4-Phenylbutyric Acid (4-PBA) and Lithium Cooperatively Attenuate Cell Death during Oxygen-Glucose Deprivation (OGD) and Reoxygenation

Abstract

Hypoxia is an important cause of brain injury in ischemic stroke. It is known that endoplasmic reticulum (ER) stress is an important determinant of cell survival or death during hypoxia. However, the signaling pathways and molecular mechanisms involved remain to be studied in more detail. To investigate whether inhibition of ER stress promotes neuroprotection pathways, we applied an in vitro oxygen-glucose deprivation (OGD) followed by reoxygenation model of human SK-N-MC neuronal cell cultures in this study. Our results showed that neuronal cell death was induced in this model during the OGD reoxygenation by the sustained ER stress, but not during OGD phase. However, treatment of the cultures with lithium with the OGD reoxygenation insult did not result in neuroprotection, whereas concomitant treatment of chemical chaperon 4-phenylbutyric acid (4-PBA) provides protective effects in ER stress-exposed cells. Moreover, 4-PBA rescued ER stress-suppressed Akt protein biosynthesis, which works cooperatively with lithium in the activation of Akt downstream signaling by inhibition of autophagy-induced cell death. Taken together, our finding provides a possible mechanism by which 4-PBA and lithium contribute to mediate neuroprotection cooperatively. This result may potentially be a useful therapeutic strategy for ischemic stroke.

Fig. 1

Effects of OGD/reoxygenation on insults to SK-N-MC neuronal cells. a Treatment with oxygen–glucose deprivation (OGD) on SK-N-MC cells for 20 h shows slight morphological changes and decreased number of neuronal cells. However, the additional 24 h of reoxygenation markedly induces cell death. b Effects of cytotoxicity induced by exposure to OGD/reoxygenation determined by the MTT assay. c Quantify cell death by lactate dehydrogenase (LDH) activity assays. d Cell viability was evaluated by treatment with or without 100 μM of lithium bicarbonate (Li₂CO₃) under the OGD/reoxygenation process by MTT assay. e Apoptosis is determined by fragmented morphology in the nucleus for DAPI fluorescence. The number of apoptotic cells are quantified by averaging cell counts in five random 400 × fields. The other data were performed in three independent experiments, and values are presented as mean ± standard error of the means (SEM). Significant differences were determined using the multiple comparisons of Dunnett’s post hoc test for *P < 0.05 and **P < 0.01. Scale bar represents 50 μm. N.S. no significant difference

Fig. 2

SK-N-MC cells respond to OGD/reoxygenation through the activation of ER stress. a Western blots showing the activation of ER stress by monitoring the phosphorylation levels of PERK and eIF-2α. b RT-PCR analysis of the splicing of XBP-1 mRNA for the indicated time during OGD and reoxygenation. c Caspase 3, 12, and PARP activation were determined by immunoblotting. d Apoptotic cells were measured by flow cytometry analysis. Results are shown from a single representative of three independent experiments, and values are presented as mean ± SEM

Fig. 3

4-Phenylbutyric acid (4-PBA) attenuates ER stress during additional reoxygenation. a Western blotting reveals that phosphorylation of Thr⁹⁸⁰-PERK and Ser⁵¹-eIF2α is elicited when cells are exposed to OGD/reoxygenation, and this up-regulation is effectively blocked by 4-PBA in a dose-dependent manner. b The levels of phosphorylated PERK and eIF2α are suppressed by 4-PBA in both OGD and additional reoxygenation treatments. c 4-PBA displays a significant inhibition in LDH release in the stage of reoxygenation. d Cell viability assay demonstrates that lithium (100 μM) combined with 4-PBA (100 μM) significantly reduces OGD/reoxygenation-induced cell death. e Western blots showing ER stress and apoptosis induction by monitoring pPERK, p-eIF2α, cleaved caspase 3, cleaved caspase 12, and cleaved PARP levels in SK-N-MC cells treated with 4-PBA (100 μM) alone, lithium (100 μM) alone, or in combination with 4-PBA and lithium during OGD/reoxygenation. All data were performed in three independent experiments, and values are presented as mean ± SEM. *P < 0.05 and **P < 0.01 compared with the H/R-only group for multiple comparisons of Dunnett’s post hoc test

Fig. 4

The combination of lithium and 4-PBA significantly reduces OGD/reoxygenation-induced autophagic cell death. a Immunoblotting was probed by specific antibodies against pAkt and total Akt during OGD and reoxygenation. b Western blot analysis shows pAkt/Akt, pGSK3β/GSK3β, p-mTOR/mTOR, and LC3 in SK-N-MC cells treated with indicated conditions. c Beclin1 siRNA (20 nmol/l pool) knockdown was confirmed via Western blot, and autophagy blockade was validated using LC3-II immunoblotting. Enhanced cell death in response to OGD/reoxygenation was determined via PARP cleavage. d Both the LY294002 (20 μM) and rapamycin (200 nM) treatments for 24 h blocked 4-PBA and lithium to prevent cell death significantly. However, treatment with chloroquine (50 μM) partially returned this inhibition to that in the LY294002-treated groups. LY294002, a specific inhibitor of PI3-kinase. Mean ± SEM of three independent experiments are shown. *P < 0.05 and **P < 0.01 when compared with 4-PBA+ lithium-treated cells using multiple comparisons of Dunnett’s post hoc test