PI3K/Akt signaling pathway is required for neuroprotection of thalidomide on hypoxic–ischemic cortical neurons in vitro

Abstract

Thalidomide, a derivative of glutamic acid, is used for immunomodulatory therapy in various diseases through inhibition of tumor necrotic factor-α (TNF-α) release. However, the effects of thalidomide in central nervous system (CNS) diseases such as stroke or hypoxic–ischemic encephalopathy (HIE) are unknown. In this study, we aimed to test whether thalidomide protects against hypoxic–ischemic neuronal damage and the possible signaling pathway involved in neuroprotection. Primary cultured cortical neurons of rats were treated with oxygen and glucose deprivation (OGD) for 3 h to mimic hypoxic–ischemic injury in vivo. Neuronal apoptosis was measured with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. The expression of total caspase-3 (C3), cleaved caspase-3 (CC3), Akt, phosphorylated-Akt (p-Akt) and Bcl-2 protein were detected by Western blots. We found that OGD treatment increased the expression of CC3 and induced neuronal apoptosis. Both neuronal apoptosis and CC3 expression peaked at 24 h after OGD. Furthermore, we found that thalidomide protected neurons against apoptosis by decreasing CC3 and increasing Bcl-2 expression in a dose-dependent manner. Meanwhile, we found that thalidomide induced p-Akt expression, which could be inhibited by PI3K specific inhibitor, LY294002. In addition, inhibition of PI3K increased CC3 but decreased Bcl-2 expression. In summary, thalidomide has anti-apoptotic effects on cortical neurons after OGD by modulating CC3 and Bcl-2 expression through activation of PI3K/Akt pathway.

Introduction

Neonatal hypoxic ischemic (HI) brain injury is a serious insult that frequently leads to high disability and mortality in neonates. One of the major mechanisms involved in brain injury after HI is neuronal loss caused by cellular apoptosis (Li et al., 2007, Qu et al., 2009). Under HI stimulation, whether neurons undergo apoptosis or survival depends on a balance between cellular apoptotic and cell survival signal transduction. Recent studies have shed light on promoting cellular survival after neonatal HI by inhibiting apoptotic neuronal loss (Lan et al., 2008, Gao et al., 2010).

Thalidomide is an important drug with a broad-spectrum of pharmacologic and immunologic effects. Over the past decades, thalidomide has been effectively and safely used in the treatment of erythema nodosum leprosum, lupus erythematosus, Crohn's disease and rheumatoid arthritis (Raje and Anderson, 1999, Housman et al., 2003, Plamondon et al., 2007, García-Carrasco et al., 2007). Moreover, thalidomide has been proven to have anti-inflammatory and immunoregulatory effects due to its ability to inhibit tumor necrotic factor-α (TNF-α) production and enhance degradation of TNF-α mRNA both in vivo and in vitro (Klausner et al., 1996, Paul et al., 2006, Nakamura et al., 2007). Recently, some studies have been carried out to examine the neuroprotective effects of thalidomide. Lee et al. (2007) have reported that thalidomide reduces ischemic injury of the spinal cord in rabbits through reduction of TNF-α expression. In addition, Hyakkoku et al. (2009) have found that thalidomide has potential neuroprotection in focal cerebral ischemia in adult mice by inhibition of oxidative stress. We have demonstrated that increased TNF-α expression can induce neuronal apoptosis in a neonatal rat stroke model (Mao et al., 2006). Taken together, whether thalidomide can protect neurons against apoptosis after HI is becoming interesting and the mechanisms of how thalidomide exerts its neuroprotective effects need to be further investigated.

Besides regulation of TNF-α expression, thalidomide can exerts its functions through modulating different signaling pathways. Previous studies have shown that thalidomide induced cell apoptosis and limb anomalies in human embryonic fibroblasts and chicken embryo by stabilization of PTEN and suppression of phosphorylated-Akt (Knobloch et al., 2008). In a monocytic leukemia cell line, thalidomide was shown to inhibit cell growth through inhibition of extracellular signal regulated kinase (ERK) 1/2 (Gockel et al., 2004). However, the possible signaling pathways involved in thalidomide function remain unclear.

Previous studies have indicated that PI3K/Akt signaling pathway had a survival role in protecting cells from caspase-mediated apoptosis (Cantley, 2002, Ma et al., 2009). Phosphorylated-Akt (p-Akt) is proven to be necessary for neuronal survival after HI since p-Akt plays a key role in upregulation of anti-apoptotic proteins such as Bcl-2 and Bcl-xL (Hsu et al., 2010). Moreover, we recently found that PI3K/Akt pathway played important roles in neuroprotection against hypoxia–ischemia brain damage (Li et al., 2008, Zhang et al., 2009). Based on the above findings, we hypothesized that thalidomide can protect neurons from HI injury through PI3K/Akt signaling pathway. In the present study, we set up an HI model with oxygen and glucose deprivation (OGD) in cultured neurons. Using this model, PI3K specific inhibitor LY294002 was used to examine the possible signaling pathways involved in the anti-apoptotic effects of thalidomide.