Licochalcone E activates Nrf2/antioxidant response element signaling pathway in both neuronal and microglial cells: therapeutic relevance to neurodegenerative disease

Abstract

Oxidative stress and neuroinflammation are hallmarks of neurodegenerative diseases, which do not play independently but work synergistically through complex interactions exacerbating neurodegeneration. Therefore, the mechanism that is directly implicated in controlling oxidative stress and inflammatory response could be an attractive strategy to prevent the onset and/or delay the progression of neurodegenerative diseases. The transcription factor nuclear factor-E2-related factor-2 (Nrf2) is the guardian of redox homeostasis by regulating a battery of antioxidant and phase II detoxification genes, which are relevant to defense mechanism against oxidative stress and inflammatory responses. In this study, we show that a recently identified Glycyrrhiza-inflata-derived chalcone, licochalcone E (Lico-E), attenuates lipopolysaccharide-induced inflammatory responses in microglial BV2 cells and protects dopaminergic SH-SY5Y cells from 6-hydroxydopamine cytotoxicity. Lico-E activates Nrf2-antioxidant response element (ARE) system and up-regulates downstream NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1). Anti-inflammatory and cytoprotective effects of Lico-E are attenuated in siRNA-mediated Nrf2-silencing cells as well as in the presence with specific inhibitor of HO-1 or NQO1, respectively. Lico-E also has neuroprotective effect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nigrostriatal dopaminergic neurodegeneration in mice, with up-regulation of HO-1 and NQO1 in the substantia nigra of the brain. This study demonstrates that Lico-E is a potential activator of the Nrf2/ARE-dependent pathway and is therapeutically relevant not only to oxidative-stress-related neurodegeneration but also inflammatory responses of microglial cells both in vitro and in vivo.

Introduction

Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by a selective loss of dopamine (DA)-producing neurons in the substantia nigra pars compacta (SNpc) and a reduction in their terminals within the striatum. Dopaminergic (DArgic) neurodegeneration in this nigrostriatal pathway causes typical motor deficits, such as resting tremor, rigidity, bradykinesia and postural instability. While the etiological link to DArgic neuronal loss remains unknown, evidences suggest crucial roles for oxidative stress, mitochondrial defects, protein aggregation and misfolding, and inflammation in the pathogenesis of PD [1], [2].

Oxidative stress has been largely associated with the development of PD due to the highly oxidative conditions in DArgic neurons. Oxidative stress activates death signaling, including mitochondria-dependent apoptosis, and finally triggers cellular demise [3]. Among various candidates that can generate oxidative stress in DArgic neurons, DA itself is a primary source of oxidative stress that causes cytotoxicity to DArgic neurons [4]. DA-induced oxidative stress can be induced by auto-oxidation forming reactive quinone species [5], [6], [7]. Quinones are unstable and highly reactive and can readily attack cellular nucleophiles and cause covalent modification of essential macromolecules.

In addition, neuroinflammatory processes play a significant role in the generation of oxidative stress and the pathogenesis of PD. Neuropathological evidence indicates that neuroinflammatory response is detectable and accompanied by neuronal loss in PD: for example, inflammatory mediators are present in the cerebrospinal fluid [8] and the brains of PD patients [9], and signs of microglial activation can be observed in the area of SNpc in Parkinson's brain [10]. Many toxic factors, including superoxide anions, nitric oxide (NO), arachidonic acid and its metabolites, chemokines, and proinflammatory cytokines, can be generated by microglial activation. Activated microglia causes increased generation of reactive oxygen species (ROS), and oxidative stress also has the potential to induce an inflammatory response. Thus, these two mechanisms — oxidative stress and inflammation — are not independent but rather work synergistically through complex interactions exacerbating neurodegeneration. Therefore, the mechanism directly implicated in controlling oxidative stress and inflammatory response could be an attractive target in preventing the onset and/or delaying the progression of PD.

The transcription factor nuclear factor-E2-related factor-2 (Nrf2) is a central protein that regulates the transcription of phase II enzymes and other enzymes that are important in the antioxidative response. Under normal conditions, Nrf2 is sequestered in the cytoplasm by binding to Kelch-like ECH-associated protein 1 (Keap1). Upon stimulation, Nrf2 dissociates from Keap1 and is translocated into the nucleus where it binds to antioxidant response element (ARE) and activates the expression of ARE-dependent genes. ARE is a cis-acting regulatory element in promoter regions of genes encoding antioxidant proteins and plays a crucial role in the transcriptional regulation of downstream genes important in the cellular response to oxidative stress, such as NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1). NQO1 catalyzes the two-electron reduction of quinone to the redox-stable hydroquinone and protects cells against oxidative damage [11], [12]. HO-1 is responsible for the conversion of heme to biliverdin and carbon monoxide and functions as an antioxidant enzyme [13], [14] and an anti-inflammatory stress protein [15].

The roots of Glycyrrhiza inflata B. (Leguminosae) are known to be a source for characteristic phenolic compounds that have various pharmacological activities, including anti-inflammatory [16], antioxidative [17] and anticarcinogenic activities [18]. Six retrochalcones or reversely constructed chalcones have been isolated: licochalcone A–E and echinatin [18], [19]. Licochalcone E (Lico-E) is a very recently characterized retrochalcone that exhibits potent cytotoxicity to human tumor cell lines and endothelial cells [20], as well as anti-inflammatory potential to reduce skin inflammation [21].

Because chalcones are powerful inducers of phase II detoxifying enzymes and cytoprotective HO-1 [22], [23], in the present study, we asked whether (a) Lico-E possesses anti-inflammatory activity in lipopolysaccharide (LPS)-induced microglial BV2 cells, and antioxidant and cytoprotective activities from 6-hydroxydopamine (6-OHDA)-induced DArgic SH-SY5Y cell death; (b) Lico-E activates Nrf2 signaling and induces NQO1 and HO-1, which mediate the cytoprotective and anti-inflammatory activities of Lico-E; and (3) Lico-E is cytoprotective against DArgic neurodegeneration in the in vivo experimental models of PD.