Hydrogen sulfide in stroke: Protective or deleterious?

Accepted Manuscript Hydrogen sulfide in stroke: Protective or deleterious? Su Jing Chan, Peter T.-H. Wong PII:

S0197-0186(16)30415-6

DOI:

10.1016/j.neuint.2016.11.015

Reference:

NCI 4002

To appear in:

Neurochemistry International

Received Date: 31 October 2016 Revised Date:

24 November 2016

Accepted Date: 28 November 2016

Please cite this article as: Chan, S.J., Wong, P.T.-H., Hydrogen sulfide in stroke: Protective or deleterious?, Neurochemistry International (2017), doi: 10.1016/j.neuint.2016.11.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

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Hydrogen sulfide in stroke: protective or deleterious?

Su Jing Chan1# and Peter T.-H. Wong2*

Institute of Medical Biology, Agency for Science, Technology and Research

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1

(A*STAR), Singapore; Departments of Radiology, Massachusetts General

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Hospital, Harvard Medical School

Department of Pharmacology, Yong Loo Lin School of Medicine, National

University of Singapore

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*Corresponding author: Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, 16, Medical Drive, #04-01, Singapore

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117600. Email: [email protected]

Present address: Departments of Radiology and Neurology, Massachusetts General

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Hospital, Harvard Medical School, Charlestown, MA 02129, USA.

Running title: Hydrogen sulfide in stroke Key words: Hydrogen sulphide, stroke, toxic effect, neuroprotection

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ACCEPTED MANUSCRIPT Abbreviations: 3MST, 3-mercaptopyruvate sulfurtransferase; ADT, 5-(4-methoxyphenyl)-3H-1,2dithiole-3-thione; AIF, apoptosis-inducing factor; Akt, protein kinase B; APAF-1, Apoptotic protease activating factor 1; Bax, Bcl-2-associated X protein; BBB, blood-

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brain-barrier; Bcl-2, B-cell lymphoma 2; BDNF, brain-derived neurotrophic factor; CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase; Cys, cysteine; eIF2α, translational initiation factor 2α; ER, endoplasmic reticulum; grp78, 78 kDa glucose-

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regulated protein; γ-GCS, γ-glutamylcysteine synthetase; GSH, glutathione; Hcy,

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homocsteine; HIF-1α, hypoxia-inducible factor-1α; HSP70, heat shock protein 70; hyperHcy, hyperhomocysteinemia; IL, interleukin; iNOS, inducible nitric oxide synthase; MAPK, mitogen-activated protein kinase; MAT, Met S-adenosyltransferase; MDA, malondialdehyde; Met, methionine; MMP-9, matrix metallopeptidase; MTHFR, methylene-tetrahydrofolate reductase; mTOR, mechanistic target of

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rapamycin; Na2S, sodium sulfide; NaHS, sodium hydrosulfide; NF-κB, nuclear factor-κB; NOX, nicotinamide adenine dinucleotide phosphate oxidase; Nrf2, nuclear

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factor-2; OGD, oxygen glucose deprivation; PARP-1, poly(ADP-ribose)polymerase-1; PERK, protein kinase-like endoplasmic reticulum kinase; PI3K, phosphatidylinositol

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3-kinase; PTEN, phosphatase and tensin homolog; PTP, protein tyrosine phosphatase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SIRT-1, silent mating type information regulator 2 homolog 1; SOD, superoxide dismutase; SQR, sulfidequinone oxidoreductase; pMCAO, permanent middle cerebral occlusion; tMCAO, transient middle cerebral occlusion; TNF-α, tumor necrotic factor α.

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ACCEPTED MANUSCRIPT Contents Abstract ……………………………………………………………………………...4 1. Introduction ……………………………………………………………………….5 2. H2S is an endogenous molecule with physiological functions ……………………5

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3. H2S biosynthesis in the brain …………………………………………………….. 7 4. Homocysteine and Cysteine in ischemic stroke …………………………………..9 5. H2S in ischemic stroke – protective or deleterious? ...............................................11

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6. Mechanisms of action …………………………………………………………….15 6.1 Deleterious mechanisms ………………………………………………...15

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6.2 Protective mechanisms

6.2.1 Anti-inflammation……………………………………………..17 6.2.2 Anti-oxidation …………………………………………………18 6.2.3 Anti-apoptosis …………………………………………………19 6.2.4 Anti-ER stress …………………………………………………21

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7. Concluding Remarks ……………………………………………………………...22

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ACCEPTED MANUSCRIPT Abstract Hydrogen sulfide is believed to be a signalling molecule in the central nervous system. It is known to increase rapidly following an ischemic insult in experimental stroke. Is it protective or deleterious? This review surveys the relevant information

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available in the literature. It appears that there is no definitive answer to this question at present. Current evidence seems to suggest that the presence of H2S in the ischemic brain may either be deleterious or protective depending on its concentration,

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deleterious when high and protective when low. Therefore, it can be inferred that either an enhancement or a reduction of its concentration may be of potential use in

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future stroke therapy.

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ACCEPTED MANUSCRIPT 1. Introduction In the past 2 decades or so, hydrogen sulfide (H2S) has been transformed from a toxic gas with an offensive odour to a gasotransmitter (Wang, 2002) with important functional roles in major organ systems such as the cardiovascular (Geng et al., 2004;

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Bian et al., 2006; Liu et al., 2012) and central nervous systems (CNS) (Abe and Kimura, 1996; Abe et al., 1996; Liu et al., 2012; Tan et al., 2010). While there seems to be a general consensus that H2S plays a protective role in the cardiovascular system

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(CVS) (Liu et al., 2011; Dongo et al., 2011; Ji et al., 2008; Lavu et al., 2011; Lefer, 2007; Sodha et al., 2009; Szabo et al., 2011), there is controversy in the ischemic

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brain. Is it protective or deleterious in stroke? This short review attempts to decipher the information available in the current literature.

