Cell Mol Neurobiol DOI 10.1007/s10571-014-0059-4
ORIGINAL RESEARCH
Beneficial Effect of Astragalosides on Stroke Condition Using PC12 Cells under Oxygen Glucose Deprivation and Reperfusion Bi-Ying Chiu · Ching-Ping Chang · Jia-Wei Lin · Jung-Sheng Yu · Wen-Pin Liu · Yao-Chin Hsu · Mao-Tsun Lin
Received: 12 January 2014 / Accepted: 2 April 2014 © Springer Science+Business Media New York 2014
Abstract Astragalosides (AST) are reported to be neuroprotective in focal cerebral ischemic models in vivo. In this study, the direct effect of AST against oxygen and glucose deprivation (OGD) including neuronal injury and the underlying mechanisms in vitro were investigated. 5 h OGD followed by 24 h of reperfusion [adding back oxygen and glucose (OGD-R)] was used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. AST (1, 100, and 200 g/mL) were added to the culture after 5 h of the OGD ischemic insult and was present during the reoxygenation phases. A key finding was that OGD-R decreased cell viability, increased lactate
B.-Y. Chiu · J.-S. Yu · Y.-C. Hsu (&) Department of Chinese Medicine, Chi Mei Medical Center, Tainan 710, Taiwan, Republic of China e-mail: [email protected] C.-P. Chang · M.-T. Lin Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan 710, Taiwan, Republic of China C.-P. Chang · M.-T. Lin The Ph.D. Program for Neural Regenerative Medicine, Taipei Medical University, Taipei 110, Taiwan, Republic of China J.-W. Lin School of Medicine, Taipei Medical University, Taipei 110, Taiwan, Republic of China J.-W. Lin Neurosurgical Department, Taipei Medical University ShuangHo Hospital, New Taipei City 23561, Taiwan, Republic of China W.-P. Liu · M.-T. Lin (&) Department of Medical Research, Chi Mei Medical Center, Tainan 710, Taiwan, Republic of China e-mail: [email protected]
dehydrogenase, increased reactive oxygen species, apoptosis, autophagy, functional impairment of mitochondria, and endoplasmic reticulum stress in PC12 cells, all of which AST treatment significantly reduced. In addition, AST attenuated OGD-R-induced cell loss through P38 MAPK activation a neuroprotective effect blunted by SB203580, a specific inhibitor of P38 MAPK. Our data suggest that both apoptosis and autophagy are important characteristics of OGD-Rinduced PC12 death and that treating PC12 cells with AST blocked OGD-R-induced apoptosis and autophagy by suppressing intracellular oxidative stress, functional impairment of mitochondria, and endoplasmic reticulum stress. Our data provide identification of AST that can concomitantly inhibit multiple cells death pathways following OGD injuries in neural cells. Keywords Astragaloside · Oxygen glucose deprivation · Reactive oxygen species · Apoptosis · Autophagy · Mitochondria Abbreviations AST ATF6 ATT DEVD-PNA ER GRP78 GSK-3β IRE1 JC-1
LC3B
Astragalosides Activating transcription factor 6 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide Ac-ASP-Glu-Val-ASP-P-nitroanilide Endoplasmic reticulum Glucose-regulated protein 78 (or Bip) Glycogen synthase kinase 3 beta Insositol-requiring kinase 1 5,5´,6,6´-tetrachloro1,1´,3,3´tetraethylbenzimidazolyl-carbocyanine iodide Microtubule-associated protein 1 light chain 3
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LDH LEHD-PNA MMP NAC OGD-R PERK RIPA ROS RPMI Δφm
Lactate dehydrogenase Ac-Leu-Glu-His-ASP-P-nitroanilide Mitochondrial membrane permeabilization N-acetylcysteine Oxygen-glucose deprivation followed by reperfusion RNA-activated protein kinase-like ER resident kinase Radioimmunoprecipitation assay Reactive oxygen species Roswell Park Memorial Institute Mitochondrial transmembrane potential
aging, immune function disorders, and other diseases (Yin et al. 2005, 2010; Liu et al. 2007, 2009; Tohda et al. 2006). However, it is not known whether AST protect against cerebral ischemia (Yin et al. 2010; Luo et al. 2004) by attenuating overproduction of ROS, impairment of mitochondrial function, endoplasmic reticulum stress (ER), and neuronal apoptosis and autophagy. To evaluate the pharmacological potential of AST and its mechanisms, we used, a rat pheochromocytoma cell line (PC12) subjected to OGD followed by reperfusion (OGDR) as an in vitro model of ischemic stroke (Qi et al. 2012; Cheng et al. 2008). The purpose of present study was attempted to identify whether AST are able to inhibit concomitantly multiple cell death pathways following such injury in neural cells.
Introduction Oxidative damage to neurons is the predominant cause of mortality and chronic disability in ischemic stroke (Chan 1996; Kato and Kogure 1999). Reactive oxygen species (ROS) are generated during mitochondrial respiration (Boveris and Chance 1973), which act as intracellular messengers to transducer signals of cell death pathways (Allen and Tresini 2008; Thannickal et al. 2000). Although the mechanisms of cell death after cerebral ischemia remain unclear, it is generally believed that ROS are involved in mitochondrial and apoptotic neuronal death signaling pathways in cerebral ischemia (Niizuma et al. 2010). A more recent report has also shown that autophagic cell death occurs following oxygen and glucose deprivation (OGD) in PC12 cells and cerebral ischemia in rats (Cui et al. 2012). Astragalosides (AST) are the main component of astragalus and commonly used in the prevention and treatment of cardiovascular and cerebrovascular diseases,
Fig. 1 Experimental groups. a Medium group: PC12 cells were treated with normal RPMI plus 21 % O2 for 29 h (□); b OGD-R group: PC12 cells were treated with glucose free RPMI plus 0.2 % for 5 h and then followed by 25 mM glucose plus 21 % O2 for 24 h (▨); c OGD-R AST group: PC12 cells were treated with glucose-free RPMI plus 0.2 % for 5 h and then followed by AST plus 25 mM glucose plus 21 % O2 (▨)
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Materials and Methods Cell Culture The PC12 cell line was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). These cells were cultured in RPMI-1640 medium supplemented with 10 % heat-inactivated horse serum, 5 % fetal bovine serum, 25 mM glucose, 2 mM glutamine, 100/mL penicillin, and 100 μg/mL streptomycin and were maintained in a humidified incubator with 5 % CO2 at 37 °C. All cell culture reagents were purchased from GIBCO (Rockville, MD, USA). Oxygen Glucose Deprivation (OGD) Model (Fig. 1) Cells were plated at a density of 1.5 9 106 cells with growth medium in 6-well plates precoated with poly-L-
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Fig. 2 Protective effects of AST on OGD-R-induced cytotoxicity in PC12 cells. a Effects of AST (50, 100, 200 g/mL) on PC12 cells morphology injury induced by OGD (5 h) followed by reperfusion (24 h) (magnification 9200). AST was adopted during the entire reperfusion phase. b PC12 cells viability was assessed by measuring the MTT reduction. The viability of medium cells (□) was defined as 100 %. #P \ 0.01 OGD alone (▨) versus medium group (□); * P \ 0.05 OGD AST (▩) versus (▨) group. See Fig. 1 legend for abbreviations. c Effects of AST (100 g/mL) on OGD-R-induced increased intracellular ROS contents in PC12 cells. Intracellular ROS was detected using the 2´,7´-dichlorofluorescein method as described
in (“Materials and Methods” section). #P\0.01 versus medium group (□); *P\0.05 versus OGD-R group (▨). Please see Fig. 1 legend for abbreviations. d Inhibition of OGD-R-induced LDH leakage by AST. PC12 cells were incubated with AST during OGD treatment and the cells were harvested 24 h later. Supernatants and cell lysates were prepared as described in “Materials and Methods” section. LDH leakage was detected with a LDH assay kit. Data are the mean ± SD from three independent experiments. Statistical comparisons were conducted using an ANOVA followed by the Tukey test. #P \ 0.01 versus medium group (□); *P \ 0.01 versus OGD-R-treated group without AST (▨)
lysine (Trevigen, Gaithersburg, MD, USA). To induce OGD, the cells were washed with phosphate-buffered saline (PBS), switched to RPMI without glucose and serum, and placed in 0.2 % O2 hypoxia chamber with a CO2 and O2 controller (ProOxc 21 Biosphrix, Red field, NY, USA) balanced with 5 % CO2/95 % N2 for 5 h at 37 °C. After the OGD, glucose was added to a final concentration of 25 mM and incubated at normal oxygen as reperfusion. AST (Fusol Material Co., Ltd, Tainan, Taiwan) were dissolved in dimethylsulfoxide (DMSO) and filtered using a 0.22-m polytetrafluoroethylene (PTEE) filter. The AST were added at the indicated concentrations and incubated with the cells after the OGD treatment. The control cells were maintained with growth medium in a normoxic atmosphere at 37 °C. AST (Mol. Formula; C28H32017; Mol. weight: 640.55; store at 2–8 °C, dry place, avoid light; purity: ≥98.0 %).
