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. 2014 Oct 28;35(3):303–312. doi: 10.1007/s10571-014-0125-y

15d-Prostaglandin J2 Protects Cortical Neurons Against Oxygen–Glucose Deprivation/Reoxygenation Injury: Involvement of Inhibiting Autophagy Through Upregulation of Bcl-2

Haidong Qin 1, Weiguo Tan 2, Zheng Zhang 1, Lei Bao 1, Hua Shen 1, Feng Wang 3, Feng Xu 4, Zizheng Wang 3,✉
PMCID: PMC11486323  PMID: 25349027

Abstract

We have previously shown that PPAR-γ agonist 15d-PGJ2 inhibited neuronal autophagy after cerebral ischemia/reperfusion injury. However, the underlying mechanism of its regulatory role in neuronal autophagy remains unclear. This study was designed to test the hypothesis that 15d-PGJ2 upregulated Bcl-2 which binds to Beclin 1, and thereby inhibits autophagy. We performed cell viability assay, cytotoxicity assay, western blot, and co-immunoprecipitation to analyze autophagy activities in vitro model of oxygen–glucose deprivation/reoxygenation (OGD/R). OGD/R induced autophagy in cultured cortical neurons. 15d-PGJ2 treatment significantly decreased LC3-II/LC3-I ratio and Beclin 1 expression, but increased p62 expression. Autophagic inhibitor 3-methyladenine decreased LC3-II levels, increased neuronal cell viability, and mimicked some protective effect of 15d-PGJ2 against OGD/R injury. OGD/R-induced autophagy coincided with decreases in Bcl-2 expression and increases in Beclin 1 expression. 15d-PGJ2 treatment upregulated Bcl-2 expression and decreased Beclin 1 expression, and inhibit the dissociation of Beclin1 from Bcl-2 significantly. Bcl-2 siRNA abrogated the effect of 15d-PGJ2 on Beclin 1, LC3-II and p62, and influence cell viability and LDH level, while scRNA did not. PPAR-γ agonist 15d-PGJ2 exerts neuroprotection partially via inhibiting neuronal autophagy after OGD/R injury. The inhibition of autophagy by 15d-PGJ2 is mediated through upregulation of Bcl-2.

Keywords: Peroxisome proliferator-activated receptor-γ, Oxygen–glucose deprivation, Autophagy, Bcl-2, Beclin 1

Introduction

Autophagy is a highly regulated process involving the bulk degradation of cytoplasmic macromolecules and organelles via the lysosomal system. It plays an important role in maintaining homeostasis and protein quality control in the neuron (Wong and Cuervo 2010). Insufficient or defective autophagy may contribute to neurodegenerative diseases (Hara et al. 2006; Komatsu et al. 2006). Also, increased autophagy has been reported in cerebral ischemic injury, including hypoxia–ischemia (Koike et al. 2008; Carloni et al. 2008; Ginet et al. 2009), global ischemia (Wang et al. 2011), and focal cerebral ischemia (Rami et al. 2008; Wen et al. 2008; Puyal et al. 2009). The role of autophagy in cerebral ischemic injury is complex, and depends on brain maturity, region, severity of insult and stage of ischemia (Xu et al. 2012). It has been reported that autophagy may be protective during ischemia, whereas it may be detrimental during reperfusion (Matsui et al. 2007). In focal cerebral ischemia/reperfusion (I/R) models of neonatal rats, the autophagy inhibitor 3-methyladenine (3-MA) reduced the lesion volumes even when given >4 h after ischemia (Puyal et al. 2009). Downregulation of Beclin 1 by RNA interference also reduced infarct volume and neurological deficits induced by focal I/R injury in adult rats (Zheng et al. 2009). Moreover, various agents, including nicotinamide adenine dinuleotide (Zheng et al. 2012), propofol (Cui et al. 2012), beta-asarone (Liu et al. 2012), transmembrane protein 166 (Li et al. 2012), edaravone (Liu et al. 2011), selenite (Mehta et al. 2012), 15d-prostaglandin J2 (15d-PGJ2) (Xu et al. 2013), have been shown to decrease I/R injury by inhibiting autophagy.