2. H2S is an endogenous molecule with physiological functions

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H2S, a colourless toxic gas well known for its rotten egg smell, was described way back in the early eighteenth century (Ramazzini et al. 1713). H2S has emerged as an endogenous signalling molecule produced in many tissues in mammalian species

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(Hosoki et al., 1997; Linden et al., 2008; Doeller et al., 2005; Kamoun, 2004; Renga, 2011; Shibuya et al., 2013). It is water soluble and, at physiological pH,

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approximately 70% are ionized to hydrosulfide anion (HS-) with an insignificant amount of sulfur anion (S2-) (Reinffenstein et al., 1992; Cuevasanta et al., 2016). H2S is slightly hydrophobic and twice as soluble in lipid membranes as in water, thus it can rapidly traverse plasma membranes and diffuse between compartments, such as from cytoplasm to mitochondria where it can be oxidized (Cuevasanta et al., 2016). H2S can be found in many tissues, including heart, liver, blood and brain. H2S levels measured in biological tissues had been decreasing with improved measuring

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ACCEPTED MANUSCRIPT techniques and advancement in technology. It was previously reported that the H2S concentration in the brain was as high as 50-160 µM (Goodwin et al., 1989; Warenycia et al., 1989; Mitchell et al., 1993; Yu et al., 2015). Ishigami et al. (2009) reported 9 µM, using a method that is likely to be superior to the traditional

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methylene blue assay by avoiding the possibility of measuring acid labile sulfur. However low H2S levels of about 14 nM has also been measured in the mouse brain using gas chromatography (Furne et al., 2008). More recently, the endogenous level

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of free H2S in the brain has been measured at 0.12 nmol/g protein which is thousands of folds lower than the levels of acid-labile H2S at about 900 nmol/g protein (Levitt et

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al., 2011). However, as H2S will be released from its acid-labile sulfur pool only when mitochondrial pH falls below 5.4, the acid-labile sulfur pool is not likely to be a main source of H2S release at physiological pH (Kimura, 2015). On the other hand, H2S can be released from bound sulfane sulfur localized in cytoplasm under both reducing and

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acidic conditions. It is widely believed that H2S release from the bound sulfane sulfur is regulated by the redox status in the cytoplasm (Kimura, 2015). In view of the low free H2S levels measured, Levitt et al. (2011) suggested that it may not directly

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influence tissue metabolism in the brain. However, as H2S is known to have a high

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turnover rate with an estimated half-life of 10 min in brain homogenate (Vitvitsky et al., 2012), the possibility of H2S involving in normal brain physiology cannot be ruled out. The physiological roles of H2S in the CNS have been described (Abe et al., 1996; Bos et al., 2015). It may function as a neuromodulator regulating ion channels and tyrosine kinase activities (Abe et al., 1996; Liu et al., 2012). Furthermore, several pathophysiological roles of H2S has been widely studied in CNS diseases, for example Parkinson disease (Hu et al., 2010; Kida et al., 2011; Tang et al., 2011), Alzheimer’s disease (Barbaux et al., 2000; Fan et al., 2013; Giuliani et al., 2013;

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ACCEPTED MANUSCRIPT Morrison et al., 1996), Huntington's disease (Paul et al., 2014) and ischemic stroke (Qu et al., 2006; Wong et al., 2006).

3. H2S biosynthesis in the brain

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H2S biosynthesis is closely linked to the transulfuration pathway through which, homocysteine (Hcy) is converted by cystathionine β-synthase (CBS, EC. 4.2.1.22) to cystathionine, which is then converted to cysteine (Cys) by cystathionine

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γ-lyase (CSE, EC. 4.4.1.1) (Qu et al., 2008). H2S is known to be synthesized endogenously by three enzymes, namely CBS, CSE and 3-mercaptopyruvate

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sulfurtransferase (3MST, EC. 3.4.1.2) in conjunction with cysteine aminotransferase (EC 2.6.1.3) (Kabil and Banerjee, 2014). CBS and CSE have drawn much interest and are better understood. CSE, which produces H2S from Cys, can be found in abundance in the cardiovascular system (Bian et al., 2006) and its mRNA levels were

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previously reported in the myocardium (Geng et al., 2004), endothelial cells (ECs) (Yang et al., 2008) and smooth muscle cells (SMCs) (Zhao et al., 2001). The expression of CSE was confirmed to be minor in the brain and in support, CSE

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inhibitor was shown not to suppress the production of H2S in the rat brain (Abe and

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Kimura, 1996; Bian et al., 2006). CSE-/- mice developed symptoms like spontaneous hypotension (Geng et al., 2004), lethal myopathy and were more susceptible to oxidative stress with Cys deficient diet (Ishii et al., 2010). Recently, Jiang et al. (2015) reported that CBS is upregulated in the cerebral cortex of CSE-/- mice following MCA ligation. Moreover, they also reported that L-cysteine-induced H2S production was increased in the ischemic cortex, when compared to the contralateral control, to the same extent in both control and CSE-/- mice.

