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

Accepted Manuscript Research report A novel Nrf2 activator, RS9, attenuates secondary brain injury after intracerebral hemorrhage in sub-acute phase Tomoki Sugiyama, Takahiko Imai, Shinsuke Nakamura, Keita Yamauchi, Shigenobu Sawada, Masamitsu Shimazawa, Hideaki Hara PII: DOI: Reference:

S0006-8993(18)30443-8 https://doi.org/10.1016/j.brainres.2018.08.021 BRES 45918

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Brain Research

Received Date: Revised Date: Accepted Date:

17 May 2018 7 August 2018 20 August 2018

Please cite this article as: T. Sugiyama, T. Imai, S. Nakamura, K. Yamauchi, S. Sawada, M. Shimazawa, H. Hara, A novel Nrf2 activator, RS9, attenuates secondary brain injury after intracerebral hemorrhage in sub-acute phase, Brain Research (2018), doi: https://doi.org/10.1016/j.brainres.2018.08.021

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

Tomoki Sugiyama1#, Takahiko Imai1#, Shinsuke Nakamura*1, Keita Yamauchi2, Shigenobu Sawada3, Masamitsu Shimazawa1, Hideaki Hara1

1

Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu

Pharmaceutical University, Gifu 501-1196, Japan. 2 3

Department of Neurosurgery, Toyohashi Medical Center, Aichi 440-8510, Japan. Department of Neurosurgery, Matsunami General Hospital, 185-1 Dendai, Kasamatsu,

Gifu 501-6062, Japan. #

Contributed equally

*Corresponding author: Shinsuke Nakamura, PhD, Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan. Tel. and Fax: +81-58-230-8126, E-mail: [email protected]

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. examined the effects of a novel Nrf2 activator, RS9, on SBI after ICH.

We ICH was

induced by injecting autologous blood collected from the jugular vein (25 µL) into the striatum of mice.

RS9 was administrated 0, 24, and 48 h after the induction of ICH.

Using the ICH model, we measured brain edema, neurological function, 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. suppressed neuronal cell death and ROS over-production in vitro. effects were cancelled by the ZnPP co-treatment.

RS9 also

These protective

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.

Keywords Heme oxygenase-1, intracerebral hemorrhage, Nrf2, oxidative stress. 1

Abbreviations ALS; amyotrophic lateral sclerosis ANOVAs; one-way analysis of variance ARE; antioxidant response element BARD; bardoxolone methyl BBB; blood-brain barrier CNS; central nervous systems DMEM; Dulbecco’s Modified Eagle’s medium DMF; dimethyl fumarate DMSO; dimethyl sulfoxide FBS; fetal bovine serum GST; glutathione s-transferase Hb; hemoglobin HO-1; heme oxygenase-1 ICH; intracranial hemorrhage Keap1; kelch-like ECH-associated protein 1 NQO-1; nicotinamide adenine dinucleotide phosphate quinone oxidoreductase 1 Nrf2; nuclear factor erythroid 2-related factor 2 PBS; phosphate-buffered saline PARP; Poly (ADP-ribose) polymerase ROS; reactive oxygen species SBI; secondary brain injury SH-SY5Y; Human neuroblastoma cell SOD1; Superoxide dismutase 1 2

ZnPP; zinc protoporphyrin-IX

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1. 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). devastating disease that accounts for 20-30% of stroke cases. month is approximately 40-50% (Feigin et al., 2003). 4

ICH is a

The mortality rate in one

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). bilirubin, and carbon monoxide.

Heme is degraded into iron (Fe2+),

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). also reported that the Nrf2 activator, bardoxolone methyl (BARD) exerted several 5

We

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). activation of Nrf2 protects against SBI in ICH.

These findings indicate that the

Therefore, we investigated whether

RS9 exerts protective effects against SBI in vivo and in vitro.

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2. Results 2.1 RS9 protected the brain from SBI in the autologous blood injection ICH model The protocol of this experiment was shown in Fig. 1A.

In order to investigate the

effects of RS9 in the autologous blood injection model, we initially measured brain edema volumes using the water content test.

The water content of the ipsilateral side,

particularly in the striatum, was significantly lower in the RS9 treatment group than in the vehicle group (Fig. 1B). We also performed the Garcia test and grid walk test to investigate the effects of RS9 on neurological deficits.