The regulation of autophagy is a very complex biological process; it is possible that energy-sensing, endoplasmic, hypoxia, reticulum (ER), and oxidative stress in cerebral ischemia are potent stimulate of neuronal autophagy (Dai et al. 2013). Multiple signaling pathways, including mammalian target of rapamycin (mTOR), AMP-activated protein kinase (AMPK), Bcl-2/Beclin 1 complex, and p53, play important roles in regulating autophagy (Yang and Klionsky 2010). mTOR, the major inhibitor signal of autophagy, upstream of mTOR, the TSC1/2 complex accepts the regulations of PI3KCI/Akt, LKB1/AMPK, MEM/ERK, and HIF-1/REDD1 and Rheb signal pathways (Dai et al. 2013; Yang and Klionsky 2010; Schmelzle and Hall 2000). While the class I phosphatidylinositol 3-kinase (PI3K)/Akt can be an upstream activator, p70S6 kinase is a downstream substrate of mTOR that may act to limit mTOR activity. AMPK is another sensor of cellular bioenergetics, especially in response to energy stress (Yang and Klionsky 2010). When activated by low energy levels, AMPK can phosphorylate and activate TSC 1/2, thereby inhibiting the mTOR. Downstream of mTOR, Beclin1 is a very important protein required for autophagy. Beclin 1 contains a Bcl-2 homology-3 (BH3) domain that is necessary and sufficient for binding to Bcl-2 and Bcl-xl. Beclin1 is tightly bound and regulated by Bcl-2; the dissociation of Beclin 1 from Bcl-2 is essential for its autophagic activity, and Bcl-2/Bcl-xl inhibits autophagy at endoplasmic reticulum (Maiuri et al. 2010). The tumor suppressor and transcription factor p53 has dual positive and negative roles in autophagy induction (Levine and Abrams 2008).

Among Beclin1-binding proteins, Bcl-2 functions as an important autophagy inhibitor. Beclin1 is tightly bound and regulated by Bcl-2, the dissociation of Beclin 1 from Bcl-2 is crucial for autophagy and regulated by some signal pathways, such as DAPK, IKK, PKC, ERK, JNK1, TLRs/MyD88/TRIF/TRAF6, and some proteins, such as HMGB1, MyD88, TRIF, BNIP3, Bad, Noxa, Puma, BimEL, Bik, Bif-1, Ambra1, nPIST, VMP1, SLAM, PINK1, and Survivin.

Our previous in vivo studies have demonstrated that PPAR-γ agonist 15d-PGJ2 exerts neuroprotection by inhibiting neuronal autophagy after cerebral I/R injury (Xu et al. 2013). However, the underlying mechanism of its regulatory role in neuronal autophagy remains unclear. As previously demonstrated, ischemia stimulates autophagy through the AMPK-mTOR pathway, whereas I/R stimulates autophagy through a Beclin 1-dependent pathway (Matsui et al. 2007). A putative PPAR response element (PPRE) has been reported in the 3′-untranslated region of the Bcl-2 gene in human cancer cells. Moreover, PPAR-γ agonists protect neurons against I/R damage by enhancing Bcl-2 (Fuenzalida et al. 2007; Wu et al. 2009). As the autophagy-inducing activity of Beclin 1 is inhibited by multidomain proteins of the Bcl-2 family, we hypothesize that PPAR-γ agonists might upregulate Bcl-2 which binds to Beclin 1 and functionally antagonizes Beclin 1-mediated autophagy. To test this hypothesis, we used an in vitro model of oxygen–glucose deprivation followed by reoxygenation (OGD/R) in the present study. Our results indicate that PPAR-γ agonist 15d-PGJ2 protects cortical neurons partially by inhibiting neuronal autophagy after OGD/R injury, and the inhibition of autophagy by 15d-PGJ2 is mediated through upregulation of Bcl-2.