Cell Viability Assay Cell viability was assessed using MTT assay (Invitrogen, Carlsbad, CA, USA). After the 24 h of reperfusion, the MTT solution was added to attain a final concentration of 0.5 mg/mL and then incubated for 1 h at 37 °C. Finally, the medium was removed, and DMSO was added to dissolve the violet crystals. Absorbance was read at 540 nm to quantify the formazan products on a microplate spectrophometer (Quantl; BioTek instruments; Winooski, VT, USA). Results are expressed as the percent of absorbance of normoxic control cells. Assessing Cell Death Cell death was assessed based on the amount of lactate dehydrogenase (LDH) with the LDH released (LDH
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Fig. 3 The level of apoptosis that increased in OGD-R was attenuated by AST (100 g/mL). Apoptotic cells were detected on a propidium iodide (PI) histogram as percentage of sub G1 population (a) PC12 cells were incubated during OGD-R and the percent of apoptotic cells was determined by flow cytometry after PI labeling, (as reported in “Materials and Methods” section). b Data are the
mean ± SD of triplicate experimetns. #P\0.01 versus medium group (□); *P \ 0.01 versus OGD-R-treated group without AST (▨). In the upper panel, the data obtained after 24 h reperfusion incubation are reported as a representative experiment. Untreated cells were used as control. Apoptotic cells are characterized by low DNA stain ability and appear below the G1 peak in the distribution
Activity Assay kit; Biovision, San Francisco, CA, USA). Culture medium was collected for measurement of LDH leakage. Ten microliters of supernatant was transferred to a 96-well plate, LDH reaction mix was added to each well, and the plates were incubated for 30 mi at room temperature (RT). The reaction was stopped, and absorbance was read at 450 nm. To assess total LDH activity, the cells were incubated with 100 l of lysis solution per well for 30 min at 37 °C, and then the lysates were centrifuged to remove cellular debris. LDH release is expressed as a percentage of total LDH.
Evaluating DNA Fragmentation
Propidium Iodide (PI) Staining for the Sub-G1 Phase The treated cells were collected and fixed in 70 % ethanol at −20 °C overnight. The next day, the cells were resuspended in a staining buffer composed of 20 μg/mL of PI, 0.1 % Triton-X, and 200 μg/mL of RNase A. After they had been incubated for 30 min in the dark, the cells were washed and analyzed using a flow cytometer (Beckman Coulter, Fullerton, CA, USA). Apoptotic cells were detected on a PI histogram as percentage of the sub-GI population.
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DNA was extracted using a genomic DNA purification kit (Wizard; Promega, Madison, WI, USA). Cell lysate was lysed in Nuclei lysis solution and digested all RNAs with RNase A solution for 30 min at 37 °C. Protein precipitation solution was added, vortexed vigorously, and kept on ice for 5 min. After centrifugation, the supernatant was transferred to a new microtube containing 600 l of isopropanol, mixed by inversion, and centrifuged for 1 min. The supernatant was discarded, and the pellet was washed in 600 l of 70 % ethanol. Finally, the pellet was air dried, then rehydrated with hydration solution, and the concentration was determined. Ten micrograms of extracted DNA was submitted to 1.8 % agarose gel electrophoresis in Tris–borate-EDTA buffer, stained with ethidium bromide, visualized using an UVtrans-illuminator, and then photographed. Caspase-3 and -9 Enzymatic Activity The enzymatic activity of caspase-3 and -9 was measured using a colorimetric assay (Biovision, Mountain View, CA, USA). The cells were harvested and then lysed in lysis buffer
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for 10 min at 4 °C. Lysates were then centrifuged for 15 min at 10,0009g at 4 °C, and the supernatants were collected for further analysis. Two hundred micrograms of extracted protein was incubated with the specific substrates of DEVD (developed/conjugated to)-P-nitroanilide (DEVD-pNA) for caspase-3 and LEHD (chromogenic/colorimetic; AC-LeuGlu-His-Asp)-pNA for caspase-9, respectively, at 37 °C for 120 min, and then the absorptions of release p-nitroaniline were assessed at a wavelength of 405 nm spectrophotometrically. The absorbances were compared with control cells to determine the percentage of caspase-3 and -9 activity changes. Assessing Mitochondrial Membrane Potential The changes in mitochondrial membrane potential were measured using flow cytometry with a fluorescent dye, JC1 (BD Mitoscreen kit; San Jose, CA, USA). In control cells, JC-1 accumulated and formed red aggregates in the mitochondria at high membrane potential. In apoptotic cells, the collapse of potential caused JC-1 to remain in a green fluorescent monomeric form. After they had been reperfused, the cells were collected and incubated in JC-1 staining solution for 15 min at 37 °C. After they had been washed, JC-1 fluorescence was measured from single excitation (488 nm) with dual emission (shift from red 590 nm to green 527 nm) using a flow cytometry.
Fig. 4 Detection of DNA fragmentation by agarose gel electrophoresis. PC12 cells were exposed to OGD-R for 24 h, after which both floating and adherent cells, were collected. Fragmented DNA was extracted from cells as described in the “Materials and Methods” section and electrophoresed on 1 % agarose gels, stained with ethidium bromide, and photographed. Medium: non-OGD-R; OGDR: untreated; OGD-RAST: OGD-R plus AST (100 g/mL). Three experiments were done and yielded similar results
Western Blotting Measuring Intracellular ROS Intracellular ROS was detected using the 2´-7´-dichlorofluorescein (DCF) method. Briefly, cells were pre-loaded with 50 μM 2´-7´-dichlorofluorescein diacetate (SigmaAldrich, St. Louis, MO, USA) for 30 min at 37 °C and then washed. After 3 h of reperfusion, the intensity of DCF fluorescence was measured by excitation/emission at 504/ 529 nm using a microplate reader (Synergy HT, MultiMode; Bio-Tek Instrumetns). Acridine Orange Staining for Autophagy Cell autophagy was detected by evaluating the development of acidic vesicular organelles (AVOs) by staining with acridine orange (AO) (Acridine Orange hydroxhloride; Sigma-Aldrich). After reperfusion, treated cells were harvested, resuspened in a concentration of 2 μg/mL of AO solution, and incubated for 15 min at 37 °C. Green (510– 530 nm, FL1) and red (650 nm, FL3) fluorescence emission illuminated with blue (488 nm) light excitation was measured using a flow cytometer. The intensity of the red fluorescence was proportional to the degree of acidity.
After they had been treated, cells were harvested by trypsinization and centrifugation. Total proteins were extracted by the modified RIPA buffer (50 mM Tris–HCl, pH 7.4, 1 % NP-40, 0.25 % Na-deoxycholate, 150 mM NaCl, 1 mM EDTA) containing protease and phosphatase inhibitors (Sigma-Aldrich) and quantified using a Bradford method colorimetric protein assay (Bio-Rad, Hercules, CA, USA). For Western blotting, protein extracts were boiled for 5 min in loading buffer, separated using SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane (Pall corporation, East Hills, NY, USA) using a wettransfer system (Bio-red). The membranes were blocked in 5 % no-fat milk for 1 h at RT in 5 % non-fat milk in PBS that contained 0.05 % Tween-20 PBS-T. They were hybridized with Caspase-3 and -8 LC3B, Bectin-1, AKT (protein kinase B), p-AKT (Ser473), Bip, JNK, p-JNK (Thr183/Tyr185), p38, p-p38 (Thr180/Tyr182), Erk 1/2, pErk 1/2 (Thr202/Tyr204), β-catenin (Cell signaling technology, Beverly, MA, USA), Caspase-9, 12 (Abcam, Cambridge, UK), p-GSK-3β (Ser389) (Millipore, Billerica, MA, USA), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies overnight at 4 °C. After they had been washed with PBS-T, the membranes were
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Fig. 5 Western blotting of caspase-3 P17 expression (a), caspase-3 activity (b), caspase-9 expression (c), and caspase-9 activity (d) in PC12 cells. For detecting expression, cells were treated with AST (100 g/mL) and subjected to western blotting using specific antibodies. For detecting activity in apoptotic PC12 cells, cells were exposed to ODG-R/for 24 h and harvested in lysis buffer. Enzymatic activity
of caspase-3 or caspase-9 was determined using a protease assay (as described in “Materials and Methods” section). Values are mean ± SD from three independent experiments for the medium group (□), the OGD-R (▨), and the OGD-R AST (100 g/mL) (▩). Comparisons were done using ANOVA and then the Tukey test. #P \ 0.01 versus Control group; *P \ 0.01 versus OGD-R group
continuously incubated with appropriate secondary antibodies coupled to horseradish peroxidase (GE healthcare, Piscataway, NJ, USA) for 1 h at RT. The blots were developed in the ECL Western detection reagents (PerkinElmer, Waltham, MA, USA) and exposed to Amersham Hyperfilm ECL (GE healthcare). Protein bands were scanned and quantified using image analysis software (Image Master Total Lab; GE Healthcare).
were done using analysis of variance (ANOVA) F test. Bonferroni’s adjusted t test was used for multiple group comparison, and an unpaired t test was used for single comparisons. Significance was set at P \ 0.05 (two-tailed).
