC3-II in L-02 cells were determined by Western blot analysis.

In such regulation. we observed enhanced pJNK expression when the cells were exposed to astin B. Astin B induces liver injury in mice To investigate whether astin B induces liver injury in vivo. 7B). decreased cell viability and dramatically increased expression of cleaved caspase-3 were observed. the serum levels of ALT and AST in mice treated with astin B were elevated about 11. whether autophagy is a secondary result of cell death that functions to remove damaged organelles or if it is directly induced by astin B in an apoptosisindependent manner. Furthermore. Owing to their special and interesting structures. In summary. AO-stained autophagic vacuoles. Changes in ROS and GSH act as intracellular signals and can evoke inflammation by the regulation of transcription factor molecules through JNK activation [27].8 L.26 times. 4. and their toxic or side effects were often overlooked. Conversely. into the cytosol. These results indicate that apoptosis and autophagy induced by astin B were interacted and that autophagy could effectively protect the cells from B-induced cell apoptosis. the participation of the Bcl-2 family (which contains both pro. autophagy prevents cell death and de facto suppresses apoptosis. we found that an astin-rich fraction (astins were 71. Our experiments showed that astin B induces the collapse of DWm with an increased release of cyt c from the mitochondria into the cytosol. The protective pathway of autophagy. Therefore alterations in the GSH level can be monitored as an indication of oxidative stress in cells [26]. Cyt c is required for the activation of caspase-9. respectively (Fig. However. Discussion Our previous work suggested that pentapeptides. astin B could also induce liver injury in vivo. thereby triggering cell apoptosis [29]. co-treatment with caspase inhibitor z-VAD-fmk led to an enhanced autophagy for astin B-treated cells evidenced by increased LC3-II and decreased p62 expression. our results clearly showed that astin B. GSH is a major antioxidant that guards cells against oxidative injury by diminishing ROS. and it activates downstream caspase-3. Generally. were most likely the principal toxic components in RAT [14]. which discharges cyt c into the cytosol. The functional relationship between apoptosis and autophagy is quite complex. Compared to the control group. we wondered whether ROS-associated inflammation would impair mitochondrial function. The increased ratio of Bax/Bcl-2 observed in cells treated with astin B indicates that astin B induces mitochondrial outer membrane permeabilization. astin B will also induce autophagy to protect cells from apoptosis and to alleviate hepatic injury to some extent. This finding indicates that apoptosis is most likely the major event that is responsible for the cell death. the inhibited cell proliferation and survival by astin B could be reversed by antioxidants. the increased activities of caspases-9 and -3 observed in treated cells suggest that the astin B-induced apoptosis may follow a caspase-dependent intrinsic mitochondrial pathway. The initiation of apoptosis induced by astin B was further characterized by an increased sub-G1 peak population during the cell cycle and a predominant population of early apoptotic cells among the dead cells. Apoptosis and autophagy are genetically regulated processes that regulate cell fate. We hope that our finding will be helpful for providing advice regarding drug safety for the clinical use of RAT and the development of new pharmaceuticals. Such cell death will lead to liver injury. enhanced LC3-II and reduced p62 in L-02 cells treated with astin B confirmed that astin B can also induce autophagy in hepatocytes. i. In several scenarios. As a result. tataricus.9 and 2. Furthermore. All these alterations indicate that the astin B stimulus induces ROS-associated inflammation in hepatocytes. only moderately increased in cells that were exposed to high concentrations. Consistent with this view. one of the cyclic pentapeptides. This result suggests that mitochondrial dysfunction results in the opening of the mitochondrial permeability transition pores. Apoptosis and necrosis are the most frequent events in xenobiotic metabolism-induced liver injury [25]. Uncontrolled ROS production and/or decreased antioxidant defenses result in oxidative stress.78% at 60 lM after 48 h exposure. astins (including astin B) can be considered to be the major hepatotoxic substances in the herbal medicine derived from A.. Although astins have been reported to kill tumor cells and prevent colitis through regulation of apoptosis [11–13]. as evidenced by diffuse hydropic degeneration of hepatocytes with moderate inflammatory cell infiltration (Fig. Although autophagy has been observed in many dying cells.31]. Moreover. such as cyt c.7. Wang et al. Astin B treatment enhanced ROS production with downregulation of GSH levels in L-02 cells. an indicator of necrosis. remains to be determined.6]. Autophagy functions as a cytoplasmic quality control mechanism to remove protein aggregates and damaged organelles. Given that the mitochondria are the major intracellular source of ROS. consistent with its action in vitro. previous studies of astins focused on their biological activities. activated caspase-9 is involved in the mitochondrial pathway. especially the cyclic ones.2% of the relative content) [14] induces apoptosis in the same fashion (data not shown). In view of the link between mitochondrial dysfunction and apoptosis. we observed the effects of orally administered astin B on the liver cells in mice. Increased pro-apoptotic proteins can actively permeabilize the outer mitochondrial membrane and promote the release of intermembrane space proteins. which has been considered to be an important pathogenic element for the initiation of hepatotoxicity [4. Astins are a class of halogenated cyclic pentapeptides contained in RAT. leading to the release of cyt c from the mitochondria. 7A). such as CGA. 3. our studies suggest that astin B will provoke oxidative stress-associated inflammation and induce hepatocyte apoptosis through a mitochondria-dependent pathway. leading to liver injury. to some extent. and it also appears that similar stimuli can induce either apoptosis or autophagy [32]. which was evidenced by the in vivo experiment. induced hepatic cell death mainly by ROS-associated apoptosis via a mitochondria-dependent pathway. Astin B substantially increased cell death. indicating that oxidative stress occurred following the astin B stimulus. Conflict of Interest The authors declare that there are no conflicts of interest. it is generally accepted that autophagy is a pro-survival and protective pathway [30. and this action partially attenuated apoptotic cell death.e. The liver injury of astin B was further confirmed by histopathological examination. astin B also positively regulated autophagy. . The inhibitory percentage reached 60. The results indicated that. while Bax is a pro-apoptotic multidomain effector protein [28]. MTT assays showed that astin B effectively inhibited cell proliferation and survival. / Chemico-Biological Interactions 223 (2014) 1–9 real events associated with autophagosomes and that astin Binduced apoptotic cell death will be accelerated in the absence of autophagy in hepatocytes. Bcl-2 is an anti-apoptotic member. On the other hand. while the leakage of LDH. After co-treatment with the autophagy inhibitor 3-MA.and anti-apoptotic proteins) is a key event because of the regulative effects of these proteins on mitochondrial dysfunction during apoptosis.

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