Materials and Methods

Primary Neuronal Culture

Primary neuronal cultures were established from the neocortices of embryonic day 15 (E15) embryos from pregnant female C57BL/6J mice as previously described (Pavlovski et al. 2012). All animal procedures were carried out according to the NIH Guidelines for Care and Use of the Laboratory Animals. The protocol was approved by the Committee on the Ethics of Animal Experiments of Fudan University. Fetuses were decapitated, and the cerebral hemispheres were dissected. Isolated cerebral tissues were pooled into a Petri dish containing Ca2+/Mg2+-free HBSS (Invitrogen, Carlsbad, CA, USA) buffered with 10 mM HEPES. Tissues were dissociated by trituration with a fine-bore pipette, and aliquots were added to polyethyleneimine (Sigma, St. Louis, MO, USA)-coated cell culture dishes containing Neurobasal medium supplemented with B-27 (Invitrogen), 2 mM l-glutamine, 0.001 % gentamicin sulfate, and 1 mM HEPES in a humidified atmosphere with 5 % CO2 at 37 °C. All experiments were performed with neurons that had been in culture for 7 days.

Oxygen–Glucose Deprivation/Reoxygenation (OGD/R) Cell Model

To induce OGD/R injury, Neurobasal medium was replaced with serum-free glucose-free Lockers buffer (154 mmol/L NaCl, 5.6 mmol/L KCl, 2.3 mmol/L CaCl2, 1 mmol/L MgCl2, 3.6 mmol/L NaHCO3, 5 mmol/L HEPES, and 5 mg/mL gentamicin, pH 7.2). Culture dishes were then placed in a hypoxic chamber (Billups-Rothenberg, Inc) equilibrated with 95 % N2/5 % CO2 at 37 °C for 2 h. After OGD, neurons were cultured in serum and glucose-supplemented original medium under normoxic conditions in a 5 % CO2 incubator for various time periods. Control groups were incubated in Neurobasal medium in a normoxic atmosphere for the same period. For the neuroprotection studies, the cultures were treated with 0.1–1 μmol/L 15d-PGJ2 (Cayman) or 10 mmol/L 3-MA (Sigma) immediately before reoxygenation. For the mechanism studies, the cultures were treated with 15d-PGJ2 at a concentration of 1 μmol/L.

Cell Viability Assay

Neuronal viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assays. Briefly, MTT solution was added to the culture medium (final concentration = 500 μg/mL) at the end of OGD/R treatment. The reaction was terminated by the addition of 10 % acidified SDS (100 μL) to the cell culture 4 h after MTT addition. The absorbance value (A) was measured at 570 nm using a multiwell spectrophotometer (Bio-Rad Laboratories). The percentage of cell death was calculated with the following formula: cell death%=1-Aof experiment well/Aof control well×100%.

Cytotoxicity Assay

Cytotoxicity was assessed by lactate dehydrogenase (LDH) assays. Briefly, after OGD/R treatment, the supernatant of the cell culture was reserved. Neurons were rinsed with PBS and lysed with 1 % triton X-100 at 37 °C for 30 min. Then samples of supernatants and cell lysates were prepared following the manufacturer’s instruction for the LDH assay using a cell viability assay kit (Nanjing Jiancheng Bioengineering Institute, A020). The absorbance value (A) at 440 nm was determined with an automatic multiwall spectrophotometer (Bio-Rad Laboratories). LDH leakage was calculated as followings: LDH leakage%=Apositive/Apositive blank/Anegative/Anegative blank×100%.