Results
Statistical Analysis
OGD-R-Induced Cell Death was Lower in AST-Treated PC12 Cells
All assays were independently done 3 times. The results are expressed as the means ± standard deviation (SD) of three independent experiments. Comparisons between groups
Cell viability, intracellular contents of ROS, and LDH (Zhang et al. 2008) were measured as indicators of OGDR-induced injury in PC12 cells. PC12 cell viability
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OGD-R-Induced Apoptosis was Lower in AST-Treated PC12 Cells The percentage of apoptotic cells (Fig. 3), DNA fragmentation (Fig. 4), and expression, and both activity and expression of caspase-3 (Fig. 5a, b) and caspase-9 (Fig. 5c, d) were measured as indicators of OGD-R-induced apoptosis in PC12 cells. These parameters were all significantly higher 24 h after OGD-R treatment. In AST-treated (200 g/ mL) PC12 cells, however, these parameters were all significantly lower (Fig. 5a–d). OGD-R-Induced Autophagy was Lower in ASTTreated PC12 Cells LC3B-II expression (Fig. 6) and percentage of autophagy (Fig. 7), both indicators of OGD-R-induced autophagy, in PC12 cells were all significantly higher 24 h after OGD-R, but in AST-treated (200 g/mL) PC12 cells, they were significantly lower. Bectin-1, a homologue of the yeast Atg6, forms a protein complex with P13 K within the autophagosome (Qu et al. 2003); however, the protein levels of Bectin-1 were not significantly higher 24 h after OGD-R (Fig. 6). OGD-R-Induced Functional Impairment of Mitochondria was Lower in AST-Treated PC12 Cells The percentage of cells with △φm collapse (Fig. 8) was almost four times higher (P\0.01 compared with controls) 24 h after OGD-R (Fig. 8) in vehicle-treated PC12 cells, but in AST-treated (200 g/mL) PC 12 cells, they were only about 1.5 times higher (P \ 0.01 compared with OGDtreated cells) (Fig. 8). Fig. 6 Western blotting of Bectin-1 and LC3B-11 expression in PC12 cells. a Cells were treated or not with AST and subjected to Western blotting using bectin-1- or LC3B-11-specific antibody. The results are the mean ± SD from three independent experiments (b, c) for the medium group (□), the OGD-R group (▨), and the OGDR AST (100 g/mL) group (▩). Comparisons were done using ANOVA and then the Tukey test. #P \ 0.01 versus medium; *P \ 0.05 versus OGD-R group
(Fig. 2a, b) was markedly lower at 24 h after OGD-R, but both ROS (Fig. 2c) and LDH contents (Fig. 2d) were markedly higher at 24 h after OGD-R. The intracellular content of ROS was also significantly lower in NAC (Nacetylcysteine; a ROS scavenger)-treated PC12 cells. AST treatment resulted in a significant increase in cell viability (Fig. 2a, b) but a significant decrease in both ROS and LDH leakage (Fig. 2c, d).
OGD-R-induced ER Stress was Lower in AST-Treated PC12 Cells Twenty-four hours after OGD-R, Bip and caspase-12 expression were measured as indicators of OGD-R-induced ER stress (Badiola et al. 2011). As compared to those of the non-OGD-R controls, vehicle-treated OGD-R PC 12 cells had significantly (P \ 0.01) higher expression of both Bip and caspase-12 (Fig. 9). AST treatment (200 g/mL) resulted in a significant decrease in these two parameters (Fig. 9). OGD-R-induced Activation of P38 MAPK was Higher in AST-Treated PC 12 Cells To test the hypothesis that AST augment neuronal viability after OGD-R by raising the expression of P38-MAPK in PC12 cells, we measured phospho-AKT, phospho-JNK,
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Fig. 7 Acridine orange staining for autophagy in PC12 cells. a Cells were treated or not with AST and subjected to acridine orange staining for autophagy. b The results are the mean ± SD from three independent experiments for the medium (□), the OGD-R (▨), and
the OGD-R AST (100 g/mL) (▩). Comparisons were done using ANOVA and then the Tukey test. #P \ 0.01 versus medium; *P \ 0.05 versus OGD-R
phospho-38, phospho-ERK 1/2, and caspase-3 p17 expression (Fig. 10). Phospho-AKT was inhibited but phospho-JNK, phospho-p38, phospoho-ERK 1/2, and caspase-3 p17 were activated in neurons subjected to OGD-R. The inhibition of phospho-AKT as well as the activation of phospho-JNK or phospho-ERK 1/2 by OGD-R was unaffected by AST treatment. On the other hand, OGD-Rinduced activation of phospho-p38 and caspase-9 p17 was respectively enhanced and inhibited by AST therapy (Fig. 10). The activation of p38-MAPK, but not ERK 1/2, JNK, or AKT, was higher in AST-treated cells (Fig. 11). In SB203580 (a P38 MAPK inhibitor)-treated cells, however, OGD-R-induced cell loss through p38 MAPK activation was inhibited (Ding et al. 2008).
