Protective effects of tanshinone IIA on SH-SY5Y cells against oAβ_1–42-induced apoptosis due to prevention of endoplasmic reticulum stress

Highlights

• Tan IIA increased cell viability and inhibited apoptosis caused by oAβ_1–42 in SH-SY5Y cells.

• Tan IIA decreased the expression of GRP78, phosphor-eIF2α, ATF6, cytochrome c, cleaved caspase-9 and cleaved caspase-3.

• Tan IIA blocked ER stress-induced apoptosis via suppression of CHOP and JNK pathways.

• Tan IIA increased oAβ_1–42-induced the ratio of Bcl-2/Bax, MMP and ATP content.

• Tan IIA suppressed oAβ_1–42-induced the activity of caspase-3/7.

Abstract

Endoplasmic reticulum (ER) stress caused by β-amyloid protein (Aβ) may play an important role in the pathogenesis of Alzheimer disease (AD). Our previous data have indicated that tanshinone IIA (tan IIA) protected primary neurons from Aβ induced neurotoxicity. To further explore the neuroprotection of tan IIA, here we study the effects of tan IIA on the ER stress response in oligomeric Aβ_1-42 (oAβ_1-42)-induced SH-SY5Y cell injury. Our data showed that tan IIA pretreatment could increase cell viability and inhibit apoptosis caused by oAβ_1-42. Furthermore, tan IIA markedly suppressed ER dilation and prevented oAβ_1-42-induced abnormal expression of glucose regulated protein 78 (GRP78), initiation factor 2α (eIF2α), activating transcription factor 6 (ATF6), as well as inhibited the activation of C/EBP homologous protein (CHOP) and c-Jun N-terminal kinase (JNK) pathways. Moreover, tan IIA ameliorated oAβ_1-42-induced Bcl-2/Bax ratio reduction, prevented cytochrome c translocation into cytosol from mitochondria, reduced oAβ_1-42-induced cleavage of caspase-9 and caspase-3, suppressed caspase-3/7 activity, and increased mitochondrial membrane potential (MMP) and ATP content. Meanwhile, oAβ_1-42-induced cell apoptosis and activation of ER stress can also be attenuated by the inhibitor of ER stress 4-phenylbutyric acid (4-PBA). Taken together, these data indicated that tan IIA protects SH-SY5Y cells against oAβ_1-42-induced apoptosis through attenuating ER stress, modulating CHOP and JNK pathways, decreasing the expression of cytochrome c, cleaved caspase-9 and cleaved caspase-3, as well as increasing the ratio of Bcl-2/Bax, MMP and ATP content. Our results strongly suggested that tan IIA may be effective in treating AD associated with ER stress.

Introduction

Alzheimer’s disease (AD) is a common dementia in the elderly. Two major hallmarks, senile plaques (SPs) and neurofibrillary tangles (NFTs), are widely known to be characteristic of AD pathology. NFTs consist of hyperphosphorylated tau protein in neurons, and SPs consist of β-amyloid protein (Aβ) in the extracellular space. Several hypotheses have been currently proposed to elucidate AD pathogenesis, and Aβ hypothesis is accepted as one of the most evidence-based (Ferreira and Klein, 2011; Musiek and Holtzman, 2015). Accumulation of Aβ in the brain leads to a series of harmful events including plaque formation, tau hyperphosphorylation, oxidative stress, endoplasmic reticulum (ER) stress and eventually neuronal apoptosis (Chen et al., 2017; Gerakis and Hetz, 2017; Guo et al., 2018; Hardy and Allsop, 1991; Lim and Han, 2018; Yao et al., 2017).