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ACCEPTED MANUSCRIPT While CBS is strongly expressed in the brain, there are some conflicting observations with respect to the cellular localization of this enzyme. Using in situ hybridization and Northern blot, Robert et al. (2003) found that the expression of CBS was strongest in the Purkinje cell layer and hippocampus. Immunohistochemical

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staining indicated that CBS localization in most parts of the mouse brain and predominantly in the cell bodies and neuronal process of Purkinje cells and Ammon’s horn neurons. Enokido et al. (2005) confirmed the ubiquitous presence of CBS but

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reported intense expression of CBS in the cerebellar molecular layer and hippocampal dentate gyrus. They further reported that CBS is also preferentially expressed in

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cerebellar Bergmann glia and in astrocytes throughout the brain. This was supported by the presence of a functional transsulfuration pathway in cultured human astrocytes (Vitvitsky et al., 2006). Similarly, Chan et al. (2015) demonstrated that CBS immunostaining colocalized with GFAP staining indicating an astrocytic localization.

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Lee et al. (2009) have also reported that in human astrocytes, CBS enzymatic activity specific to H2S production was higher than those in microglia, SH-SY5Y neuroblastoma, and embryonic NT-210 carcinoma by 7, 10 and 11-fold, respectively,

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thus providing further support that CBS has a preponderant expression in astrocytes. 3MST produces H2S from 3-mercaptopyruvate which is produced from Cys

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and α-ketoglutarate via the action of CAT. This pathway was discovered following reports that CBS-/- mice still have the capacity to synthesize H2S in the brain (Shibuya et al., 2009a). 3MST is believed to produce H2S more efficiently than CBS by utilizing bound sulfane sulphur (Shibuya et al., 2009b). 3MST expression was first described to be mainly localized in neurons in many areas of the mouse brain and spinal cord. Specifically in the cortex, 3MST was found in pyramidal neurons located in layers II/III and V and in the layers of I-VI of the neocortical areas (Shibuya et al.,

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ACCEPTED MANUSCRIPT 2009a). However, it has also been reported that 3MST expression was found mainly in GFAP-positive cells, but negative in NeuN-positive cells using double immunofluorescent staining (Zhao et al. 2013). The predominant localization of 3MST expression remains to be resolved.

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Based on current evidence, it may be concluded that CBS is a predominant H2S synthesizing enzyme in the CNS. The most kinetically efficient reaction is the βreplacement of Cys with Hcy to H2S and cystathionine, which may contribute up to

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95% of the net H2S production by CBS in the brain at high substrate concentrations (Fig. 1) (Kabil et al., 2011). However, significant contribution from 3MST may not be

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ruled out. Interestingly, aspartate, an inhibitor of the CAT/3MST pathway, had been reported to abolish the post-ischemic dysfunction of the blood-brain-barrier (BBB) (Jiang et al., 2015).

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4. Homocysteine and Cysteine in ischemic stroke

Hcy can be derived from methionine (Met) which is an essential amino acid. First,

Met

was

converted

to

S-adenosylmethionine

(SAM)

by

Met

S-

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adenosyltransferase (MAT). SAM was then demethylated to S-adenosylhomocysteine (SAH) and further hydrolyzed into Hcy. However, Hcy can be remethylated to Met by

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Met synthase (MS) (Stipanuk, 2004). MS works with vitamin B12 as coenzyme and 5methyl-tetrahydrofolate as the methyl donor. As 5-methyl-tetrahydrofolate is a product of folate metabolism that requires methylene-tetrahydrofolate reductase (MTHFR), the conversion of Hcy to methionine is vitamin B12 dependent and folate (Gorelick, 2002) (Fig. 1). In addition, Hcy can be metabolized by the transulfuration pathway to Cys by CBS with coenzyme vitamin B6. Hence, elevated levels of Hcy or hyperhomocysteinemia (hyperHcy) may result from vitamin B and folate deficiency,

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ACCEPTED MANUSCRIPT as well as genetic factors such as C667T substitution of the MTHFR gene (Li et al., 2014; Morita et al., 1998) or CBS deficiency (Clarke et al., 1991). Accumulating evidence suggests that Hcy may lead to adverse consequences such as endothelial dysfunction, posttranslational modification of protein, increased

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oxidative stress, increase in nitric oxide availability and platelet activation (Santilli et al., 2016). MTHFR polymorphism (C677T) is associated with increased risk for ischemic stroke (Cronin et al., 2005; Linnebank et al., 2005; Sazci et al., 2006; Kim et

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al., 2007; Sawula et al., 2009). Numerous clinical studies have reported that hyperHcy

1998; Kawasaki et al., 1999)

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is an independent risk factor of stroke (Ansari et al., 2014; Selhub and D’Angelo,

High Hcy is directly associated with increased cerebral arterial resistance (Lim et al., 2009). It may mediate atherosclerotic progression as evidenced by its association with increases in aortic arch plague thickness and area in patients with

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ischemic stroke or transient ischemic attack (Sen et al., 2010). As a result, there had been, in the past decade, a number of clinical studies that focused mainly on lowering of Hcy level in ischemic stroke patients using vitamin B6, B12 and/or folate

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supplementation, including the Norwegian Vitamin Trial (NORVIT), the Western

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Norway B Vitamin Intervention Trial (WENBIT) and Women’s Antioxidant and Folic Acid Cardiovascular Study (WAFCS), but no beneficial effects had been demonstrated (Terwecoren et al., 2009). The VITAmins TO Prevent Stroke (VITATOPS) trial that involved 8164 patients from 1998 to 2008, also showed no beneficial effects of such treatment in stroke patients (VITATOPS, 2010). Moreover, the 5 years follow-up trial, Heart Outcomes Prevention Evaluation 2 (HOPE 2) involving 5522 participants demonstrated that vitamin treatment could not reduce stroke severity or disability (Saposnik et al., 2009). A meta-analysis of randomized

