Beneficial Effect of Astragalosides on Stroke Condition Using PC12 Cells under Oxygen Glucose Deprivation and Reperfusion

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 µg/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 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-R-induced 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.

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, 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 (OGD-R) 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.

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, 100µ/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)

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 (▨)

Cells were plated at a density of 1.5 × 10⁶ cells with growth medium in 6-well plates precoated with poly-l-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-µm 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 (µQuantl; 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 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 µl 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.

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.

Evaluating DNA Fragmentation

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 µl of isopropanol, mixed by inversion, and centrifuged for 1 min. The supernatant was discarded, and the pellet was washed in 600 µl 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 UV-trans-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 for 10 min at 4 °C. Lysates were then centrifuged for 15 min at 10,000×g 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-Leu-Glu-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, JC-1 (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.

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 (Sigma-Aldrich, 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, Multi-Mode; 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.

Western Blotting

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 wet-transfer 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, p-Erk 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 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).

Statistical Analysis

All assays were independently done 3 times. The results are expressed as the means ± standard deviation (SD) of three independent experiments. Comparisons between groups 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

OGD-R-Induced Cell Death was Lower in AST-Treated PC12 Cells

Cell viability, intracellular contents of ROS, and LDH (Zhang et al. 2008) were measured as indicators of OGD-R-induced injury in PC12 cells. PC12 cell viability (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 (N-acetylcysteine; 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).

Fig. 2.

Protective effects of AST on OGD-R-induced cytotoxicity in PC12 cells. a Effects of AST (50, 100, 200 µg/mL) on PC12 cells morphology injury induced by OGD (5 h) followed by reperfusion (24 h) (magnification ×200). 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 µg/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 (▨)

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 µg/mL) PC12 cells, however, these parameters were all significantly lower (Fig. 5a–d).

Fig. 3.

The level of apoptosis that increased in OGD-R was attenuated by AST (100 µg/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

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; OGD-R: untreated; OGD-R+AST: OGD-R plus AST (100 µg/mL). Three experiments were done and yielded similar results

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 µg/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 µg/mL) (▩). Comparisons were done using ANOVA and then the Tukey test. ^# P < 0.01 versus Control group; *P < 0.01 versus OGD-R group

OGD-R-Induced Autophagy was Lower in AST-Treated 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 µg/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).

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 OGD-R + AST (100 µg/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. 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 µg/mL) (▩). Comparisons were done using ANOVA and then the Tukey test. ^# P < 0.01 versus medium; *P < 0.05 versus OGD-R

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 µg/mL) PC 12 cells, they were only about 1.5 times higher (P < 0.01 compared with OGD-treated cells) (Fig. 8).

Fig. 8.

Assessment of mitochondrial membrane potential (△φm) collapse in PC12 cells. a Cells were treated or not with AST (100 µg/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

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 µg/mL) resulted in a significant decrease in these two parameters (Fig. 9).

Fig. 9.

Western blotting of Bip and caspase-12 expression in PC12 cells. a Cells were treated or not with AST (100 µg/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 AST-treated 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

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, 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-R-induced 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).

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 µg/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

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 µg/mL), both AST (100 µg/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

Discussion

We assessed the direct effect of AST against OGD-R-induced acute neuronal injury and its underling mechanisms in vitro. 5 h of OGD and then 24 h of reperfusion (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 µg/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 (Siesjö 1982; Yoshida et al. 1982). Excessive ROS expression activates ischemic neuronal apoptotic pathways (Niizuma et al. 2010), which 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 GSK-3β dephosphorylation, β-catenin degradation, and caspase-3 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 MAPK-mediated 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/β-catenin 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 β-catenin 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 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 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 PC12 death, and the AST treatment blocked OGD-R-induced 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.

Glossary

Abbreviations

AST

Astragalosides

ATF6

Activating transcription factor 6

ATT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

DEVD-PNA

Ac-ASP-Glu-Val-ASP-P-nitroanilide

ER

Endoplasmic reticulum

GRP78

Glucose-regulated protein 78 (or Bip)

GSK-3β

Glycogen synthase kinase 3 beta

IRE1

Insositol-requiring kinase 1

JC-1

5,5′,6,6′-tetrachloro1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide

LC3B

Microtubule-associated protein 1 light chain 3

LDH

Lactate dehydrogenase

LEHD-PNA

Ac-Leu-Glu-His-ASP-P-nitroanilide

MMP

Mitochondrial membrane permeabilization

NAC

N-acetylcysteine

OGD-R

Oxygen-glucose deprivation followed by reperfusion

PERK

RNA-activated protein kinase-like ER resident kinase

RIPA

Radioimmunoprecipitation assay

ROS

Reactive oxygen species

RPMI

Roswell Park Memorial Institute

Δφm

Mitochondrial transmembrane potential