Treatment with Bcl-2 siRNA

The Bcl-2 (sc-29215) siRNA and the control siRNA (sc-37007) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif, USA). For the transfection procedure, cells were grown to 50 % confluence and Beclin 1, or control siRNAs were transfected using the Lipofectamine 2000 reagent (Invitrogen, Calif, USA) according to the manufacturer’s instructions.

Western Blot Analysis and Co-immunoprecipitation (Co-IP) Assay

Western blot was used to analyze protein expression in the neurons. In brief, samples were homogenized in lysis buffer (150 mM NaCl, 1 % SDS, 1 % Triton, 1 % Na-deoxycholate, 1 mmol/L EDTA, 50 mmol/L Tris–Cl pH 7.4, and protease inhibitor cocktail) (Roche, Basel, Switzerland). Protein concentrations were determined using a BCA kit (Piece, Rockford, IL, USA). Samples were boiled in SDS-PAGE loading buffer for 5 min and were analyzed by loading equivalent amounts of total proteins (30 μg) onto 10 % SDS-polyacrylamide gels. Proteins were subsequently transferred to a nitrocellulose membrane, which was then incubated with 5 % skimmed milk in Tris-buffered saline with 0.1 % Tween 20 (TBST) for 1 h at room temperature. Afterward, the membranes were incubated with the primary antibodies against LC3 (Abcam, 1:1000); Beclin 1 (Santa Cruz, 1:500); Bcl-2 (Santa Cruz, 1:500); AMPK and phospho-AMPK (Cell Signaling, 1:200), mTOR and phospho-mTOR (Cell Signaling, 1:200); p70S6K and phospho-p70S6K (Cell Signaling, 1:200); and β-actin (Sigma, 1:5000) overnight at 4 °C. After washing with TBST, membranes were then incubated with fluorescence secondary antibodies (LI-COR Biosciences, Lincoln, Nebraska USA), and the signal was read with an Odyssey® Western Blot Analysis system (LI-COR Biosciences, Lincoln, Nebraska USA). The signal intensity of primary antibody binding was quantitatively analyzed with Sigma Scan Pro 5 and was normalized to a loading control β-actin. The influence of 15d-PGJ2 on the dissociation of Beclin1–Bcl2 heterodimer was detect by co-IP assay. The interactions were determined following the instrument of the co-immunoprecipitation Kit (Thermo scientific, #23600).

Statistical Analysis

Data are expressed as the mean ± SD. Statistical analyses were performed using one-way ANOVA tests followed by Dunnett t test. A p value of less than 0.05 was considered to be significant.

Results

15d-PGJ2 Protects Primary Cortical Neurons Against OGD/R-Induced Cell Death

We subjected primary cortical neurons to 2 h of OGD followed by reoxygenation for 2–24 h, and then measured OGD/R-induced MTT reduction and LDH release. Neuronal cell viability declined progressively as the reoxygenation time prolonged (Fig. 1a). About only 42 % of cells remained viable at 24 h. Meanwhile, LDH leakage was markedly increased from 2 to 24 h after reoxygenation, with a maximum effect at 12 h (Fig. 1b).

Fig. 1.

Fig. 1

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15d-PGJ2 protects primary cortical neurons against OGD/R-induced cell death. Primary cortical neurons were subjected to 2 h OGD followed by reoxygenation for 2–24 h. a Cell viability was analyzed with a MTT assay. b Cytotoxicity was assessed by LDH assays. c, d Neuroprotection effects of PGJ2. Neuronal cultures were treated with different concentrations of 15d-PGJ2 (0.1, 0.5, 1 μM) immediately before reoxygenation. Cell viability and LDH leakage was analyzed at 24 h after OGD/R. Data are means from three independent experiments. *p < 0.05 versus control, #p < 0.05 versus OGD/R