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ACCEPTED MANUSCRIPT controlled trials also indicated that B-vitamin supplementation is not associated with a lower risk of stroke based on relative and absolute measures (Zhang et al., 2013a). Interestingly, unlike C677T polymorphism, CBS polymorphism is not known to increase risk for ischemic stroke (Pezzini et al., 2002; Sawula et al., 2009). One

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possible explanation may be that in the absence of CBS, Hcy could not be converted to Cys and H2S which has been suggested to activate the arachidonic acid cascade by phosphorylating phospholipase A2 and increased generation of thromboxane A2 may

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promote atherothrombosis (Santilli et al., 2016). In a small cohort study, it has also been reported that higher plasma Cys levels were linked to early stroke deterioration

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and poor clinical outcome 3 months post stroke in acute stroke patients, while similar associations were not found for the excitotoxic amino acids glutamate and aspartate (Wong et al., 2006). As discussed in the previous section, Cys and Hcy are the major

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precursors of H2S via the action of CBS in the brain.

5. H2S in ischemic stroke – protective or deleterious? The role of H2S in ischemic stroke has only been studied in animal stroke

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models. Wong et al. (2006) reported that pre-stroke loading of Cys dose dependently

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increased infarct volume. This effect of Cys loading was shown to be completely abolished by the co-administration of a CBS inhibitor aminooxyacetic acid, indicating that it required the conversion of Cys to H2S. Qu et al. (2006) reported increases in plasma and brain levels of H2S and in CBS activity 24 hours after permanent middle cerebral artery occlusion (pMCAO). Consistently, Wang et al. (2014) reported a small increase in H2S level in mice subjected to transient middle cerebral artery occlusion (tMCAO) with 45 min or 3 hours reperfusion. Qu et al. (2006) further reported that exogenously administered H2S in the form of sodium hydrosulfide (NaHS, 180

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ACCEPTED MANUSCRIPT µmol/kg, i.p.) increased infarct volume in pMCAO rats, but not at 90 µmol/kg. Such increase was attenuated by an NMDA receptor antagonist. Importantly, administration of CBS inhibitors resulted in a reduction in infarct volume suggesting that production of endogenous H2S contributed to ischemic injuries (Qu et al., 2006; Hadadha et al.,

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2015). Consistently, Dai et al. (2016) measured hippocampal H2S levels in rats subjected to global ischemia (4 vessel occlusion) and found a 50% increase when compared to sham controls (at about 14 nmol/g). This increase was abolished by the

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administration of hydroxylamine (a non-selective CBS inhibitor) but markedly enhanced (about 70% to 37 nmol/g) by NaHS at a low dose of 14 µmol/kg.

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The mechanism of action for H2S increase could be due to a rapid increase in CBS expression after stroke onset, as the more active truncated CBS was found elevated markedly in the cortex and striatum (Chan et al., 2015). Similarly, in an in vitro setting, CBS was observed to increase in primary astrocytes subjected to oxygen

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glucose deprivation (OGD). These authors concluded that CBS inhibition might be a viable approach in the treatment of acute stroke. A recent report demonstrated that a novel, more selective CBS inhibitor can attenuate cellular H2S levels and reduce

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ischemic infarct volume in tMCAO rats (McCune et al., 2016).

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Using a global cerebral ischemia-reperfusion model in rats (permanent bilateral occlusion of the vertebral arteries and transient (15 min) bilateral occlusion of the common carotid arteries), Ren et al. (2010) reported up-regulation in CBS expression at 12 hours after reperfusion but down-regulation at 24 hours after reperfusion with concomitant increases and decreases, respectively, in CBS activities and H2S levels in both the hippocampus and cortex. They further assessed the neuronal injuries in the hippocampus on Day 7 after reperfusion, with or without a single administration of NaHS at 30 min prior to global ischemia with dosages

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ACCEPTED MANUSCRIPT ranging from 25 to 180 µmol/kg. They found that neuronal injuries were markedly exacerbated by NaHS treatment at 180 µmol/kg but no effects were observed at 90 µmol/kg, entirely consistent with previous findings (Qu et al., 2006). However, NaHS at 25 µmol/kg offered a significant level of neuroprotection when compared to the

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saline-treated controls. This laboratory subsequently reported neuroprotective effects of NaHS at the same low dose in a tMCAO model (Li et al., 2015). In support, Li et al. (2011) also reported protective effects of NaHS on hippocampal neurons in rats

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subjected to bilateral common carotid artery occlusion for 15 days and NaHS was

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administered daily at 100 µmol/kg. In contrast to other studies, hippocampal H2S levels were reported to be decreased in the occluded rats and NaHS treatment attenuated this decrease. This difference may be due to the timing of the measurements and experimental conditions. More recently, Wang et al. (2014) also reported protective effects of NaHS at 25 µmol/kg. A summary of the effects of H2S

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or H2S donors on ischemic injuries in selected in vivo studies is presented in Table 1. Taken together, it is conceivable that the initial increase in H2S production

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through rapid up-regulation of CBS may be an endogenous tissue response to counter the effects of the ischemic insult, and thus a small dose of exogenous H2S may