(OGD-R) were used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. AST (1, 100, and 200 μg/mL) were added to the cultures 0 min before the reperfusion insult and was present during the reperfusion phase. We found that AST attenuated OGD-R-induced loss of cell viability and increased expression of LDH in a dose-dependent way. Then, we evaluated that the effect of AST on OGD-R-induced increased expression of ROS, apoptosis, and others at a contration of 100 g/mL in present study. The percentages of apoptotic cells, DNA fragmentation, and the expression or activity of ROS, caspase-3, or caspase-9 were all significantly higher 24 h post-OGD-R in cells not treated with AST, but they were all significantly lower in cells that had been treated with AST. Both our present data and findings of Fan et al. (2011) showed that OGDR caused PC12 cell death primarily by apoptosis rather than necrosis. The excessive accumulation of ROS in reperfusion injury has been previously described (Siesjo¨ 1982; Yoshida et al. 1982). Excessive ROS expression activates ischemic neuronal apoptotic pathways (Niizuma et al. 2010), which
Discussion We assessed the direct effect of AST against OGD-Rinduced acute neuronal injury and its underling mechanisms in vitro. 5 h of OGD and then 24 h of reperfusion
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Fig. 8 Assessment of mitochondrial membrane potential (△φm) collapse in PC12 cells. a Cells were treated or not with AST (100 g/ mL) and subjected to △φm assessment. b The results are the mean ± SD from three independent experiments. Medium group (□): the non-OGD-R; OGD-R (▨): 5 h untreated OGD and then 24 h of
reperfusion; OGD-R AST group (▩): 5 h of AST-treated OGD and then 24 h of reperfusion. Comparisons were done using ANOVA and then the Tukey test. #P \ 0.01 versus Control; *P \ 0.01 versus OGD-R
suggests that AST may protect against reperfusion injury via the intrinsic pathways. Reperfusion after stroke causes ROS expression, which induces dysfunction of the AKT cell-signaling pathway; in addition, ROS upregulate JNK and ERK activity (Zhao 2009). Inhibiting P13K-AKT directly dephosphorylates GSK-3β and degrades β-catenin, and indirectly upregulates cytochrome c release and downregulates caspase-3 activity. AST treatment did not affect the OGD-R-induced inhibition of AKT or the activation of JNK and ERK 1/2 in PC12 cells, but it did significantly prevent OGD-R-induced inhibition of both GSK-3β and β-catenin. AST protected PC12 cells against reperfusion injury by modulating GSK3β dephosphorylation, β-catenin degradation, and caspase3 activity, but not by acting through a JNK-, ERK 1/2-, or AKT-independent pathway. Activating β-catenin-mediated signaling by inhibiting GSK 3β provides a potential mechanism for p38 MAPKmediated survival of specific types of tissue (Thornton et al. 2008). Adiponectin regulates the proliferation of adult
neural stem cells by activating a p38-MAPK/GSK-3β/βcatenin signaling cascade (Zhang et al. 2011). Icarlin, a flavonoid, increases neuronal survival after OGD-R in cortical neuron culture by activating the MAPK/p38 pathway, not the MAPK/JNK pathways (Wang et al. 2009). In rats, estrogen inhibits cerebral ischemia by upregulating the expression of Wnt 3 proteins, which causes nuclear βcatenin to accumulate, which, in turn, inhibits apoptosis and increases neuronal cell survival (Zhang et al. 2008). Wnt 3 proteins are extracellular factors important in the developed and mature central nervous system. Wnt signaling increases neurogenesis and improves neurological function after focal ischemic injury (Shruster et al. 2012). We also found that AST attenuated reperfusion injury in PC12 cells by activating a p38-MAPK/GSK-3β/β- catenin signaling cascade. The beneficial effects of AST in reducing reperfusion injury can be significantly attenuated by inhibiting P38 MAPK with SB203580. Intrinsic apoptosis is regulated by mitochondria (Galluzzi et al. 2009). If the cell death sentence has been
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Fig. 9 Western blotting of Bip and caspase-12 expression in PC12 cells. a Cells were treated or not with AST (100 g/mL) and subjected to Western blotting using Bip- or caspase-12-specific antibody. b, c The results are the mean ± SD from three independent experiments. Medium: non-OGD-R group (□): OGD-R group (▨): 5 h untreated OGD and then 24 h of reperfusion; OGD-R AST (▩): 5 h ASTtreated OGD and then 24 h of reperfusion. Comparisons were done using ANOVA and then the Tukey test. #P \ 0.01 versus medium; * P \ 0.05 versus OGD-R
initiated, mitochondrial membrane permeabilization (MMP) occurs, and cells trespass the frontier between life and death (Kroemer and Reed 2000). MMP has a number of consequences that contribute to cell death; they include: (a) losing mitochondrial transmembrane potential (Δφm); (b) overproducing ROS; and (c) releasing into the cytosol, proteins that in healthy cells are secured within the mitochondrial intermembrane space (Galluzzi et al. 2009). As shown in the present study, OGD-R-induced upregulated the percentage of cells with Δφm collapse the intracellular expression of ROS, and AST treatment significantly attenuated the expression and activity of caspase 9 in PC12 cells. This finding indicated that AST protected PC12 cells against reperfusion injury by reducing MMP. Disturbed calcium homeostasis and accumulated misfolded proteins in the ER are considered contributory
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components of cell death after ischemia (Badiola et al. 2011). In response to ER stress, GRP78 dissociates and binds to the unfolded proteins to facilitate refolding, which allows the activation of PERD (RNA-activated protein kinase-like ER resident kinase), inositol requiring kinase (IRE1), and activating transcription factor 6 (ATF6). These three ER-transmembrane effector proteins lead to the unfolded protein response (UPR). UPR activation can help the cell to cope with ER stress, but if the stress is persistent, it can also contribute to cell death (Rao et al. 2004). In addition, caspase-12 activation has been associated with the ER stress-induced cell damage (Nakagawa et al. 2000; Shibata et al. 2003). Our data showed that AST treatment attenuated OGD-R-induced cell injury in PC12 cells by reducing ER stress (as reflected by decreased expression of both GRP78 and caspase-12). Neuronal injury in rat model of permanent focal cerebral ischemia is associated with the activation of autophagic pathways (Wen et al. 2008). Inhibiting autophagy prevents hippocampal pyramidal neuron death after hypoxic-ischemic injury (Koike et al. 2008). Propofol prevents autophagic cell death after OGD-R in PC12 cells and cerebral ischemia–reperfusion injury in rats (Cui et al. 2012). However, in the absence of direct evidence that activation of autophagy directly contributes to cell death. We should not necessarily equate that AST shared with propofol the same beneficial effects in preventing autophagic cell death after OGD-R in PC12 cells. Autophagy can also be activated as a protective mechanism. In addition, autophagic cell death is also associated with specific morphologic ultrastructual changes that have not been documented here. The Fas pathway (Fas is a death receptor) is involved in apoptosis after ischemia. Fas, Fas-associated death domain, and pro-caspase-8 form a protein complex that is referred to as the death-inducing signaling complex (DISC), which activates procaspase-8 in the apoptosome (Niizuma et al. 2010). Caspase-8 activation is followed by activation of caspase-3 after cerebral ischemia (Jin et al. 2001). However, neither OGD-R nor AST treatment-induced caspase-8 activation in the PC12 cells in this study. The present study examined the therapeutic effect of AST in PC12 cells in an in vitro model of ischemic stroke. We found that 5 h of ischemia and then 24 h of reperfusion downregulated cell viability and upregulated LDH and ROS expression, and augmented apoptosis, autophagy, functional impairment of mitochondria, and ER stress in PC12 cells, all of which AST treatment during reperfusion significantly attenuated. In addition, AST attenuated OGD-R-induced cell loss by activating P38 MAPK, and SB203580, a specific inhibitor of P38 MAPK, inhibited this neuroprotective effect. Our findings suggest that both apoptosis and autophagy are important characteristics of OGD-R-induced
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Fig. 10 Western blotting of phospho-AKT (a), phospho-38 (b), phospho-JNK, phospho-ERK 1/2, and caspase-3 p17 expression (c) in PC12 cells. Cells were treated with OGD-R and OGD-R and AST (100 g/mL). The results are the mean ± SD from three independent
experiments. a Phospho-AKT expression: #P \ 0.05 versus medium group. b Phospho-p38 expression: #P \ 0.01 versus medium group; *P \ 0.05 versus OGD-R group
PC12 death, and the AST treatment blocked OGD-Rinduced apoptosis and autophagy by suppressing intracellular oxidative stress, functional impairment of mitochondria, and ER stress. Our data provide support for a concept that has been only poorly analyzed so far in the ischemia/reperfusion studies; the role of concomitant activation of multiple cell death pathways following such injuries in neural cells. We also provide identification of AST
that can concomitantly inhibit multiple cell death pathways following OGD injuries in neural cells. Acknowledgments This work was supported by Grant NSC 1012314-B-218-001-MY3 from the Taiwan National Council of Science (Taipei) (to C. P. Chang) and the grant MFHR10115 from Chi Mei Medical Center (Tainan, Taiwan) (to B. Y. Chio). Conflict of interest
The authors declared no conflicts of interest.