To examine the neuroprotective effect of 15d-PGJ2 against OGD/R-induced cell death, different concentrations of 15d-PGJ2 (0.1, 0.5, 1 μM) were added to the culture medium immediately before reoxygenation. As shown in Fig. 1c, neuronal cell viability was significantly decreased at 24 h after OGD/R. 15d-PGJ2 at 0.5–1.0 μM significantly increased neuronal cell viability (Fig. 1c). The ability of 15d-PGJ2 to protect against OGD/R-induced neuronal cell death was confirmed by LDH assays (Fig. 1d). 15d-PGJ2 treatment effectively suppressed OGD/R-induced LDH release from primary cortical neurons from 0.5 to 1.0 μM. Taken together, these results demonstrated that 15d-PGJ2 protected primary cortical neurons against OGD/R-induced cell death in a concentration-dependent manner.

15d-PGJ2 Inhibits Neuronal Autophagy After OGD/R Injury

To evaluate the involvement of neuronal autophagy in OGD/R cell model, a time course analysis of autophagic marker LC3, Beclin 1, and p62 was conducted in primary cultured neurons at 2, 6, 12, and 24 h after OGD/R. Western blot analysis showed the ratio of LC3-II/LC3-I and the expression of Beclin 1 increased significantly from 6 to 24 h after reoxygenation. Conversely, the expression of p62 decreased since 2 h and lasted up to 24 h (Fig. 2a). These findings suggest an enhancement of neuronal autophagy in this OGD/R model.

Fig. 2.

Fig. 2

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15d-PGJ2 inhibits neuronal autophagy after OGD/R injury. a, b Increased LC3-II and Beclin 1 expression and decreased p62 expression in primary cortical neurons after OGD/R injury. Cultured cortical neurons were exposed to OGD/R injury. Cells were harvested for western blot analysis at various times after OGD/R. c, d 15d-PGJ2 decreased LC3-II and Beclin 1 expression and increased p62 expression in primary neurons. 15d-PGJ2 (0.1 to 1 μM) was added to the culture medium immediately before reoxygenation. Cells were harvested for western blot analysis at 24 h after OGD/R. Optical densities of respective protein bands were analyzed with Sigma Scan Pro 5 and normalized to the loading control (β-actin). Data are means from three independent experiments. *p < 0.05 versus control, #p < 0.05 versus OGD/R

To determine the influence of 15d-PGJ2 on autophagy in cultured neurons after OGD/R injury, we added 15d-PGJ2 (0.1 to 1 μM) to the culture medium immediately before reoxygenation. As shown in Fig. 2c, 2 h of OGD followed by 24 h reoxygenation resulted in a significant increase in the ratio of LC3-II/LC3-I and the expression of Beclin 1 compared to control group. Conversely, the expression of p62 decreased significantly compared to control group. Treatment with 15d-PGJ2 at 0.5–1 μM significantly decreased LC3-II/LC3-I ratio and Beclin 1 expression, but increased p62 expression.

Inhibition of Autophagy Plays a Role in 15d-PGJ2 Neuroprotective Effects Against OGD/R Injury

To conclude if inhibition of autophagy plays a role in the neuroprotection of 15d-PGJ2, we use 3-MA to investigate whether autophagy inhibitors mimic some neuroprotective effects. First, we examined the effect of 3-MA on the protein levels of LC3. Western blot analysis showed that 10 mM of 3-MA significantly decreased LC3-II levels at 24 h after OGD/R injury (Fig. 3a, b). Then, we examined whether 3-MA could provide neuroprotection against OGD/R injury. 3-MA treatment significantly increased neuronal cell viability(Fig. 3c), and decreased LDH leakage in a certain extent (Fig. 3d). These results indicated that 3-MA could inhibit autophagy activation and attenuates ischemia-reoxygenation injury in vitro. In a nutshell, we suggest that inhibition of autophagy might play a role in 15d-PGJ2 neuroprotective effects against OGD/R injury.

Fig. 3.