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enhance such a protective effect but a large dose may become deleterious. However, this does not readily agree with the observations that CBS inhibition can attenuate ischemic injuries (Qu et al., 2006; Wang et al., 2014; Hadadha et al., 2015; McCune et al., 2016). One possible explanation is that, under certain endogenous conditions such as high substrate availability and CBS up-regulation, production of H2S triggered by the ischemic insult may quickly exceed the neuroprotective range. This is consistent with the observed association of higher plasma Cys levels with early stroke deterioration and poor clinical outcome (Wong et al., 2006). A similar paradox has 13

ACCEPTED MANUSCRIPT been observed whereby the inhibition of endogenous synthesis of H2S and the use of H2S donors both induce anti-cancer effects (Yagdi et al. 2016). It should be noted that Cheung et al. (2007) reported that NaHS at a low concentration of 25 µM exacerbated glutamate-induced cell death in mature cultured

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cortical neurons while at high concentrations (>200 µM) caused necrotic cell death by itself. This argues against the idea that H2S can be neuroprotective in the presence of glutamate excitotoxicity under ischemic conditions. In addition, Chan et al. (2015)

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observed a marked increase in cell death mediated by OGD when cells were manipulated to produce endogenously a high level of H2S, but failed to observe any

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protective effects when cells were manipulated to produce only a low level of H2S. As fine tuning H2S production at low concentration can be challenging, many investigators turned to the development of H2S releasing compounds (Li et al., 2008). Marutani et al. (2012) performed an innovative study in which they combined a H2S

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donor (ACS48) with an NMDA antagonist (memantine) to produce a H2S releasing NMDA antagonist (S-memantine). They did not observe any protective effects using sodium sulfide (Na2S, up to 50 µM) as H2S donor in SH-SY5Y cells subjected to

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OGD. Furthermore, they showed that both Na2S (50–100 µM) and ACS48 (25–100

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µM) are toxic to primary cortical neurons. However, they observed that both ACS48 and memantine at 10 or 50 µM offered protection against OGD-induced cell death in SH-SY5Y cells and primary neurons. Importantly, S-memantine showed effects greater than either ACS48 or memantine at the same concentration. In mice subjected to bilateral carotid artery occlusion and reperfusion, various treatment at a dose of 25 µmol/kg demonstrated that only the S-memantine group showed significantly improved survival rate while both S-memantine and memantine groups showed improved neurological score. Na2S and ACS48 treated group showed no significant 14

ACCEPTED MANUSCRIPT effects when compared to the vehicle-treated group. These authors concluded that H2S releasing NMDA antagonist may represent a novel therapeutic approach for ischemic brain injury. These results may also suggest that the effects being observed may be dependent on the H2S donor used in addition to being concentration dependent. More

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detailed investigations are necessary in order to gain further insight.

6. Mechanisms of action

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In the event of a stroke, the cessation of blood flow leads to a rapid loss of energy and oxygen to sustain cell survival. In the ischemic core, necrotic cell death is

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generally considered non-rescuable by therapeutic intervention. However, in the surrounding penumbra, tissue injuries trigger inflammation, oxidative stress, endoplasmic reticulum (ER) stress, free radical formation and mitochondrial dysfunction leading to apoptotic cell death. These processes are thus the potential

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therapeutic targets. One might expect that the actions of H2S would either be harmful when it enhances the deleterious mechanisms, or beneficial when it reverses those mechanisms.

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6.1 Deleterious mechanisms

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H2S is oxidized in the mitochondria by sulfide-quinone oxidoreductase (SQR) which is believed to be instrumental in maintaining very low levels of sulfide in cells (Theissen et al., 2003). By the action of SQR, electrons are transferred from H2S to coenzyme Q (Fig. 1). In this way, H2S can act as a substrate for the respiratory electron transport chain for ATP synthesis. In addition, it may also inhibit mitochondrial phosphodiesterase 2A to increase cAMP levels (Modis et al., 2013). Both actions may stimulate mitochondrial energy metabolism but may not be significant under ischemic conditions for lack of oxygen. However, SQR activity was

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ACCEPTED MANUSCRIPT reported to be non-detectable in brain mitochondria and neuroblastoma cells (Lagoutte et al., 2010). On the other hand, H2S can also act as an inhibitor of cytochrome c oxidase (complex IV) (Bouillaud and Blachier, 2011). This inhibition is reversible with a Ki of 0.2 µM for the isolated enzyme as compared to 10-30 µM

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reported in isolated mitochondria or cultured cells (Cuevasanta et al., 2016). Therefore, at concentrations used in most in vitro studies described earlier, H2S may be expected to adversely influence mitochondrial functions. In accidental exposure to

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H2S, the toxic actions of H2S is characterized by the inhibition of mitochondrial oxidative phosphorylation via inhibition of complex IV (Guo et al. 2012). However,

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Dorman et al. (2002) reported that exposure to inhaled H2S up to 400 ppm by volume (5 times the toxicity threshold) for 3 hours inhibited lung and liver cytochrome oxidase activity but not in the hindbrain. This may suggest that inhibition of complex IV may not be as important a mechanism in the brain as in other organs. However, a

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“high” level of endogenously produced H2S may have very different consequences. H2S may activate NMDA receptor function through either cAMP production or reduction at redox modulatory sites (Qu et al., 2008). It has been demonstrated that

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NaHS (>200 µM) caused cell death in mature mouse primary neurons that were