The ER is the main compartment involved in protein folding and secretion and is drastically affected in AD neurons. The accumulation of unfolded or misfolded proteins in the ER activates a cellular stress response known as the unfolded protein response (UPR) and initiates the removal of toxic misfolded proteins as a way to protect the cell (Gerakis and Hetz, 2017; Hosoi et al., 2009). The UPR is initiated by three stress sensor proteins, including inositol requiring enzyme 1 (IRE1), (PKR)-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6). These are normally maintained in an inactive state via binding with an ER chaperone, glucose regulated protein 78 (GRP78). However, ER stress triggers the release of GRP78 from the complexes, and the stress sensors recognize the misfolded proteins in the ER and activate a complex signaling network of UPR (Bertolotti et al., 2000). For example, extended phosphorylation of PERK subsequently induces the phosphorylation of α subunit of eukaryotic translation initiation factor (eIF2α) at ser51. Phosphorylated eIF2α also promotes the translation of mRNAs such as ATF4 (Duran-Aniotz et al., 2014; Urra et al., 2013). ATF4 acts as the upstream activator of genes involved in amino acid metabolism and transportation, as well as genes related to apoptosis, including C/EBP homologous protein (CHOP) and members of the B-cell lymphoma 2 (Bcl-2) protein family (Chen et al., 2018; Gu et al., 2010; Vattem and Wek, 2004).

Recently, soluble oligomeric Aβ (oAβ) were reported to be more highly toxic to synapses than fibrillary Aβ (fAβ) (Evans et al., 2008; Kittelberger et al., 2012; Sakono and Zako, 2010). oAβ may be formed early on in the disease process both inside and outside synaptic terminals (Kokubo et al., 2005; Takahashi et al., 2004), and intracellular and extracellular Aβ may interact (LaFerla et al., 2007; Oddo et al., 2006). Previous studies indicated that oAβ, but not fAβ, induced cell apoptotic death by activating ER stress (Chafekar et al., 2007; Costa et al., 2012). Data from in vitro and in vivo experiments confirmed that Aβ promotes the expression of the markers of ER stress and increases the level of effectors of ER stress related apoptosis pathways such as CHOP, c-Jun N-terminal kinase (JNK), and caspase-12 (Barbero-Camps et al., 2014; Song et al., 2003; Tseng et al., 2008). In contrast, blockage of ER stress is indicated to effectively ameliorate cells damage induced by Aβ (Lee et al., 2010). These data demonstrate that ER stress is one of the significant molecular mechanisms involved in Aβ cytotocixity.

There have been continuous efforts to develop drugs or nutraceuticals to treat or prevent AD. Numerous candidate treatment agents have been examined, including various kinds of herbal and natural products. Tanshinone IIA (tan IIA) is an active lipophilic component extracted from the root of Salvia miltiorrhiza Bunge (Danshen) and exerts multiple neuroprotective potentials relevant to AD, such as anti-Aβ, antioxidant, anti-inflammation, anti-apoptosis, and acetylcholinesterase inhibition (Akaberi et al., 2016; Dong et al., 2012; Jeon et al., 2011; Jiang et al., 2014; Kong et al., 2017; Liu et al., 2016; Maione et al., 2017; Seo et al., 2017; Wang et al., 2013) (Fig. 1). Our previous study demonstrated that pretreatment of tan IIA protected primary neurons from Aβ_25–35 induced neurotoxicity, reduced the cleavage of p35 into p25 and thus inhibited the cyclin-dependent kinase 5 pathway. Additionally, the compound can significantly reduce phosphorylated tau levels in neurons and improve the impairment of the cell ultrastructure, including nuclear condensation, fragmentation, and neurofibril collapse (Shi et al., 2012). In addition, we found that tan IIA also suppresses Aβ_1–42 induced apoptosis in cortical neurons by activating the Bcl-xL pathway (Qian et al., 2012). Recently, our experimental data confirmed that tan IIA prevents streptozotocin (STZ) induced memory deficits in mice (Liu et al., 2016). It has been proposed that tan IIA protects SH-SY5Y cells against glutamate toxicity by reducing oxidative stress and regulating apoptosis and MAPK pathways (Li et al., 2017). Moreover, tan IIA enhanced insulin sensitivity and improved glucose metabolic disorders by attenuating ER stress induced insulin resistance in L6 myotubes mice (Hwang et al., 2012). Tan IIA also significantly attenuated oxidative stress injury and decreased ER stress mediated apoptosis in the protection of kidney hypothermic preservation (Zhang et al., 2012). However, the effects of tan IIA on ER stress mediated apoptosis in AD are lacking. Therefore, in the present study, we investigate the potentially beneficial effects of tan IIA on preventing ER stress and apoptosis induced by oAβ_1–42 in SH-SY5Y cells and attempted to explore its underlying mechanisms.