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Fig. 11 Western blotting of phopho-p38, phospho-GSk-3β, β-catenin, LC3B-II, caspase-12, caspase-9, caspase-3 expression in PC12 cells. a cells were treated with OGD-R, AST, SB203580, both OGD-R and AST (100 g/mL), both AST (100 g/mL) and SB203580, both OGD-R and SB203580, or all three.b The results are the mean ± SD from three independent experiments. #P \ 0.01 versus group 1; *P \ 0.05 versus group 5
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Cell Mol Neurobiol reactive oxygen species by mitogenic growth factors and TGFbeta1. FASEB J 14:1741–1748 Thornton TM, Pedraza-Alva G, Deng B, Wood CD, Aronshtam A, Clements JL, Sabio G, Davis RJ, Matthews DE, Doble B, Rincon M (2008) Phosphorylation by p38 MAPK as an alternative pathway for GSK3beta inactivation. Science 320:667–670 Tohda C, Tamura T, Matsuyama S, Komatsu K (2006) Promotion of axonal maturation and prevention of memory los in mice by extracts of astragalus morgholicus. Br J Pharmacol 149:532–541 Wang L, Zhang L, Chen ZB, Wu JY, Zhang X, Xu Y (2009) Icariin enhances neuronal survival after oxygen and glucose deprivation by increasing SIRT1. Eur J Pharmacol 609:40–44 Wen YD, Sheng R, Zhang LS, Han R, Zhang X, Zhang XD, Han F, Fukunaga K, Qin ZH (2008) Neuronal injury in rat model of permanent focal cerebral ischemia is associated with activation of autophagic and lysosomal pathways. Autophagy 4:762–769 Yin YY, Li WP, Gong HL, Zhu FF, Li WZ, Wu GC (2010) Protective effect of astragaloside on focal cerebral ischemia/reperfusion injury in rats. Am J Chin Med 38:517–527 Yin YY, Li WP, Wang SB, He T, Hing L (2005) Effect of extract of astragalus on inflammatory factor and apoptosis after focal
cerebral ischemia/reperfusion injury in rats. Chin Pharmacol Bull 12:1486–1489 Yoshida S, Abe K, Busto R, Watson BD, Kogure K, Ginsberg MD (1982) Influence of transient ischemia on lipid-soluble antioxidants, free fatty acids and energy metabolites in rat brain. Brain Res 245:307–316 Zhang QG, Wang R, Khan M, Mahesh V, Brann DW (2008) Role of Dickkopf-1, an antagonist of the Wnt/beta-catenin signaling pathway, in estrogen-induced neuroprotection and attenuation of tau phosphorylation. J Neurosci 28:8430–8441 Zhang D, Guo M, Zhang W, Lu XY (2011) Adiponectin stimulates proliferation of adult hippocampal neural stem/progenitor cells through activation of p38 mitogen-activated protein kinase (p38MAPK)/glycogen synthase kinase 3β (GSK-3β)/β-catenin signaling cascade. J Biol Chem 286:44913–44920 Zhao H (2009) Ischemic postconditioning as a novel avenue to protect against brain injury after stroke. J Cereb Blood Flow Metab 29:873–875 Zheng Z, Kim JY, Ma H, Lee JE, Yenari MA (2008) Antiinflammatory effects of the 70 kDa heat shock protein in experimental stroke. J Cereb Blood Flow Metab 28:53–63
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ORIGINAL RESEARCH
Beneficial Effect of Astragalosides on Stroke Condition Using PC12 Cells under Oxygen Glucose Deprivation and Reperfusion Bi-Ying Chiu · Ching-Ping Chang · Jia-Wei Lin · Jung-Sheng Yu · Wen-Pin Liu · Yao-Chin Hsu · Mao-Tsun Lin
Received: 12 January 2014 / Accepted: 2 April 2014 © Springer Science+Business Media New York 2014
Abstract Astragalosides (AST) are reported to be neuroprotective in focal cerebral ischemic models in vivo. In this study, the direct effect of AST against oxygen and glucose deprivation (OGD) including neuronal injury and the underlying mechanisms in vitro were investigated. 5 h OGD followed by 24 h of reperfusion [adding back oxygen and glucose (OGD-R)] was used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. AST (1, 100, and 200 g/mL) were added to the culture after 5 h of the OGD ischemic insult and was present during the reoxygenation phases. A key finding was that OGD-R decreased cell viability, increased lactate
B.-Y. Chiu · J.-S. Yu · Y.-C. Hsu (&) Department of Chinese Medicine, Chi Mei Medical Center, Tainan 710, Taiwan, Republic of China e-mail: [email protected] C.-P. Chang · M.-T. Lin Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan 710, Taiwan, Republic of China C.-P. Chang · M.-T. Lin The Ph.D. Program for Neural Regenerative Medicine, Taipei Medical University, Taipei 110, Taiwan, Republic of China J.-W. Lin School of Medicine, Taipei Medical University, Taipei 110, Taiwan, Republic of China J.-W. Lin Neurosurgical Department, Taipei Medical University ShuangHo Hospital, New Taipei City 23561, Taiwan, Republic of China W.-P. Liu · M.-T. Lin (&) Department of Medical Research, Chi Mei Medical Center, Tainan 710, Taiwan, Republic of China e-mail: [email protected]
dehydrogenase, increased reactive oxygen species, apoptosis, autophagy, functional impairment of mitochondria, and endoplasmic reticulum stress in PC12 cells, all of which AST treatment significantly reduced. In addition, AST attenuated OGD-R-induced cell loss through P38 MAPK activation a neuroprotective effect blunted by SB203580, a specific inhibitor of P38 MAPK. Our data suggest that both apoptosis and autophagy are important characteristics of OGD-Rinduced PC12 death and that treating PC12 cells with AST blocked OGD-R-induced apoptosis and autophagy by suppressing intracellular oxidative stress, functional impairment of mitochondria, and endoplasmic reticulum stress. Our data provide identification of AST that can concomitantly inhibit multiple cells death pathways following OGD injuries in neural cells. Keywords Astragaloside · Oxygen glucose deprivation · Reactive oxygen species · Apoptosis · Autophagy · Mitochondria Abbreviations AST ATF6 ATT DEVD-PNA ER GRP78 GSK-3β IRE1 JC-1
LC3B
Astragalosides Activating transcription factor 6 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide Ac-ASP-Glu-Val-ASP-P-nitroanilide Endoplasmic reticulum Glucose-regulated protein 78 (or Bip) Glycogen synthase kinase 3 beta Insositol-requiring kinase 1 5,5´,6,6´-tetrachloro1,1´,3,3´tetraethylbenzimidazolyl-carbocyanine iodide Microtubule-associated protein 1 light chain 3
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LDH LEHD-PNA MMP NAC OGD-R PERK RIPA ROS RPMI Δφm
Lactate dehydrogenase Ac-Leu-Glu-His-ASP-P-nitroanilide Mitochondrial membrane permeabilization N-acetylcysteine Oxygen-glucose deprivation followed by reperfusion RNA-activated protein kinase-like ER resident kinase Radioimmunoprecipitation assay Reactive oxygen species Roswell Park Memorial Institute Mitochondrial transmembrane potential
aging, immune function disorders, and other diseases (Yin et al. 2005, 2010; Liu et al. 2007, 2009; Tohda et al. 2006). However, it is not known whether AST protect against cerebral ischemia (Yin et al. 2010; Luo et al. 2004) by attenuating overproduction of ROS, impairment of mitochondrial function, endoplasmic reticulum stress (ER), and neuronal apoptosis and autophagy. To evaluate the pharmacological potential of AST and its mechanisms, we used, a rat pheochromocytoma cell line (PC12) subjected to OGD followed by reperfusion (OGDR) as an in vitro model of ischemic stroke (Qi et al. 2012; Cheng et al. 2008). The purpose of present study was attempted to identify whether AST are able to inhibit concomitantly multiple cell death pathways following such injury in neural cells.
Introduction Oxidative damage to neurons is the predominant cause of mortality and chronic disability in ischemic stroke (Chan 1996; Kato and Kogure 1999). Reactive oxygen species (ROS) are generated during mitochondrial respiration (Boveris and Chance 1973), which act as intracellular messengers to transducer signals of cell death pathways (Allen and Tresini 2008; Thannickal et al. 2000). Although the mechanisms of cell death after cerebral ischemia remain unclear, it is generally believed that ROS are involved in mitochondrial and apoptotic neuronal death signaling pathways in cerebral ischemia (Niizuma et al. 2010). A more recent report has also shown that autophagic cell death occurs following oxygen and glucose deprivation (OGD) in PC12 cells and cerebral ischemia in rats (Cui et al. 2012). Astragalosides (AST) are the main component of astragalus and commonly used in the prevention and treatment of cardiovascular and cerebrovascular diseases,
Fig. 1 Experimental groups. a Medium group: PC12 cells were treated with normal RPMI plus 21 % O2 for 29 h (□); b OGD-R group: PC12 cells were treated with glucose free RPMI plus 0.2 % for 5 h and then followed by 25 mM glucose plus 21 % O2 for 24 h (▨); c OGD-R AST group: PC12 cells were treated with glucose-free RPMI plus 0.2 % for 5 h and then followed by AST plus 25 mM glucose plus 21 % O2 (▨)
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Materials and Methods Cell Culture The PC12 cell line was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). These cells were cultured in RPMI-1640 medium supplemented with 10 % heat-inactivated horse serum, 5 % fetal bovine serum, 25 mM glucose, 2 mM glutamine, 100/mL penicillin, and 100 μg/mL streptomycin and were maintained in a humidified incubator with 5 % CO2 at 37 °C. All cell culture reagents were purchased from GIBCO (Rockville, MD, USA). Oxygen Glucose Deprivation (OGD) Model (Fig. 1) Cells were plated at a density of 1.5 9 106 cells with growth medium in 6-well plates precoated with poly-L-
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Fig. 2 Protective effects of AST on OGD-R-induced cytotoxicity in PC12 cells. a Effects of AST (50, 100, 200 g/mL) on PC12 cells morphology injury induced by OGD (5 h) followed by reperfusion (24 h) (magnification 9200). AST was adopted during the entire reperfusion phase. b PC12 cells viability was assessed by measuring the MTT reduction. The viability of medium cells (□) was defined as 100 %. #P \ 0.01 OGD alone (▨) versus medium group (□); * P \ 0.05 OGD AST (▩) versus (▨) group. See Fig. 1 legend for abbreviations. c Effects of AST (100 g/mL) on OGD-R-induced increased intracellular ROS contents in PC12 cells. Intracellular ROS was detected using the 2´,7´-dichlorofluorescein method as described
in (“Materials and Methods” section). #P\0.01 versus medium group (□); *P\0.05 versus OGD-R group (▨). Please see Fig. 1 legend for abbreviations. d Inhibition of OGD-R-induced LDH leakage by AST. PC12 cells were incubated with AST during OGD treatment and the cells were harvested 24 h later. Supernatants and cell lysates were prepared as described in “Materials and Methods” section. LDH leakage was detected with a LDH assay kit. Data are the mean ± SD from three independent experiments. Statistical comparisons were conducted using an ANOVA followed by the Tukey test. #P \ 0.01 versus medium group (□); *P \ 0.01 versus OGD-R-treated group without AST (▨)
lysine (Trevigen, Gaithersburg, MD, USA). To induce OGD, the cells were washed with phosphate-buffered saline (PBS), switched to RPMI without glucose and serum, and placed in 0.2 % O2 hypoxia chamber with a CO2 and O2 controller (ProOxc 21 Biosphrix, Red field, NY, USA) balanced with 5 % CO2/95 % N2 for 5 h at 37 °C. After the OGD, glucose was added to a final concentration of 25 mM and incubated at normal oxygen as reperfusion. AST (Fusol Material Co., Ltd, Tainan, Taiwan) were dissolved in dimethylsulfoxide (DMSO) and filtered using a 0.22-m polytetrafluoroethylene (PTEE) filter. The AST were added at the indicated concentrations and incubated with the cells after the OGD treatment. The control cells were maintained with growth medium in a normoxic atmosphere at 37 °C. AST (Mol. Formula; C28H32017; Mol. weight: 640.55; store at 2–8 °C, dry place, avoid light; purity: ≥98.0 %).