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inhibited by NMDA receptor antagonists (Cheung et al., 2007). The H2S-induced cell death was not dependent on caspase-3 but instead associated with activation of calcium-dependent calpains, Ca2+ mobilization and lysosomal rupture. Moreover, NMDA receptor blockers were reported to inhibit H2S-induced cell death in neurons (Cheung et al., 2007; Garcia-Bereguiain et al., 2008) and to reduce infarct volume in an in vivo rat stroke model (Qu et al., 2006), suggesting that H2S may induce cell death via the activation of NMDA receptors leading to calcium overload, thus enhancing receptor-mediated glutamate excitotoxicity in the ischemic brain. Using 16

ACCEPTED MANUSCRIPT microarray analyses on RNA from primary cultures of mouse cortical neurons exposed to NaHS or NMDA (200 µM, 5-24 hours), Chen et al. (2011) reported that the global gene profiles overlapped extensively between the two treatments. The gene families involved included those related to cell death, endoplasmic reticulum stress,

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calcium homeostasis and heat shock proteins among others. The analyses also revealed dysfunction of the ubiquitin-proteasome system following H2S treatment but

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not NMDA treatment.

6.2.1 Anti-inflammation

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6.2 Protective mechanisms

Therapeutic hypothermia (2 – 6°C reduction of core temperature) is wellrecognized to protect against brain injuries including stroke (Zhang et al., 2013b; Yenari et al., 2012; Adbullah and Husin, 2011; van der Worp et al., 2010). Exposure

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to gaseous H2S at 80 ppm can reduce core body temperature, heart rate and respiration rate (Blackstone et al., 2005), and protects against lethal hypoxia in mice (Blackstone and Roth, 2007). Using continuous exposure at 70 (first 2 hours) and then 50 (46

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hour) ppm, Florian et al. (2008) reported marked reduction in infarct volume,

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decreased caspase 12, nuclear factor-κB (NF-κB) and grp78 in the peri-infarct region of aged rats after focal ischemia (tMCAO). This group further demonstrated that the pro-inflammatory annexin A1, which was up-regulated after stroke, was downregulated in the peri-infarct cortex (Joseph et al. 2012). Most recently, Sandu et al. (2016) reported increased density of newly formed blood vessels in the peri-infarct cortex but they did not observe enhanced neurogenesis in the infarcted area. These findings suggest that hypothermia may afford protection through angiogenesis and reduced inflammation.

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ACCEPTED MANUSCRIPT Dysfunction of the BBB is not uncommon after stroke, affecting more than one third of patients and may be linked to poor clinical outcome (Warach and Latour, 2004). Local inflammatory responses to ischemic injuries contribute significantly to BBB disruption (Huang et al., 2013). Wang et al. (2014) reported that a slow-release

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H2S donor, 5-(4-methoxyphenyl)-3H-1,2-dithiole-3-thione (ADT, Jia et al., 2013), protected BBB integrity and reduced brain edema in the affected regions in tMCAO mice probably by suppressing post-ischemic inflammation as indicated by reduction

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in inflammation-induced matrix metallopeptidase (MMP)-9 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). Moreover, ADT inhibited nuclear

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translocation of the NF-κB thereby inhibited the expression of pro-inflammatory markers inducible nitric oxide synthase (iNOS) and interleukin (IL)-1β, and enhanced the expression of anti-inflammatory markers arginase-1 and IL-10. IL-10 and arginase -1 can activate microglia of the M2 phenotype which is associated with

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neuroprotection (Hu et al., 2012). On the contrary, it should be noted that Jiang et al. (2015) reported that inhibition of H2S production was able to maintain BBB integrity following transient ischemia in both control and CSE-/- mice. The related mechanism

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was not investigated. Most recently, ADT was also reported to attenuate tissue

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plasminogen activator induced cerebral haemorrhage following tMCAO (Liu et al., 2016). A summary of the proposed anti-inflammatory effects of H2S is presented in Fig. 2.

6.2.2 Anti-oxidation

H2S can protect neurons from oxidative stress by scavenging free radicals and other reactive species with antioxidant potency similar to glutathione (GSH) (Whiteman et al., 2005; Qu et al., 2008). In addition, it has been reported that H2S protects through enhancing γ-glutamylcysteine synthetase (γ-GCS) activity and

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ACCEPTED MANUSCRIPT cystine transport, both can lead to increased level of intracellular GSH (Kimura et al., 2004; Kimura et al., 2010). This group further demonstrated in a neuronal cell line (HT22) that the H2S-mediated protection against non-receptor mediated oxidative glutamate toxicity involved the activation of ATP-dependent K+ (KATP) and Cl-

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(CFTR) channels (Kimura et al., 2006). It should be noted that the protection was observed at 100-300 µM of NaHS, as opposed to cell death at >200 µM observed by

reflect differences in cell types used in these studies.

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Cheung et al. (2007) in cultured neurons. The contrasting effects observed, perhaps,

Also using HT22 cells, Yu et al. (2015) observed that NaHS (up to 250 µM)

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afforded dose-dependent protection through prevention of mitochondrial dysfunction and ROS production. These authors further reported that NaHS reversed the ischemiainduced rises in the level of malondialdehyde (MDA) and falls in Cu/Zu superoxide dismutase (SOD) and GSH peroxidase activities in tMCAO rats at a dose range of 2.5

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– 5 mg/kg (about 45 – 90 µmol/kg). Li et al. (2015) reported similar findings at a dose of 50 µmol/kg but aggravation of oxidative stress at a high dose of 200 µmol/kg. Most

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recently, Ji et al. (2016) reported increased levels of MDA together with the inflammatory tumor necrotic factor (TNF)-α, and up-regulation of nuclear factor-2