In both tests, RS9 suppressed

the aggravation of neurological deficits 48 and 72 h after the induction of ICH (Fig. 1C). Moreover, we investigated the neural damage after ICH onset by using cresyl violet staining.

In early phase, there was no difference between both groups (Fig. 2A).

However, the neural damage area in RS9 treatment group was lower than that of vehicle group in sub-acute phase after ICH induction (Fig. 2B). These results suggest that RS9 protected the neuron and attenuated the aggravation of SBI after ICH.

2.2 RS9 accelerated antioxidant defense system after ICH In order to confirm the mechanism of action of RS9 for minimizing the deterioration of brain edema and neurological deficits in the autologous blood injection model, we performed western blotting 8 and 72 h after ICH. such as HO-1, NQO-1 and SOD1.

We focused on antioxidant enzyme

The expression of NQO1 was not different between

both groups (data not shown). On the other hand, the expression of HO-1 and SOD1in the peri-hematoma region was significantly increased at 72 h after the induction of ICH compared to the vehicle group.

Moreover, the expression of phosphorylation Akt was

significantly increased at 8 h after ICH induction compared to the vehicle group (Fig. 7

3B). These results indicated that the protective effects of RS9 against ICH damage via promoting the antioxidant defense system such as HO-1 and SOD1expression.

2.3 The RS9 treatment suppressed cell death in the in vitro hemin injury model RS9 exerted protective effects in the autologous blood injection model in vivo by up-regulating the expression of HO-1.

In order to elucidate the mechanisms

responsible for the effects of RS9, we performed a cell death evaluation using the in vitro hemin injury model (Fig. 4A). Hemin is a similar material to heme, which has iron atom and is the highly reactive regent.

Previous reports show that hemin induces

the neuronal toxicity through ROS over-production. (Zhou et al., 2017) Hemin treatment for 24 h induced cell death in a dose-dependent manner (Fig. 4B).

In

contrast, the RS9 treatment decreased SH-SY5Y cell death in a dose-dependent manner after the hemin treatment (Fig. 4C). We then measured ROS production in the same model using the CM-H2DCFDA probe. 4D).

RS9 (10 µM) decreased ROS production (Fig.

These results indicate that RS9 protects neuronal cells against oxidative damage

caused by the excess production of ROS. Moreover, we investigated the effect of RS9 treatment on proteins expression in in vitro injur model.

Antioxidant enzymes (HO-1, SOD1 and NQO1) and an apoptosis

marker (Poly (ADP-ribose) polymerase; PARP) were changed by RS9 and hemin co-treatment, however, there were no differences between both group (Supplemental Fig).

2.4 HO-1 was a crucial factor in RS9 protective effects In the in vivo experiment, we found that the protective effects of RS9 might be related 8

to the up-regulated expression of HO-1.

Therefore, we investigated whether the

cytoprotective effects of RS9 depend on HO-1 using ZnPP, a HO inhibitor.

As

expected, ZnPP dose-dependently suppressed the cytoprotective effects of RS9 without harmful effects (Fig. 5).

9

3. Discussion A few days after ICH, heme and hemin derived from the hematoma induce oxidative stress and cause neuronal cell death (Duan et al., 2016; Wang and Dore, 2007).

This

damage has been suggested to aggravate functional outcomes after ICH (Chen-Roetling et al., 2015a). outcomes.

We focused on the Nrf2 pathway as an approach to improve patient

Previous studies showed that the activation of Nrf2 exerts protective effects

on neurons and glial cells (Chen-Roetling et al., 2015b; Iizumi et al., 2016).

Moreover,

Nrf2 activators, such as nicotinamide, have also been shown to exert neuroprotective effects against ICH and the up-regulated expression of HO-1 improved the death rate (Chen-Roetling et al., 2017; Wei et al., 2017).

These findings indicated that the

activation of Nrf2, an antioxidant response signal, contributed to the new approach in the treatment of CNS diseases.