Cell Viability Assay Cell viability was assessed using MTT assay (Invitrogen, Carlsbad, CA, USA). After the 24 h of reperfusion, the MTT solution was added to attain a final concentration of 0.5 mg/mL and then incubated for 1 h at 37 °C. Finally, the medium was removed, and DMSO was added to dissolve the violet crystals. Absorbance was read at 540 nm to quantify the formazan products on a microplate spectrophometer (Quantl; BioTek instruments; Winooski, VT, USA). Results are expressed as the percent of absorbance of normoxic control cells. Assessing Cell Death Cell death was assessed based on the amount of lactate dehydrogenase (LDH) with the LDH released (LDH
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Fig. 3 The level of apoptosis that increased in OGD-R was attenuated by AST (100 g/mL). Apoptotic cells were detected on a propidium iodide (PI) histogram as percentage of sub G1 population (a) PC12 cells were incubated during OGD-R and the percent of apoptotic cells was determined by flow cytometry after PI labeling, (as reported in “Materials and Methods” section). b Data are the
mean ± SD of triplicate experimetns. #P\0.01 versus medium group (□); *P \ 0.01 versus OGD-R-treated group without AST (▨). In the upper panel, the data obtained after 24 h reperfusion incubation are reported as a representative experiment. Untreated cells were used as control. Apoptotic cells are characterized by low DNA stain ability and appear below the G1 peak in the distribution
Activity Assay kit; Biovision, San Francisco, CA, USA). Culture medium was collected for measurement of LDH leakage. Ten microliters of supernatant was transferred to a 96-well plate, LDH reaction mix was added to each well, and the plates were incubated for 30 mi at room temperature (RT). The reaction was stopped, and absorbance was read at 450 nm. To assess total LDH activity, the cells were incubated with 100 l of lysis solution per well for 30 min at 37 °C, and then the lysates were centrifuged to remove cellular debris. LDH release is expressed as a percentage of total LDH.
Evaluating DNA Fragmentation
Propidium Iodide (PI) Staining for the Sub-G1 Phase The treated cells were collected and fixed in 70 % ethanol at −20 °C overnight. The next day, the cells were resuspended in a staining buffer composed of 20 μg/mL of PI, 0.1 % Triton-X, and 200 μg/mL of RNase A. After they had been incubated for 30 min in the dark, the cells were washed and analyzed using a flow cytometer (Beckman Coulter, Fullerton, CA, USA). Apoptotic cells were detected on a PI histogram as percentage of the sub-GI population.
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DNA was extracted using a genomic DNA purification kit (Wizard; Promega, Madison, WI, USA). Cell lysate was lysed in Nuclei lysis solution and digested all RNAs with RNase A solution for 30 min at 37 °C. Protein precipitation solution was added, vortexed vigorously, and kept on ice for 5 min. After centrifugation, the supernatant was transferred to a new microtube containing 600 l of isopropanol, mixed by inversion, and centrifuged for 1 min. The supernatant was discarded, and the pellet was washed in 600 l of 70 % ethanol. Finally, the pellet was air dried, then rehydrated with hydration solution, and the concentration was determined. Ten micrograms of extracted DNA was submitted to 1.8 % agarose gel electrophoresis in Tris–borate-EDTA buffer, stained with ethidium bromide, visualized using an UVtrans-illuminator, and then photographed. Caspase-3 and -9 Enzymatic Activity The enzymatic activity of caspase-3 and -9 was measured using a colorimetric assay (Biovision, Mountain View, CA, USA). The cells were harvested and then lysed in lysis buffer
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for 10 min at 4 °C. Lysates were then centrifuged for 15 min at 10,0009g at 4 °C, and the supernatants were collected for further analysis. Two hundred micrograms of extracted protein was incubated with the specific substrates of DEVD (developed/conjugated to)-P-nitroanilide (DEVD-pNA) for caspase-3 and LEHD (chromogenic/colorimetic; AC-LeuGlu-His-Asp)-pNA for caspase-9, respectively, at 37 °C for 120 min, and then the absorptions of release p-nitroaniline were assessed at a wavelength of 405 nm spectrophotometrically. The absorbances were compared with control cells to determine the percentage of caspase-3 and -9 activity changes. Assessing Mitochondrial Membrane Potential The changes in mitochondrial membrane potential were measured using flow cytometry with a fluorescent dye, JC1 (BD Mitoscreen kit; San Jose, CA, USA). In control cells, JC-1 accumulated and formed red aggregates in the mitochondria at high membrane potential. In apoptotic cells, the collapse of potential caused JC-1 to remain in a green fluorescent monomeric form. After they had been reperfused, the cells were collected and incubated in JC-1 staining solution for 15 min at 37 °C. After they had been washed, JC-1 fluorescence was measured from single excitation (488 nm) with dual emission (shift from red 590 nm to green 527 nm) using a flow cytometry.
Fig. 4 Detection of DNA fragmentation by agarose gel electrophoresis. PC12 cells were exposed to OGD-R for 24 h, after which both floating and adherent cells, were collected. Fragmented DNA was extracted from cells as described in the “Materials and Methods” section and electrophoresed on 1 % agarose gels, stained with ethidium bromide, and photographed. Medium: non-OGD-R; OGDR: untreated; OGD-RAST: OGD-R plus AST (100 g/mL). Three experiments were done and yielded similar results
Western Blotting Measuring Intracellular ROS Intracellular ROS was detected using the 2´-7´-dichlorofluorescein (DCF) method. Briefly, cells were pre-loaded with 50 μM 2´-7´-dichlorofluorescein diacetate (SigmaAldrich, St. Louis, MO, USA) for 30 min at 37 °C and then washed. After 3 h of reperfusion, the intensity of DCF fluorescence was measured by excitation/emission at 504/ 529 nm using a microplate reader (Synergy HT, MultiMode; Bio-Tek Instrumetns). Acridine Orange Staining for Autophagy Cell autophagy was detected by evaluating the development of acidic vesicular organelles (AVOs) by staining with acridine orange (AO) (Acridine Orange hydroxhloride; Sigma-Aldrich). After reperfusion, treated cells were harvested, resuspened in a concentration of 2 μg/mL of AO solution, and incubated for 15 min at 37 °C. Green (510– 530 nm, FL1) and red (650 nm, FL3) fluorescence emission illuminated with blue (488 nm) light excitation was measured using a flow cytometer. The intensity of the red fluorescence was proportional to the degree of acidity.