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(Nrf2) in tMCAO mice following pretreatment with inhaled H2S (40 ppm) for 7 days. Under oxidative stress, Nrf2 is translocated to the nucleus where it initiates transcription of antioxidative genes such as γ-GCS that catalyzes GSH synthesis (Solis et al., 2002). A summary of the proposed anti-oxidative effects of H2S is presented in Fig. 2. 6.2.3 Anti-apoptosis In the acute phase following an ischemic insult, apoptosis is in full swing due to activation of pro-apoptotic pathways and suppression of anti-apoptotic pathways. Ji 19

ACCEPTED MANUSCRIPT et al. (2016) reported the up-regulation of heat shock protein 70 (HSP70) by H2S via the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B)/Nrf2 pathway. HSP70 inhibits apoptosis by preventing recruitment of procaspase-9 to the apoptotic protease activating factor (APAF)-1 apoptosome (Beere et al., 2000). Wei et al.,

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(2014) reported the up-regulation of brain-derived neurotrophic factor (BDNF) in PC12 cells by NaHS. BDNF, acting via its receptor TrkB, causes Akt activation as well as PTEN (an inhibitor of PI3K) inactivation, thus activating the PI3K/Akt

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pathway. Activation of this pathway also up-regulated the expression of HIF-1α which promotes cell survival in ischemia (Kumar and Choi, 2015). Interestingly, Dai et

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al. (2016) observed that mild hypothermia, which did not alter H2S levels, had the same effect as low dose NaHS. However, the mild hypothermia effect was still attenuated by a CBS inhibitor. It was suggested that mild hypothermia acted via a H2S-independent mechanism which is influenced by the level of endogenous H2S.

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Up-regulation of silent mating type information regulator 2 homolog 1 (SIRT1 or sirtuin 1) by NaHS was also observed in PC12 cells (Li et al. 2015). SIRT-1 is a NAD-dependent histone deacetylase that can inhibit the pro-apoptotic p53, which acts

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through Bax activation. This is consistent with earlier observations that NaHS

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increased bcl-2 and decreased bax and caspase 3 expressions (Li et al., 2012). Among the many effects of Bax activation is the release of apoptosis-inducing factor (AIF) from mitochondria. Consistently, OGD-induced nuclear translocations of AIF and poly(ADP-ribose)polymerase-1 (PARP-1) were attenuated by NaHS in tMCAO rats (Yu et al., 2015). Thus NaHS is able to suppress PARP-1/AIF signaling to reduce caspase-independent cell death (Hong et al., 2004). NaHS was also reported to attenuate phosphorylation of p38 mitogen-activated protein kinase (MAPK) in PC12 cells subjected to OGD and its protective effects were reversed by p38 MAPK

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ACCEPTED MANUSCRIPT activation (Li et al., 2016). This demonstrated that the p38 MAPK pathway also contributes to OGD-induced cell death in these cells. Consistently, H2S has been shown to attenuate lipopolysaccharides-induced NO release and TNF-α production via the p38 MAPK pathway in primary microglia cultures (Hu et al., 2007). A

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schematic diagram of the proposed anti-apoptotic mechanisms of H2S is presented in Fig. 3. It should be noted that some of these pathways have multiple and varied effects.

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6.2.4 Anti-ER stress

One of the important functions of the endoplasmic reticulum (ER) is the

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folding of newly translated proteins and only properly folded proteins can be transported to the Golgi apparatus. Under ischemic conditions, the folding capacity of the ER is compromised and accumulation of unfolded protein would lead to ER stress response. Wei et al. (2014) reported that H2S inhibited Hcy-induced ER stress via up-

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regulation of the BDNF-TrkB pathway. The enzyme protein tyrosine phosphatase (PTP)1B has been implicated in ER stress signalling (Gu et al., 2004). It is located on the cytoplasmic face of the ER and dephosphorylates its substrate protein kinase-like

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endoplasmic reticulum kinase (PERK) at Tyr619. Krishnan et al. (2011) reported that

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H2S can inactivate PTP1B through sulfhydration of a Cys215 residue at the active site, thus leaving PERK in the active form to phosphorylate translational initiation factor 2 (eIF2α), which in turn inhibits global protein translation. In this way, H2S seems to be able to aid in inhibiting protein translation in response to ER stress. Most recently, Kabil et al. (2016) reported that the production of H2S can increase under ER stress conditions through a switch in substrate preference of CSE from cystathionine to Cys.

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ACCEPTED MANUSCRIPT 7. Concluding remarks There seems not to be a definitive answer to the question posed in the title of this review. Current evidence seems to suggest that the presence of H2S in the ischemic region may either be deleterious or protective depending on its concentration,

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deleterious when “high” and protective when “low”. Therefore, it can be inferred that either an enhancement or a reduction of its concentration may be of potential use in future stroke therapy. It is entirely possible that patients may be stratified by a suitable

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biomarker or biomarkers to determine which of the two may be an appropriate approach. One such possible biomarker may be the plasma Cys and Hcy levels (Wong

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et al., 2006). However, clinical studies will not be possible, in the case of H2S reduction, without the availability of suitable selective CBS inhibitors (McCune et al., 2015). On the other hand, a controlled delivery of H2S at “low” concentrations can be challenging. It has been demonstrated (Toombs et al., 2010) that intravenous infusion

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(over 1 min) of a parenteral formulation of Na2S (0.005 – 0.2 mg/kg) into human subjects did not cause any significant changes in heart rate and radial artery pressure over 30 min post-infusion. Increased plasma Na2S (at doses >0.03 mg/kg) and