In the present study, we used RS9 as a novel Nrf2

activator. We speculated that RS9 may improve outcomes after ICH because it is known to exert protective effects in an ischemic stroke model (Yamauchi et al., 2016). Therefore, we herein investigated whether RS9 exerts protective effects in in vivo and in vitro ICH models. The present results demonstrated that RS9 exerted protective effects in ICH. reduced brain edema, particularly at the striatum (Fig. 1B).

RS9

Furthermore, RS9

prevented the deterioration of neurological deficits in two behavioral tests (Fig. 1C) and attenuated the neural damage (Fig. 2).

A previous study suggested that the

mechanisms underlying brain edema formation are dependent on the disruption of BBB integrity (Keep et al., 2014).

Under hemorrhagic conditions, several reactions are

induced, for example, the activation of metalloproteases (MMPs), neutrophil infiltration, and the activation of glial cells, such as microglia and astrocytes (Wang and Dore, 2007). 10

These reactions subsequently lead to inflammation and are related to cerebral edema formation after ICH.

On the other hand, the activation of Nrf2 maintains BBB

integrity and inhibits the activation of MMPs (Sajja et al., 2015).

Moreover, the Nrf2

signal also protects against astrocyte damage caused by Hb and promotes the removal of residual blood by increasing HO-1 activity (Chen-Roetling et al., 2015b; Chen-Roetling et al., 2017; Lan et al., 2017; Stokum et al., 2015).

Previous studies showed that Nrf2

activation maintained BBB integrity and promoted the hematoma clearance through the modulating microglia function (Chen et al., 2015; Zhao et al., 2015a; Zhao et al., 2014). Our results indicated that a correlation exists between the protective effects of RS9 against cerebral edema and the amelioration of neuronal deficits the ICH mouse model. Based on these results, improvements in functional outcomes may be attributed to reductions in brain edema.

As described above, the Nrf2 pathway plays a very

important role in the protection of the BBB, particularly HO

In glial cells, HO

converts heme into bilirubin and carbon monoxide, which exert antioxidant effects and remove blood components (Araujo et al., 2012; Chen-Roetling et al., 2017).

This

enzyme has two isoforms: HO-1/2. HO-1 is an inducible type that is up-regulated in response to oxidative stress and exerts antioxidant effects.

In contrast, HO-2 is the

constituent type that is expressed under normal conditions (Itoh et al., 2010; Suzuki et al., 2013; Yamazaki et al., 2015).

A previous study showed that nicotinamide exerted

neuroprotective effects in ICH by up-regulating the expression of HO-1 (Satoh et al., 2006). Therefore, we focused on HO-1 in order to elucidate the mechanisms of action of RS9.

As expected, HO-1 was significantly up-regulated around the hematoma after

ICH (Fig. 3B).

Moreover, SOD1 and phosphorylation Akt (p-Akt) were also increased

(Fig. 3B). These proteins are strongly related to Nrf2 signal. 11

SOD1is the antioxidant

factor in downstream of Nrf2 pathway, it is activated by antioxidant such as (-) epicatchin (Lan et al., 2017).

On the other hand, Akt exists in upstream of Nrf2

pathway. Previous reports demonstrated that increasing the expression of p-Akt leads to suppress the Nrf2 degradation and accelerate the antioxidant defense system (Xin et al., 2018).

In stroke condition, astaxanthin attenuated the subarachnoid hemorrhage

damage via increasing p-Akt expression (Zhang et al., 2014), and reducing the expression of p-Akt ultimately leads to cell apoptosis (Zheng et al., 2013).

These

results support our hypothesis that RS9 exerts neuroprotective effects in a manner that is dependent on the Akt-Nrf2-antioxidant pathway. In order to clarify the mechanisms responsible for the effects of RS9 in vivo, we performed a cell death evaluation using the in vitro hemin injury model.

The

SH-SY5Y cell line, a human neuroblastoma cell, was used in the present study.

This

cell line has been employed to evaluate neuronal cell death in CNS disorders, such as Alzheimer’s disease and Parkinson’s disease (Bir et al., 2014; Mengke et al., 2016). We demonstrated that RS9 reduced cell death in the in vitro hemin injury model without cytotoxicity under normal conditions (Fig. 4C).

These results indicate that RS9

reduces neuronal cell death around hematomas in ICH without side effects. In ICH conditon, various stresses, including oxidative stress and endoplasmic reticulum stress, contribute to cell damage (Qu et al., 2016).

