A novel Nrf2 activator, RS9, attenuates secondary brain injury after intracerebral hemorrhage in sub-acute phase

Highlights

• RS9 decreased brain edema and neurological deficit after ICH.

• RS9 up-regulated HO-1 and SOD-1 expression after ICH.

• RS9 decreased neuronal cell death after in vitro hemin injury.

• RS9 has a protective effect for SBI after ICH.

Abstract

The poor prognosis of intracranial hemorrhage (ICH) is attributed to secondary brain injury (SBI), which is caused by oxidative stress. Blood components induce reactive oxygen species (ROS) over-production and cause cytotoxicity. We focused on the antioxidant system and investigated nuclear factor-erythroid 2-related factor 2 (Nrf2), which is a transcription factor that controls several antioxidant enzymes. We examined the effects of a novel Nrf2 activator, RS9, on SBI after ICH. ICH was induced by injecting autologous blood collected from the jugular vein (25 µL) into the striatum of mice. RS9 (0.2 mg/kg, i.p.) was administrated 0, 24, and 48 h after the induction of ICH. Using the ICH model, we measured brain edema, neurological function, neuronal damage and antioxidant proteins expression. We then investigated the mechanisms responsible for the effects of RS9 in vitro using the SH-SY5Y cell line. We used zinc protoporphyrin (ZnPP), a heme oxygenase-1 (HO-1) inhibitor, to elucidate the relationship between HO-1 expression and cell death in vitro in a hemin injury model. RS9 decreased brain edema, improved neurological deficits, decreased neuronal damage area and up-regulated HO-1 and superoxide dismutase 1 (SOD) expressions in the ICH mouse model. RS9 also suppressed neuronal cell death and ROS over-production in vitro. These protective effects were cancelled by the ZnPP co-treatment. Our results suggest that the activation of Nrf2 by RS9 exerts neuroprotective effects that are mediated by the attenuation of oxidative stress, and also that RS9 is an effective therapeutic candidate for the treatment for SBI after ICH.

Introduction

The central nervous system (CNS) is highly sensitive to oxidative stress, and the disruption of the redox system is known to induce neuronal damage (Liu et al., 2017). A main factor in oxidative stress is reactive oxygen species (ROS), which cause neuronal cell death and accelerate several neurological disorders in Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease (D'Ambrosi et al., 2017, Dias et al., 2013). The removal of excess ROS produced in CNS disorders represents a potential approach to improve patient outcomes.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that controls antioxidant responses and plays important roles in attenuating oxidative stress in order to maintain homeostasis (Itoh et al., 2010, Suzuki et al., 2013, Yamazaki et al., 2015). Nrf2 induces the expression of cytoprotective and antioxidant genes, such as heme oxygenase 1 (HO-1), nicotinamide adenine dinucleotide phosphate quinone oxidoreductase 1 (NQO-1), and glutathione s-transferase (GST) (Zhang et al., 2013). Under normal conditions, Nrf2 binds the cytoskeletal protein, Kelch-like ECH-associated protein 1 (Keap1) with the N-terminal Nrf2-ECH homology 2 (Neh2) domain of Nrf2. After exposure to oxidative stress, Nrf2 dissociates from Keap1 and the complex is translocated into the nucleus. Nrf2 subsequently binds to the antioxidant response element (ARE) and activates antioxidant proteins, which exert cytoprotective and anti-inflammatory effects (Itoh et al., 2010).

A recent study demonstrated that oxidative stress is involved in stroke (Lee and Won, 2014), particularly in intracerebral hemorrhage (ICH) (Duan et al., 2016). ICH is a devastating disease that accounts for 20–30% of stroke cases. The mortality rate in one month is approximately 40–50% (Feigin et al., 2003). The main cause of ICH is the vascular burden caused by persistently high blood pressure conditions. Vascular fragility due to arteriosclerosis and congenital cerebral arteriovenous malformation is also considered to contribute to ICH (Sutherland and Auer, 2006, Wang and Dore, 2007).

Brain injury after ICH is divided into two types: primary brain injury (PBI) and secondary brain injury (SBI). PBI is mainly caused by the mass effect of a hematoma within the brain parenchyma, and induces neuronal cell death via mechanical damage within a few hours of the onset of ICH (Aronowski and Hall, 2005, Siaw-Debrah et al., 2017). SBI is caused by hematoma components within a few days (Wang and Dore, 2007). Hematoma components induce neuronal damage and are one of the factors aggravating SBI (Hua et al., 2007, Liu et al., 2010, Liu et al., 2017). In the pathology of SBI, blood components, such as hemoglobin (Hb), heme, and hemin, induce neuronal injury by accelerating the production of ROS (Duan et al., 2016, Qu et al., 2016). Previous studies showed that hemin induced oxidative stress and neuronal injury (Siaw-Debrah et al., 2017, Zhou et al., 2017). Heme is degraded into iron (Fe²⁺), bilirubin, and carbon monoxide. Fe²⁺ generates ROS, such as the hydroxyl radical, through the Fenton reaction, which leads to oxidative stress (Satoh et al., 2006, Thomas et al., 2009).

The activation of Nrf2 induces the up-regulation of downstream antioxidant enzymes, including HO-1 (Inoue et al., 2017). In recent years, the Nrf2 pathway has been attracting increasing attention as a therapeutic target for oxidative stress in CNS disorders. Dimethyl fumarate (DMF), an Nrf2 activator, has been applied to the treatment of multiple sclerosis in the clinical stage (Linker and Haghikia, 2016). We also reported that the Nrf2 activator, bardoxolone methyl (BARD) exerted several protective effects, and ameliorated cerebral ischemia reperfusion injury and hemorrhagic infract under the administration of warfarin (Imai et al., 2016, Takagi et al., 2014). The mechanism of activating Nrf2 involves BARD interacting with Keap1 in order to prevent Nrf2 ubiquitination and accelerating translocation into the nucleus (Ichikawa et al., 2009). RS9 (C₃₂H₄₃NO₆: (1a, 2a, 21b)-2-cyano-21-hydroxy-3, 12-dioxo-1, 2-epoxyolean-9(11)-en-28-oate) is a novel Nrf2 activator that is biotransformed from BARD (Nakagami et al., 2015). A previous study demonstrated that RS9 exerts cytoprotective effects by suppressing blood-retinal barrier hyper-permeability in mice and rabbits (Nakagami et al., 2016). In addition, our findings showed that RS9 exerted protective effects in a light-induced retinal damage model and ischemic stroke model (Inoue et al., 2017, Yamauchi et al., 2016).

Some studies in the ICH field reported that DMF and nicotinamide improved ICH injury in vivo (Iniaghe et al., 2015, Wei et al., 2017). These findings indicate that the activation of Nrf2 protects against SBI in ICH. Therefore, we investigated whether RS9 exerts protective effects against SBI in vivo and in vitro.