Cytoprotective and anti-inflammatory effects of spinasterol via the induction of heme oxygenase-1 in murine hippocampal and microglial cell lines

Abstract

Spinasterol, which is isolated from the aerial parts of Aster scaber Thunb. (Asteraceae), is involved in various biological activities. In this study, we report the efficacy of spinasterol in effectively modulating the regulation of antioxidative and anti-inflammatory activity through the upregulation of heme oxygenase (HO)-1 in murine hippocampal HT22 cells and BV2 microglia. We showed that spinasterol increased the cellular resistance of HT22 cells to oxidative injury caused by the glutamate-induced cytotoxicity by extracellular signal-regulated kinase (ERK) pathway-dependent expression of HO-1. Furthermore, spinasterol suppressed the lipopolysaccharide (LPS)-induced expression of pro-inflammatory enzymes and inflammatory mediators in BV2 microglia. Spinasterol also suppressed the production of nitric oxide (NO), prostaglandin E2 (PGE₂), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) through extracellular signal-regulated kinase (ERK) pathway-dependent expression of HO-1. These results suggest that spinasterol has a therapeutic potential against neurodegenerative diseases that are caused by oxidative stress and neuroinflammation.

Introduction

A number of diseases that lead to injury of the central nervous system (CNS) are caused by oxidative stress and inflammation in the brain [1]. Oxidative stress or the accumulation of reactive oxygen species (ROS) plays a central role in neuronal injury, cell death, and pathogenesis of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and stroke, thereby leading to neuronal death and dysfunction [2], [3], [4]. Oxidative glutamate toxicity is a form of nerve cell death, in which glutamate inhibits cystine uptake via the cystine/glutamate antiporter system Xc⁻, thereby eventually leading to programmed cell death due to glutathione depletion and accumulation of ROS [5], [6]. Immortalized mouse hippocampal HT22 cells have been used as in vitro models for studying the mechanism of oxidative glutamate toxicity. This is because neuronal cell death is induced by oxidative damage and not by the activation of ionotropic glutamate receptors, thereby excluding excitotoxicity as a cause of glutamate-induced cell death [7], [8].

Several neurodegenerative disorders such as Alzheimer's and Parkinson's diseases are characterized by neuroinflammation of the pathologically affected tissue [9], [10]. Microglia, which are brain macrophages, are the primary immune cells of the brain that are activated on injury to the brain, and they release various pro-inflammatory cytokines and inflammatory mediators such as NO, prostaglandins, TNF-α, and IL-1β [11], [12], [13], [14]. BV2, an immortalized murine microglia cell lines are widely used as a model of microglia in vitro, because of this cell line retains most of the morphological and functional properties described for primary microglia [15], [16]. In previous studies on microglial responses in CNS inflammation, microglial activation was induced by a variety of agents, including the bacterial product LPS, and pro-inflammatory cytokines such as interferon-γ (IFN-γ) and TNF-α [17], [18], [19]. Therefore, regulation of oxidative damage and neuroinflammation would be an effective therapeutic approach for alleviating the progression of neurodegenerative diseases.

Heme oxygenase (HO) is an enzyme that catalyzes the degradation of the heme group to produce carbon monoxide (CO), biliverdin, and free iron. HO and its enzymatic by-products appear to play important roles in regulating biological responses, including oxidative stress, inflammation, and cell proliferation [20]. Among the 3 reported HO isoforms (HO-1, -2, -3), HO-1 is highly inducible and expressed in many cell types, including neuronal cells [5], [8], [21]. The expression of HO-1 also has cytoprotective effects against glutamate-induced oxidative cytotoxicity in HT22 cells [5], [22]. Due to its antioxidative effects, HO-1 is considered to be a novel target for the treatment of inflammatory diseases [23], [24], [25], [26]. Studies have shown that the anti-inflammatory action of HO-1 is mediated by the inhibition of the production of pro-inflammatory cytokines and chemokines such as TNF-α, IL-1β, IL-6, and MIP-1β in activated macrophages [27], [28]. Furthermore, HO-1 and its by-product–carbon monoxide–can suppress the expression of the pro-inflammatory enzymes cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), thereby decreasing the production of COX-2-derived PGE₂ and iNOS-derived NO [29], [30].

Mitogen-activated protein kinase (MAPK) pathway is one of the most common signaling pathways that participate in transducing a variety of extracellular signals to evoke cellular responses. There are 3 members of the MAPK subfamily, namely, extracellular signal-regulated kinases (ERK), p38 kinase, and c-Jun N-terminal kinase (JNK) [31], [32], [33]. MAPK activation leads to the phosphorylation of several transcription factors and is responsible for the regulation of genes involved in the control of fundamental cellular processes such as proliferation, stress responses, apoptosis, and immune defense [34]. MAPK activation modulates the expression of several genes and proteins, including HO-1 [35].

Spinasterol, which is a biologically active compound isolated from the aerial parts of Aster scaber Thunb. (Asteraceae), exhibits various pharmacological activities, including anti-tumor, antiulcerogenic, and anti-carcinogenic activities [36], [37], [38]. Several published studies have shown that spinasterol also has anti-inflammatory effects [39]. However, the mechanisms underlying the anti-inflammatory effect of spinasterol remain to be elucidated. As a part of our ongoing research to identify phytochemicals isolated from natural sources that can induce HO-1 expression in vitro [8], [40], spinasterol was shown to induce significant expression of HO-1 in mouse hippocampal HT22 cells and BV2 microglia cells. In this study, we showed that spinasterol increased cellular resistance of HT22 to oxidative injury caused by glutamate-induced cytotoxicity by the ERK pathway-dependent expression of HO-1. Using BV2 microglia as the model, we also investigated the possible involvement of HO-1 in the anti-inflammatory activity of spinasterol, and examined whether spinasterol-mediated HO-1 expression correlates with the inhibition of LPS-induced pro-inflammatory mediators such as NO, PGE₂, TNF-α, and IL-1β. Further, we provided evidence to support the view that HO-1 plays an important role in mediating the antioxidative and anti-inflammatory effects of spinasterol.