Under these stress

conditions, intracellular calcium levels increase and ROS are generated excess (Kiselyov and Muallem, 2016), ultimately causing neuronal cell death.

ROS

over-production is generated from blood components, such as heme and hemin. Therefore, ROS appears to be closely related to SBI.

Previous studies showed that the

scavenging of ROS improved outcomes after ICH (Kumar and Bandyopadhyay, 2005; 12

Qu et al., 2016).

RS9 might scavenge ROS and improve SBI after ICH because of

RS9 decreased ROS production (Fig. 4C).

These effects of RS9 have been suggested

to be associated with the up-regulated expression of HO-1.

ZnPP, a HO inhibitor,

cancelled the protective effects of RS9 in a dose-dependent manner (Fig. 5). These results support our hypothesis that the protective effects of RS9 in neuronal cells may depend on the up-regulated expression of HO-1 via the Nrf2/ARE pathway. Collectivity, the present results show that RS9 protected the brain against SBI by promoting the antioxidant defense system such as HO-1 and SOD1.

Considering the

drug concentration, the Nrf2 activation of RS9 (0.2 mg/kg) may be superior to other Nrf2 activator such as DMF (10 or 15 mg/kg), sulforaphane (0.5 mg/kg) and nicotinamide (300 mg/kg) (Iniaghe et al., 2015; Wei et al., 2017; Xin et al., 2018; Zhao et al., 2015b).

However, the detailed mechanisms remain unclear; for example, what

brain region is HO-1 up-regulated in and what types of cell lines express HO-1 after the administration of RS9?

Previous studies showed that HO-1 was up-regulated both

neurons and glial cells in the brain (Chen-Roetling et al., 2015a; Schipper, 2004). Moreover, BARD, which is a similar compound to RS9, activates Nrf2 in neurons and astrocytes (Takagi et al., 2014).

Based on these findings, we hypothesize that RS9

up-regulates HO-1 expression in neurons and astrocytes, which may protect against SBI after ICH. In conclusion, RS9, a novel Nrf2 activator, prevented the aggravation of brain edema and neuronal damage in vivo.

It also protected against neuronal cell death following

hemin exposure. RS9 up-regulated the antioxidant enzyme such as SOD1 and HO-1 expression via the Akt- Nrf2 pathway and may protect the BBB and neuronal cells against SBI in ICH. Thus, our study suggests that RS9may be an effective therapeutic 13

candidate for the treatment for SBI after ICH.

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4. Experimental procedures 4.1 Animals and experiments All experimental designs and procedures were performed in accordance with the ARRIVE (Animal Research; Reporting In Vivo Experiments) guidelines, basic experiment guidelines (Hemorrhagic Stroke Academia Industry Roundtable, 2018) and approved by the animal experiment committees of Gifu Pharmaceutical University, Japan (Ethic nos. 2015-245, 2015-302, 2016-036, 2017-104).

In all experiments, we

used male ddY mice (12 weeks old, body weight; 40-50 g) purchased from Japan SLC, Ltd. (Hamamatsu, Japan). Animals were housed in an air-controlled room at 24±2 °C under a 12-h light-dark cycle with free access to food and water. All experimental procedures and the evaluation of outcomes were blinded to all operators (T. Sugiyama, K. Yamauchi, and S. Sawada). We used 70 mice in this study. The number of mice used in each experiment is as follow: (1) brain water content assay and behavior test, 27 mice; (2) western blotting, 24 mice, (3) neuronal damage area assay by cresyl violet staining, 9 mice. The mice were excluded from samples according to following exclusion criteria; injected autologous blood was leaked.

4.2 RS9 treatment Mice were randomly divided into two groups receiving vehicle (phosphate-buffered saline: PBS; Wako Pure Chemical, Osaka, Japan) or RS9 at 0.2 mg/kg/day (supplied from Daiichi Sankyo Co., Ltd. Tokyo, Japan). The dosage of drugs was selected based on a previous study (Yamauchi et al., 2016). RS9 was initially dissolved in dimethyl sulfoxide (DMSO, Nacalai Tesque, Kyoto, Japan) and diluted by PBS. The final 15

concentration of DMSO was less than 0.1% of solution. RS9 was administered intraperitoneally (i.p.) 0, 24, and 48 h after the induction of ICH. The vehicle group was i.p. administered the same amount of PBS.