Fig. 3

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Inhibition of autophagy reduced OGD/R-induced neuronal injury. a 3-MA (10 mM) blocked autophagy in neurons at 24 h after OGD/R. b Cell viability showing that autophagy inhibitor 3-MA could provide neuroprotection against OGD/R injury. c 3-MA had little effect on LDH leakage. Data are means from three independent experiments. *p < 0.05 versus control, #p < 0.05 versus OGD/R

15d-PGJ2 Inhibits Neuronal Autophagy through Bcl-2/Beclin 1 Pathway after OGD/R Injury

Previous studies have suggested that mTOR and Bcl-2/Beclin 1 complex might play important roles in regulating autophagy. To explore the potential mechanism underlying the regulation of 15d-PGJ2 on autophagy in neurons upon OGD/R injury, we focused on the AMPK-mTOR-p70S6 K and Bcl-2/Beclin pathway. While OGD/R2 induced increases in phosphorylation of AMPK, we showed that phosphorylation of AMPK decreased with the prolongation of reoxygenation time after OGD. At 24 h after reoxygenation, it went back to normal levels (Fig. 4a, b). In addition, OGD/R2 induced decreases in phosphorylation of mTOR and phosphorylation of p70S6K. However, either phosphorylation of mTOR or phosphorylation of p70S6K was restored to normal levels at 24 h after reoxygenation (Fig. 4a, b). Treatment with 15d-PGJ2 at 1 μM did not affect phosphorylation of AMPK, phosphorylation of mTOR and phosphorylation of p70S6K at 24 h after OGD/R (Fig. 4c, d).

Fig. 4.

Fig. 4

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Inhibition of autophagy by 15d-PGJ2 is independent of mTOR signaling pathway. a, b Phosphorylation of AMPK decreased with the prolongation of perfusion time after OGD, whereas phosphorylation of mTOR and p70S6 K increased. At 24 h after reoxygenation, the phosphorylation went back to normal levels. c, d Treatment with 15d-PGJ2 (1 μM) did not affect phosphorylation of AMPK, phosphorylation of mTOR and phosphorylation of p70S6 K at 24 h after OGD/R. Optical densities of respective protein bands were analyzed with Sigma Scan Pro 5 and normalized to the loading control (β-actin). Data are means from three independent experiments. *p < 0.05 versus control,**p < 0.05 versus control

In contrast, Bcl-2 expression declined in a time-dependent manner after OGD/R. Meanwhile, Beclin 1 expression dramatically increased (Fig. 5a, b). Treatment with 15d-PGJ2 at 1 μM upregulated Bcl-2 expression and decreased Beclin 1 expression at 24 h after OGD/R (Fig. 5c, d). To identify the binding effect of Bcl-2 on Beclin 1, cell extracts were co-immunoprecipitated with anti-Bcl-2 antibody and followed by western blotting with anti-Beclin 1 antibody, we confirmed that 15d-PGJ2 inhibit the dissociation of Beclin1 from Bcl-2 (Fig. 5c). To test the hypothesis that 15d-PGJ2 upregulated Bcl-2 which binds with Beclin 1 and thereby, inhibits autophagy, we transfected neuronal cells with Bcl-2 siRNA or control scRNA and subjected cells to OGD2/R24 in the presence or absence of 15d-PGJ2. In OGD/R-treated neurons, 15d-PGJ2 decreased the expression of Beclin 1 and LC3-II. Bcl-2 siRNA abrogated the effect of 15d-PGJ2 on Beclin 1 and LC3-II, while scRNA did not (Fig. 5e, f). Further, 15d-PGJ2 increased neuronal cell viability and decreased LDH level, which could also be abrogated by Bcl-2 siRNA(Fig. 5g, h). These results indicate that 15d-PGJ2 inhibits autophagy through upregulation of Bcl-2.

Fig. 5.