After they had been treated, cells were harvested by trypsinization and centrifugation. Total proteins were extracted by the modified RIPA buffer (50 mM Tris–HCl, pH 7.4, 1 % NP-40, 0.25 % Na-deoxycholate, 150 mM NaCl, 1 mM EDTA) containing protease and phosphatase inhibitors (Sigma-Aldrich) and quantified using a Bradford method colorimetric protein assay (Bio-Rad, Hercules, CA, USA). For Western blotting, protein extracts were boiled for 5 min in loading buffer, separated using SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane (Pall corporation, East Hills, NY, USA) using a wettransfer system (Bio-red). The membranes were blocked in 5 % no-fat milk for 1 h at RT in 5 % non-fat milk in PBS that contained 0.05 % Tween-20 PBS-T. They were hybridized with Caspase-3 and -8 LC3B, Bectin-1, AKT (protein kinase B), p-AKT (Ser473), Bip, JNK, p-JNK (Thr183/Tyr185), p38, p-p38 (Thr180/Tyr182), Erk 1/2, pErk 1/2 (Thr202/Tyr204), β-catenin (Cell signaling technology, Beverly, MA, USA), Caspase-9, 12 (Abcam, Cambridge, UK), p-GSK-3β (Ser389) (Millipore, Billerica, MA, USA), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies overnight at 4 °C. After they had been washed with PBS-T, the membranes were
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Fig. 5 Western blotting of caspase-3 P17 expression (a), caspase-3 activity (b), caspase-9 expression (c), and caspase-9 activity (d) in PC12 cells. For detecting expression, cells were treated with AST (100 g/mL) and subjected to western blotting using specific antibodies. For detecting activity in apoptotic PC12 cells, cells were exposed to ODG-R/for 24 h and harvested in lysis buffer. Enzymatic activity
of caspase-3 or caspase-9 was determined using a protease assay (as described in “Materials and Methods” section). Values are mean ± SD from three independent experiments for the medium group (□), the OGD-R (▨), and the OGD-R AST (100 g/mL) (▩). Comparisons were done using ANOVA and then the Tukey test. #P \ 0.01 versus Control group; *P \ 0.01 versus OGD-R group
continuously incubated with appropriate secondary antibodies coupled to horseradish peroxidase (GE healthcare, Piscataway, NJ, USA) for 1 h at RT. The blots were developed in the ECL Western detection reagents (PerkinElmer, Waltham, MA, USA) and exposed to Amersham Hyperfilm ECL (GE healthcare). Protein bands were scanned and quantified using image analysis software (Image Master Total Lab; GE Healthcare).
were done using analysis of variance (ANOVA) F test. Bonferroni’s adjusted t test was used for multiple group comparison, and an unpaired t test was used for single comparisons. Significance was set at P \ 0.05 (two-tailed).
Results
Statistical Analysis
OGD-R-Induced Cell Death was Lower in AST-Treated PC12 Cells
All assays were independently done 3 times. The results are expressed as the means ± standard deviation (SD) of three independent experiments. Comparisons between groups
Cell viability, intracellular contents of ROS, and LDH (Zhang et al. 2008) were measured as indicators of OGDR-induced injury in PC12 cells. PC12 cell viability
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OGD-R-Induced Apoptosis was Lower in AST-Treated PC12 Cells The percentage of apoptotic cells (Fig. 3), DNA fragmentation (Fig. 4), and expression, and both activity and expression of caspase-3 (Fig. 5a, b) and caspase-9 (Fig. 5c, d) were measured as indicators of OGD-R-induced apoptosis in PC12 cells. These parameters were all significantly higher 24 h after OGD-R treatment. In AST-treated (200 g/ mL) PC12 cells, however, these parameters were all significantly lower (Fig. 5a–d). OGD-R-Induced Autophagy was Lower in ASTTreated PC12 Cells LC3B-II expression (Fig. 6) and percentage of autophagy (Fig. 7), both indicators of OGD-R-induced autophagy, in PC12 cells were all significantly higher 24 h after OGD-R, but in AST-treated (200 g/mL) PC12 cells, they were significantly lower. Bectin-1, a homologue of the yeast Atg6, forms a protein complex with P13 K within the autophagosome (Qu et al. 2003); however, the protein levels of Bectin-1 were not significantly higher 24 h after OGD-R (Fig. 6). OGD-R-Induced Functional Impairment of Mitochondria was Lower in AST-Treated PC12 Cells The percentage of cells with △φm collapse (Fig. 8) was almost four times higher (P\0.01 compared with controls) 24 h after OGD-R (Fig. 8) in vehicle-treated PC12 cells, but in AST-treated (200 g/mL) PC 12 cells, they were only about 1.5 times higher (P \ 0.01 compared with OGDtreated cells) (Fig. 8). Fig. 6 Western blotting of Bectin-1 and LC3B-11 expression in PC12 cells. a Cells were treated or not with AST and subjected to Western blotting using bectin-1- or LC3B-11-specific antibody. The results are the mean ± SD from three independent experiments (b, c) for the medium group (□), the OGD-R group (▨), and the OGDR AST (100 g/mL) group (▩). Comparisons were done using ANOVA and then the Tukey test. #P \ 0.01 versus medium; *P \ 0.05 versus OGD-R group
(Fig. 2a, b) was markedly lower at 24 h after OGD-R, but both ROS (Fig. 2c) and LDH contents (Fig. 2d) were markedly higher at 24 h after OGD-R. The intracellular content of ROS was also significantly lower in NAC (Nacetylcysteine; a ROS scavenger)-treated PC12 cells. AST treatment resulted in a significant increase in cell viability (Fig. 2a, b) but a significant decrease in both ROS and LDH leakage (Fig. 2c, d).
OGD-R-induced ER Stress was Lower in AST-Treated PC12 Cells Twenty-four hours after OGD-R, Bip and caspase-12 expression were measured as indicators of OGD-R-induced ER stress (Badiola et al. 2011). As compared to those of the non-OGD-R controls, vehicle-treated OGD-R PC 12 cells had significantly (P \ 0.01) higher expression of both Bip and caspase-12 (Fig. 9). AST treatment (200 g/mL) resulted in a significant decrease in these two parameters (Fig. 9). OGD-R-induced Activation of P38 MAPK was Higher in AST-Treated PC 12 Cells To test the hypothesis that AST augment neuronal viability after OGD-R by raising the expression of P38-MAPK in PC12 cells, we measured phospho-AKT, phospho-JNK,
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Fig. 7 Acridine orange staining for autophagy in PC12 cells. a Cells were treated or not with AST and subjected to acridine orange staining for autophagy. b The results are the mean ± SD from three independent experiments for the medium (□), the OGD-R (▨), and
the OGD-R AST (100 g/mL) (▩). Comparisons were done using ANOVA and then the Tukey test. #P \ 0.01 versus medium; *P \ 0.05 versus OGD-R
phospho-38, phospho-ERK 1/2, and caspase-3 p17 expression (Fig. 10). Phospho-AKT was inhibited but phospho-JNK, phospho-p38, phospoho-ERK 1/2, and caspase-3 p17 were activated in neurons subjected to OGD-R. The inhibition of phospho-AKT as well as the activation of phospho-JNK or phospho-ERK 1/2 by OGD-R was unaffected by AST treatment. On the other hand, OGD-Rinduced activation of phospho-p38 and caspase-9 p17 was respectively enhanced and inhibited by AST therapy (Fig. 10). The activation of p38-MAPK, but not ERK 1/2, JNK, or AKT, was higher in AST-treated cells (Fig. 11). In SB203580 (a P38 MAPK inhibitor)-treated cells, however, OGD-R-induced cell loss through p38 MAPK activation was inhibited (Ding et al. 2008).