4.3 Autologous blood injection mouse model The experimental ICH model was a modification of a previously described method (Zhu et al., 2014). Mice were randomly divided into three groups, anesthetized with 2.0-3.0% isoflurane (Mylan Inc., Canonsburg, PA), and maintained with 1.0-1.5% isoflurane in 70% N2O/30% O2 with a facemask (Soft Lander, Sin-ei Industry Co., Ltd., Saitama, Japan).

In experiments, operators were blinded to these groups. Under deep

anesthesia, autologous blood was slowly sampled from the left jugular vein.

A small

burr hole was drilled into the skull (2 mm lateral to the bregma at a depth of 3.5 mm), and a 22s-G 100-μL injection needle (Hamilton, 700 series, Hamilton, Reno, NV, USA) was slowly inserted, and collected blood (25 µL) was injected into the left brain striatum.

The speed of administration was 2 μL/30 sec. The syringe remained in

place for 5 min and was then slowly withdrawn for 5 min. After surgery, mice were housed under preoperative conditions.

4.4 Edema volume quantification The water content test was performed by a modification to a previously described method (Lei et al., 2016; Qin et al., 2015; Wang et al., 2015; Zhu et al., 2014). Brain edema was calculated in the water content test 72 h after the induction of ICH. Under deep anesthesia, mice were sacrificed and brain tissue was immediately removed. Brain tissue was cut at the center of the brain and divided into the brain striatum and 16

cortex. Each section was placed into 1.5-mL tubes and tissue weights were measured. Tissue was subsequently dried at 100 °C for 24 h in order to obtain dry weights.

The

percentage of the brain water content was calculated using the following formula: (wet weight - dry weight)/(wet weight) × 100

4.5 Neurological assessment A neurological assessment was performed in a quiet and dimly lit room 24, 48, and 72 h after the induction of ICH and operators were blinded to the treatment groups. Neurological function was evaluated by the Garcia test and Grid walk test, as previously described (Garcia et al., 1995; Lee et al., 2011; Yamauchi et al., 2016).

The Garcia

score was calculated by the following six tests: spontaneous activity, symmetry in the movement of the four limbs, forepaw outstretching, climbing ability, body proprioception, and response to vibrissae touch.

Each test scored from 0 to 3. A lower

score indicated that a severe neurological deficit was expressed in this test.

The grid

walk test was also performed in order to evaluate the function of the forepaw. Mice were placed on a grid of 0.24 mm (length, width) with 10 mm2 and freely walked in the open space for 2 min.

The number of failures was counted by operators under blind

conditions and calculated.

4.6 Neuronal damage area evaluation Mice were euthanized and transcardially perfused with cold saline for 2 min at room temperature.

After that, the perfusate was changed to 0.1 M phosphate buffer (PB; ph

7.4) containing 4% paraformaldehyde (PFA, Wako Pure Chemicals) for 2 min. Then brains were immersed in PFA overnight at 4°C. 17

Next, the brains were immersed in

25% sucrose in 0.1 M PB for 24 h and frozen in liquid nitrogen. Coronal sections were cut on a cryostat at -20°C and stored at -80°C until use, the size is 15 µm.

The

sections were washed with 0.01 M PBS, and were stained with Cresyl Violet to estimate the lesion volume. Then the sections were observed using BIOREVO BZ-X710 (Keyence, Osaka, Japan) and lesional volume was analyzed with Image-J software version 1.43h by measuring band intensities..

4.7 Western blotting Western blotting was performed by a modification to a previously described method (Imai et al., 2016; Takagi et al., 2014). Mice were sacrificed and peri-hematoma tissues were removed immediately, 8, 24, and 72 h after the induction of ICH. Tissue from the peri-hematoma side was homogenized in 10 mL/g tissue with ice-cold RIPA lysis buffer containing protease inhibitor and phosphatase inhibitors -II and -III (Sigma Aldrich Co., St. Louis, MO, USA). The lysate was centrifuged at 12,000 × g at 4 °C for 20 min.