Fig. 5

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Inhibition of autophagy by 15d-PGJ2 is mediated through Bcl-2/Beclin 1 pathway. a, b Bcl-2 expression decreased in a time-dependent manner after OGD/R, whereas Beclin 1 expression dramatically increased. c, d Treatment with 15d-PGJ2 (1 μM) could upregulate Bcl-2 expression and decrease Beclin 1 expression at 24 h after OGD/R. Cell extracts from neurons at 24 h after OGD/R with or without 15d-PGJ2(1 μM) treated were immunoprecipitated with anti-Bcl-2 antibody, the relative abundance of Beclin 1 in Bcl-2 immunoprecipitates were determined by western blotting, and compare to the level of Beclin 1 in the cell extracts, the statistic result suggested that 15d-PGJ2 inhibited the dissociation of Beclin 1 from Bcl-2. e, f Cells were tranfected with Bcl-2 siRNA or control scRNA and subjected to OGD2/R24 in the presence or absence of 15d-PGJ2. In OGD/R-treated neurons, 15d-PGJ2 (1 μM) decreased the expression of Beclin 1 and LC3-II and increase the expression of p62. Bcl-2 siRNA abrogated the effect of 15d-PGJ2 on Beclin 1, LC3-II and p62, while scRNA did not. Optical densities of respective protein bands were analyzed with Sigma Scan Pro 5 and normalized to the loading control (β-actin). Data are means from three independent experiments. g Cell viability was analyzed with a MTT assay. h Cytotoxicity was assessed by LDH assays.*p < 0.05 versus control, **p < 0.015 versus control, #p < 0.05 versus OGD/R, *p < 0.05 versus Bcl-2 scRNA, **p < 0.01 versus Bcl-2 scRNA

Discussion

The present study has proved that 15d-PGJ2 protects cortical neurons in primary culture against OGD/R injury. We further show that inhibition of autophagy plays a role in 15d-PGJ2 neuroprotective effects against OGD/R injury. Finally, we demonstrate that 15d-PGJ2 inhibits neuronal autophagy through upregulating Bcl-2 after OGD/R injury.

In the mammalian brain, there is very little detectable autophagy, but accumulating evidence indicates that autophagy is enhanced following cerebral ischemia, normally the basal level of autophagy are judged by the autophagosome-related marker LC3-II, Beclin 1 and lysosomal activity including cathepsin D, LAMP-1(Xu et al. 2012). The role of autophagy in cerebral ischemia remains unclear. Although Autophagy might play dual roles in cerebral ischemia, depending on the severity and stages of cerebral ischemia (Xu et al. 2012). Autophagy is protective when activated by mild hypoxia or ischemia, it is essential for neuronal development as well as remodeling and defective autophagy may be critical in neurodegenerative diseases(Wong and Cuervo 2010). However, overactivation of autophagy caused by severe hypoxia or ischemia might be detrimental (Xu and Zhang 2011). In addition, In I/R heart, autophagy may be protective during ischemia, whereas it may be detrimental during reperfusion. Autophagy is induced during reperfusion after cerebral ischemia. Moreover, most studies suggest that autophagy might play a detrimental role during reperfusion (Puyal et al. 2009; Zheng et al. 2009, 2012; Cui et al. 2012; Liu et al. 2012, , 2011; Li et al. 2012 Mehta et al. 2012; Xu et al. 2013). However, most chemical inhibitors of autophagy are not entirely specific. For instance, the autophagy inhibitor 3-MA has potential pro-apoptotic side-effects due to inhibition of the anti-apoptotic PI3 K/Akt pathway (Xu et al. 2012).