(OGD-R) were used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. AST (1, 100, and 200 μg/mL) were added to the cultures 0 min before the reperfusion insult and was present during the reperfusion phase. We found that AST attenuated OGD-R-induced loss of cell viability and increased expression of LDH in a dose-dependent way. Then, we evaluated that the effect of AST on OGD-R-induced increased expression of ROS, apoptosis, and others at a contration of 100 g/mL in present study. The percentages of apoptotic cells, DNA fragmentation, and the expression or activity of ROS, caspase-3, or caspase-9 were all significantly higher 24 h post-OGD-R in cells not treated with AST, but they were all significantly lower in cells that had been treated with AST. Both our present data and findings of Fan et al. (2011) showed that OGDR caused PC12 cell death primarily by apoptosis rather than necrosis. The excessive accumulation of ROS in reperfusion injury has been previously described (Siesjo¨ 1982; Yoshida et al. 1982). Excessive ROS expression activates ischemic neuronal apoptotic pathways (Niizuma et al. 2010), which
Discussion We assessed the direct effect of AST against OGD-Rinduced acute neuronal injury and its underling mechanisms in vitro. 5 h of OGD and then 24 h of reperfusion
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Fig. 8 Assessment of mitochondrial membrane potential (△φm) collapse in PC12 cells. a Cells were treated or not with AST (100 g/ mL) and subjected to △φm assessment. b The results are the mean ± SD from three independent experiments. Medium group (□): the non-OGD-R; OGD-R (▨): 5 h untreated OGD and then 24 h of
reperfusion; OGD-R AST group (▩): 5 h of AST-treated OGD and then 24 h of reperfusion. Comparisons were done using ANOVA and then the Tukey test. #P \ 0.01 versus Control; *P \ 0.01 versus OGD-R
suggests that AST may protect against reperfusion injury via the intrinsic pathways. Reperfusion after stroke causes ROS expression, which induces dysfunction of the AKT cell-signaling pathway; in addition, ROS upregulate JNK and ERK activity (Zhao 2009). Inhibiting P13K-AKT directly dephosphorylates GSK-3β and degrades β-catenin, and indirectly upregulates cytochrome c release and downregulates caspase-3 activity. AST treatment did not affect the OGD-R-induced inhibition of AKT or the activation of JNK and ERK 1/2 in PC12 cells, but it did significantly prevent OGD-R-induced inhibition of both GSK-3β and β-catenin. AST protected PC12 cells against reperfusion injury by modulating GSK3β dephosphorylation, β-catenin degradation, and caspase3 activity, but not by acting through a JNK-, ERK 1/2-, or AKT-independent pathway. Activating β-catenin-mediated signaling by inhibiting GSK 3β provides a potential mechanism for p38 MAPKmediated survival of specific types of tissue (Thornton et al. 2008). Adiponectin regulates the proliferation of adult
neural stem cells by activating a p38-MAPK/GSK-3β/βcatenin signaling cascade (Zhang et al. 2011). Icarlin, a flavonoid, increases neuronal survival after OGD-R in cortical neuron culture by activating the MAPK/p38 pathway, not the MAPK/JNK pathways (Wang et al. 2009). In rats, estrogen inhibits cerebral ischemia by upregulating the expression of Wnt 3 proteins, which causes nuclear βcatenin to accumulate, which, in turn, inhibits apoptosis and increases neuronal cell survival (Zhang et al. 2008). Wnt 3 proteins are extracellular factors important in the developed and mature central nervous system. Wnt signaling increases neurogenesis and improves neurological function after focal ischemic injury (Shruster et al. 2012). We also found that AST attenuated reperfusion injury in PC12 cells by activating a p38-MAPK/GSK-3β/β- catenin signaling cascade. The beneficial effects of AST in reducing reperfusion injury can be significantly attenuated by inhibiting P38 MAPK with SB203580. Intrinsic apoptosis is regulated by mitochondria (Galluzzi et al. 2009). If the cell death sentence has been
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Fig. 9 Western blotting of Bip and caspase-12 expression in PC12 cells. a Cells were treated or not with AST (100 g/mL) and subjected to Western blotting using Bip- or caspase-12-specific antibody. b, c The results are the mean ± SD from three independent experiments. Medium: non-OGD-R group (□): OGD-R group (▨): 5 h untreated OGD and then 24 h of reperfusion; OGD-R AST (▩): 5 h ASTtreated OGD and then 24 h of reperfusion. Comparisons were done using ANOVA and then the Tukey test. #P \ 0.01 versus medium; * P \ 0.05 versus OGD-R
initiated, mitochondrial membrane permeabilization (MMP) occurs, and cells trespass the frontier between life and death (Kroemer and Reed 2000). MMP has a number of consequences that contribute to cell death; they include: (a) losing mitochondrial transmembrane potential (Δφm); (b) overproducing ROS; and (c) releasing into the cytosol, proteins that in healthy cells are secured within the mitochondrial intermembrane space (Galluzzi et al. 2009). As shown in the present study, OGD-R-induced upregulated the percentage of cells with Δφm collapse the intracellular expression of ROS, and AST treatment significantly attenuated the expression and activity of caspase 9 in PC12 cells. This finding indicated that AST protected PC12 cells against reperfusion injury by reducing MMP. Disturbed calcium homeostasis and accumulated misfolded proteins in the ER are considered contributory
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components of cell death after ischemia (Badiola et al. 2011). In response to ER stress, GRP78 dissociates and binds to the unfolded proteins to facilitate refolding, which allows the activation of PERD (RNA-activated protein kinase-like ER resident kinase), inositol requiring kinase (IRE1), and activating transcription factor 6 (ATF6). These three ER-transmembrane effector proteins lead to the unfolded protein response (UPR). UPR activation can help the cell to cope with ER stress, but if the stress is persistent, it can also contribute to cell death (Rao et al. 2004). In addition, caspase-12 activation has been associated with the ER stress-induced cell damage (Nakagawa et al. 2000; Shibata et al. 2003). Our data showed that AST treatment attenuated OGD-R-induced cell injury in PC12 cells by reducing ER stress (as reflected by decreased expression of both GRP78 and caspase-12). Neuronal injury in rat model of permanent focal cerebral ischemia is associated with the activation of autophagic pathways (Wen et al. 2008). Inhibiting autophagy prevents hippocampal pyramidal neuron death after hypoxic-ischemic injury (Koike et al. 2008). Propofol prevents autophagic cell death after OGD-R in PC12 cells and cerebral ischemia–reperfusion injury in rats (Cui et al. 2012). However, in the absence of direct evidence that activation of autophagy directly contributes to cell death. We should not necessarily equate that AST shared with propofol the same beneficial effects in preventing autophagic cell death after OGD-R in PC12 cells. Autophagy can also be activated as a protective mechanism. In addition, autophagic cell death is also associated with specific morphologic ultrastructual changes that have not been documented here. The Fas pathway (Fas is a death receptor) is involved in apoptosis after ischemia. Fas, Fas-associated death domain, and pro-caspase-8 form a protein complex that is referred to as the death-inducing signaling complex (DISC), which activates procaspase-8 in the apoptosome (Niizuma et al. 2010). Caspase-8 activation is followed by activation of caspase-3 after cerebral ischemia (Jin et al. 2001). However, neither OGD-R nor AST treatment-induced caspase-8 activation in the PC12 cells in this study. The present study examined the therapeutic effect of AST in PC12 cells in an in vitro model of ischemic stroke. We found that 5 h of ischemia and then 24 h of reperfusion downregulated cell viability and upregulated LDH and ROS expression, and augmented apoptosis, autophagy, functional impairment of mitochondria, and ER stress in PC12 cells, all of which AST treatment during reperfusion significantly attenuated. In addition, AST attenuated OGD-R-induced cell loss by activating P38 MAPK, and SB203580, a specific inhibitor of P38 MAPK, inhibited this neuroprotective effect. Our findings suggest that both apoptosis and autophagy are important characteristics of OGD-R-induced
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Fig. 10 Western blotting of phospho-AKT (a), phospho-38 (b), phospho-JNK, phospho-ERK 1/2, and caspase-3 p17 expression (c) in PC12 cells. Cells were treated with OGD-R and OGD-R and AST (100 g/mL). The results are the mean ± SD from three independent
experiments. a Phospho-AKT expression: #P \ 0.05 versus medium group. b Phospho-p38 expression: #P \ 0.01 versus medium group; *P \ 0.05 versus OGD-R group
PC12 death, and the AST treatment blocked OGD-Rinduced apoptosis and autophagy by suppressing intracellular oxidative stress, functional impairment of mitochondria, and ER stress. Our data provide support for a concept that has been only poorly analyzed so far in the ischemia/reperfusion studies; the role of concomitant activation of multiple cell death pathways following such injuries in neural cells. We also provide identification of AST
that can concomitantly inhibit multiple cell death pathways following OGD injuries in neural cells. Acknowledgments This work was supported by Grant NSC 1012314-B-218-001-MY3 from the Taiwan National Council of Science (Taipei) (to C. P. Chang) and the grant MFHR10115 from Chi Mei Medical Center (Tainan, Taiwan) (to B. Y. Chio). Conflict of interest
The authors declared no conflicts of interest.
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Fig. 11 Western blotting of phopho-p38, phospho-GSk-3β, β-catenin, LC3B-II, caspase-12, caspase-9, caspase-3 expression in PC12 cells. a cells were treated with OGD-R, AST, SB203580, both OGD-R and AST (100 g/mL), both AST (100 g/mL) and SB203580, both OGD-R and SB203580, or all three.b The results are the mean ± SD from three independent experiments. #P \ 0.01 versus group 1; *P \ 0.05 versus group 5
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