The supernatants of the samples were collected. The protein

concentration was compared with BSA known the protein concentration and calculated using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equivalent protein samples were separated according to molecular weight by electrophoresis using a 5-20% gradient sodium dodecyl sulfate-polyacrylamide gel (SuperSep Ace; Wako Pure Chemicals, Osaka, Japan) and transferred to a polyvinylidene fluoride membrane (Immobilon-P: Millipore Corporation, Billerica, MA, USA). After blocking by blocking one P (Nacalai Tesque) for 30 min, membranes were incubated with the primary antibody overnight.

The following antibodies were

used: rabbit anti-HO-1 (1:200, Santa Cruz, Dallas, TX, USA) and rabbit anti-SOD1 18

(1:1000, Cell Signaling Technology, MA, USA) and rabbit anti-total Akt (1:1000, Cell Signaling Technology) and phospshorylation-Akt (1:1000, Cell Signaling Technology) and mouse anti-β-actin (1:2000, Sigma Aldrich).

The following secondary antibodies

were used: anti-rabbit IgG (1:2000, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and anti-mouse IgG (1:2000, Pierce Biotechnology, Rockford, IL, USA). Immunoactive bands were visualized using a Lumino imaging analyzer (LAS-4000; Fujifilm, Tokyo, Japan).

Multi Gauge software (Fujifilm) was used to analyze

differences in band intensity.

4.8 Cell culture Human neuroblastoma (SH-SY5Y) cells (European Collection of Cell Culture, Wiltshire, UK) were maintained using Dulbecco’s Modified Eagle’s medium (DMEM, Nacalai Tesque) containing 10% fetal bovine serum (FBS) (VALEANT, Costa Mesa, CA, USA), 100 units/mL penicillin (Meji Seika, Tokyo, Japan), and 100 units/mL streptomycin (Meji Seika) in a humidified atmosphere of 5% CO2 at 37 °C. Cells were passaged by trypsinization every 2-4 days.

4.9 Cell death assay A cell death assay was performed by a modification to a previously described method (Tsujii et al., 2015). SH-SY5Y cells were seeded at 1 × 104 cells per well on a 96-well plate (number 3072, Falcon®, Becton Dickinson and Company, Franklin Lakes, NJ, USA) with medium containing 10% FBS and incubated for 24 h in a humidified atmosphere containing 5% CO2 at 37 °C. Medium was then replaced with DMEM containing 1% FBS. Cells were treated with RS9 (0.1-10 nM), hemin (1, 10, 25 or 50 19

μM), and zinc protoporphyrin Ⅸ(ZnPP: 0.1-1 µM, Santa Cruz) for 24 h. Cell death was evaluated by co-staining with Hoechst 33342 (Molecular Probes, Eugene, OR, USA) and propidium iodide (PI; Molecular Probes). After the incubation, 8 µM of Hoechst 33342 and 0.2 µM of PI were added to the medium and incubated for 15 min. All images were taken using an Olympus IX 70 inverted epifluorescence microscope (Olympus, Tokyo, Japan). At least 300 cells were present in each image and counted by a blind observer.

4.10 Measurement of ROS production ROS production was measured by a modification to a previously described method (Kuse et al., 2014).

Intracellular radical activation in SH-SY5Y cells was measured 24

h after the hemin incubation using the CM-H2DCFDA probe (Thermo Fisher Scientific). SH-SY5Y cells were seeded at 1 × 104 cells per well on a 96-well plate with medium containing 10% FBS and incubated for 24 h in a humidified atmosphere containing 5% CO2 at 37 °C. Medium was then replaced with DMEM containing 1% FBS. Cells were treated with RS9 (0.1-10 nM) and hemin (10 μM) for 24 h. The CM-H2DCFDA probe was added to the cultured medium at a final concentration of 10 µM and incubated at 37 °C for 30 min. Fluorescence was subsequently measured using a Varioscan Flash 2.4 microplate reader (Thermo Scientific) at 485 nm (excitation) and 535 nm (emission) at the end of the hemin incubation and after 30 min. Medium was then changed for DMEM containing 1% FBS to remove the CM-H2DCFDA probe. The number of cells was counted by co-staining with Hoechst 33342 and PI and ROS production per cell was calculated.