Recently, in vitro models of OGD/R have also been used to explore the role of autophagy. Mo et al. (Mo et al. 2012) used an ischemic stroke model of PC12 cell exposed to OGD/R (2 h oxygen–glucose deprivation followed by 24 h reoxygenation). The authors showed that β-Asarone protects PC12 cells against OGD/R-induced injury partly due to attenuating Beclin 1-dependent autophagy. Shi et al. (Shi et al. 2012) reported that the OGD/R model (6 h oxygen–glucose deprivation followed by reoxygenation) in cultured cortical neurons mimicked the in vivo situation. They demonstrated that excessive activation of autophagy contributes to neuronal death in cerebral ischemia. Our present study also employed an enhancement of neuronal autophagy in this OGD/R model (2 h oxygen–glucose deprivation followed by reoxygenation). In OGD/R-treated cultured neurons, the LC3-II and Beclin 1 increased significantly from 6 to 24 h after reoxygenation, which was accompanied with decreased level of p62. In addition, blockade of autophagy by 3-MA significantly increased neuronal cell viability, and decreased LDH leakage. These data suggest that autophagy overactivation might contribute to neuron death after OGD/R injury. However, some other researchers thought that autophagy in the reoxygenation phase removed the damaged mitochondria, and thus protected against cerebral I/R injury (Zhang et al. 2013). This discrepancy might be attributed to limited autophagy caused by moderate stress.

Although ample evidence demonstrated enhanced autophagy in neuronal death following cerebral ischemia, the signaling pathways regulating its activation remain poorly defined. Carloni et al. (Carloni et al. 2010) demonstrated that in neonatal hypoxia–ischemia autophagy can be part of an integrated prosurvival signaling which includes the PI3K-Akt-mTOR axis. Wang et al. (Wang et al. 2012) demonstrated that nicotinamide phosphoribosyltransferase promotes neuronal survival by inducing autophagy via regulating the TSC2-mTOR-S6K1 signaling pathway during cerebral ischemia. Matsui et al. (Matsui et al. 2007) showed that, in the heart, ischemia stimulates autophagy through an AMPK-mTOR pathway, whereas I/R stimulates autophagy through a Beclin 1-dependent but AMPK-independent pathway.

The role of PPAR-γ activation in autophagy remains controversial. Zhou et al. (Zhou et al. 2009) demonstrated that PPAR-γ activation induces autophagy in breast cancer cells. Rovito et al. (Rovito et al. 2013) showed that PPAR-γ agonists not only inhibited Akt-mTOR pathways, but also induced phosphorylation of Bcl-2 promoting its dissociation from Beclin 1 which resulted in autophagy induction. However, Jiang et al. (Jiang et al. 2010) showed that disruption of PPARgamma signaling resulted in mouse prostatic intraepithelial involving active autophagy. Recently, (Mahmood et al. 2011) showed that PPAR-γ induces apoptosis and inhibits autophagy in atherosclerosis. Consistent with our previous in vivo study, we showed that PPAR-γ agonist 15d-PGJ2 inhibits neuronal autophagy after OGD/R injury. Because AMPK is no longer activated during reoxygenation, autophagy during reoxygenation is likely to be mediated by Beclin 1 pathway. In direct contrast to results in breast cells, PPAR-γ agonist 15d-PGJ2 upregulated Bcl-2 and inhibit the dissociation of Beclin1–Bcl2 heterodimer in neurons after OGD/R injury. Bcl-2 upregulation blocks autophagic cell death by binding to Beclin 1 and thereby, downregulating levels of cellular autophagy.

In conclusion, the present study demonstrates that PPAR-γ agonist 15d-PGJ2 exerts neuroprotection partially by inhibiting neuronal autophagy after OGD/R injury, and the inhibition of autophagy by 15d-PGJ2 is mediated through upregulation of Bcl-2 and inhibited the dissociation of Beclin1–Bcl2 heterodimer.

Acknowledgments

This work was supported by National Natural Science Foundation of China 81000488 (F.X.), Fudan University Young Teacher Capability Enhancement Program 20520133268 (F.X.), and Nanjing Medical Science and Technology Development Program YKK11124 (H.D.Q.).

Conflict of interest

The authors have declared that no competing interests exist.

Footnotes

Haidong Qing, Weiguo Tan, Feng Xu and Zizheng Wang contributed equally to this work.

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