20

4.11 Statistical analysis All data are shown as the mean ± standard error of the mean (SEM). Quantitative variables were statistically analyzed using the Student’s two-tailed t-test or Mann-Whitney U test for two-group comparisons and a one-way analysis of variance (ANOVAs) followed by Dunnett’s test or Tukey’s test for multiple pair-wise comparisons. Significance was less than 0.05.

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Acknowledgemnts We would like to thank Yasuhiro Nakagami (Daiichi Sankyo Co., Ltd.) for suggesting instructions regarding this work.

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e97423.

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Figure legends Figure 1. Protective effects of RS9 at 72 h in the autologous blood injection mouse model Experimental groups included the vehicle group (n = 13) and RS9 (0.2 mg/kg) group (n = 14). Mice were sacrificed 72 h after the induction of ICH in each group. Protocol for the induction of ICH.

(A)

Mice were subjected to 72 h of ICH and treated

with vehicle or 0.2 mg/kg of RS9 intraperitoneally after the induction of ICH and once a day.

(B) The brain water content was evaluated 72 h after the induction of ICH (#p <

0.05 vs. vehicle, the Student’s t-test).

(C) Neurological scores were evaluated 24, 48,

and 72 h after the induction of ICH (#p < 0.05 vs. vehicle, the Mann-Whitney U test). Data are expressed as the mean ± S.E.M..

Figure 2. RS9 protected the neural damage after ICH Experimental groups included the vehicle group (24 h, n = 4, 72 h, n = 5) and RS9 (0.2 mg/kg) group (24 h, n = 5, 72 h, n = 5). Mice were sacrificed at 24 h or 72 h after the induction of ICH. (A) The results at 24 h after ICH induction. representative images of cresyl violet staining.

Right graph shows the quantitative

data of neuronal damage area (% of total brain area). induction.

Left images show the

(B) The results at 72 h after ICH

Left images show the representative images of cresyl violet staining.

Right graph shows the quantitative data of neuronal damage area (% of total brain area). (#p < 0.05 vs. vehicle, the Student’s t-test). expressed as the mean ± S.E.M..

30

Scar bar shows 1 mm.

Data are

Figure 3. Proteins expression at 8 and 72 h in the autologous blood injection mouse model Experimental groups included the vehicle group (n = 5) and RS9 (0.2 mg/kg) group (n = 5). Mice were sacrificed 8 and 72 h after the induction of ICH. Brain tissues were obtained from the peri-hematoma area on the ipsilateral side of autologous blood injection mice model.

(A) The representative bands. (B) The quantitative data.

(**p

< 0.01 vs. Control, #p < 0.05, vs. Vehicle, Tukey’s test). Data are expressed as the mean ± S.E.M..

Figure 4. Protective effects of RS9 at 24 h in the in vitro hemin injury model (A) The protocol used in the present study was shown. SH-SY5Y cells were treated with the RS9 (0.1-10 nM) co-treatment with hemin (10 µM). (B) The quantitative data of cell death after hemin treatment.

(C) Hoechst 33342 and PI staining was performed 24 h

after the incubation. RS9 attenuated SH-SY5Y cell death caused by hemin. (**p < 0.01 vs. Control, #p < 0.05, ##p < 0.01 vs. Vehicle, Dunnett’s test, n = 5). (D) An ROS assay was performed 24 h after the incubation. RS9 decreased ROS production 24 h after the incubation. (**p < 0.01 vs. Vehicle, *p < 0.05 vs. RS9 (10 nM),

##

p < 0.01 vs. Vehicle,

Dunnett’s test, n = 5). Data are expressed as the mean ± S.E.M..

Figure 5. Effects of a HO-1 inhibitor in the RS9 treatment at 24 h in the in vitro hemin injury model ZnPP was added 1 h before the treatment with hemin (10 μM) and RS9 (10 nM). Hoechst 33342 and PI staining was performed 24 h after the incubation.

ZnPP

suppressed the protective effects of RS9. (**p < 0.01 vs. Control, ##p < 0.01 vs. Vehicle, 31

$$

p < 0.01 vs. the RS9 treatment group, Tukey’s test, n = 5).

mean ± S.E.M..

32

Data are expressed as the

33

34

35

36

37

38

Highlight ・ 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.

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