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thiosulfate (at 0.2 mg/kg) were observed in the first 5 min after infusion. A majority

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of subjects who received a dose >0.06 mg/kg reported detecting an odour of “rotten egg” in their breath. H2S was detected to have increased to about 3000 ppb in the exhaled air of the subjects who received 0.15 or 0.2 mg/kg within 2 min of infusion, ~3-fold above baseline. Developments of slow-release H2S donors (Huang et al., 2016; Hu et al., 2016) and nanoparticle delivery systems (Wang et al., 2016) are emerging. Finally, it should be duly mentioned that the validity of the use of exogenous H2S to study its physiological functions or therapeutic potential has been questioned (Olson et al., 2014; Haouzi, 2016). Major concerns include (1) the use in in vitro

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ACCEPTED MANUSCRIPT studies at high (µM) concentrations which were previously believed to be physiological; (2) the uncertainty of the H2S concentrations achieved in the ischemic brain following administration in most in vivo studies; and (3) the administration of “low” doses may not yield effective concentrations due to the high capacity in blood

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and other tissues to trap and oxidize H2S, while “high” doses may readily become toxic. In this review, all reported findings are taken without any consideration with

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respect to these criticisms.

Acknowledgement

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The authors acknowledge funding support from the Biomedical Research Council of Singapore (BMRC 01/1/21/19/169) and the National Research Foundation (NRF-CRP3-2008-

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1).

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ACCEPTED MANUSCRIPT Figure captions: Fig. 1 The biosynthesis and oxidation of H2S. H2S is predominantly produced from Cys and Hcy by CBS in the brain. CBS catalzyes

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reactions 1 to 4. Both substrates are produced from Met. As MS is Vitamin B12 and 5-MTHF dependent and CBS is Vitamin B6 dependent, MTHFR polymorphism (C677T), Vitamin B, folate and CBS deficiency can lead to hyperHcy. SAM is an allosteric regulator of CBS that activates reactions 1 and 2. H2S is oxidized by mitochondrial SQR to persulfide (RSSH),

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which is then further oxidized to sulphite. The action of SQR generates electrons which are captured by ubiquinone and transferred to the electron transport chain. CBS, cystathionine

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β-synthase; CSE, cystathionine γ-lyase; ETHE1, persulfide dioxygenase; Lan, lanthionine; MAT, methionine adenosyltransferase; MS, methionine synthase; MT, methioninetransferases; MTHFR, methylene-tetrahydrofolate reductase; SAH, Sadenosylhomocysteine; SAHase, S-adenosylhomocysteinase; SAM, S-adenosylmethionine;

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Ser, serine; SQR, sulphur-quinone oxidoreductase.

Fig. 2 Proposed anti-inflammatory and anti-oxidative stress mechanisms of H2S. Ischemia leads to inflammation and oxidative stress. The main anti-inflammatory action of

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H2S is to down-regulate the expression of inflammatory genes, which is complemented by the up-regulation of some anti-inflammatory genes. In oxidative stress, H2S acts by promoting the

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production of GSH. H2S itself has antioxidant potency similar to that of GSH. γ-GCS, γ-

glutamylcysteine synthetase; GSH, glutathione; IL, interleukin; iNOS, inducible nitric oxide synthase; MDA, malondialdehyde; MMP-9, matrix metallopeptidase; NF-κB, nuclear factor-κB; NOX, nicotinamide adenine dinucleotide phosphate oxidase; Nrf2, nuclear factor-2; SOD, superoxide dismutase; TNF-α, tumor necrotic factor α.

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ACCEPTED MANUSCRIPT Fig. 3 Proposed anti-apoptotic mechanism of H2S.

Arrows (→) indicate a positive effect through activation, up-regulation, release or translocation leading to an increase in activity. T-bars (┴) indicates a negative effect through inactivation, down-regulation or inhibition leading to a reduction in activity.

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AIF, apoptosis-inducing factor; Akt/PKB, protein kinase B; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; BDNF, brain-derived neurotrophic factor; HIF-1α, hypoxia-inducible factor-1α; HSP70, heat shock protein 70; MAPK, mitogen-

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activated protein kinase; mTOR, mechanistic target of rapamycin; Nrf2, nuclear factor-2; PARP-1, poly(ADP-ribose)polymerase-1; PI3K, phosphatidylinositol 3-

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information regulator 2 homolog 1.

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kinase; PTEN, phosphatase and tensin homolog; SIRT-1, silent mating type

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Qu et al., 2006

Treatment agent

Time and route of administration

NaHS

1h prior to MCAO (i.p.)

Dosage 90 µmol/kg

Animals species

Stroke model

Wistar rat

pMCAO

180 µmol/kg 25 µmol/kg

Li et al., 2012

NaHS

SD rat

30 min prior to ligation (i.p.) 180 µmol/kg

NaHS

3h post treatment (i.p.)

50 µmol/kg

global cerebral ischemiareperfusion

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Ren et al., 2010

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Reference

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Table 1 Summary of the effects of H2S or H2S donors on ischemic injuries in selected in vivo studies

SD rat

pMCAO

Na2S 1

ACS48

Memantine2 S-memantine3

1 min after reperfusion (i.p.)

25 µmol/kg

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Murutani et al., 2012

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200 µmol/kg

C57BL/6J mice

Stroke outcome No significant effect on infarct volume Significant increase in infarct volume Reduced rat hippocampal neuron injury Aggravated rat hippocampal neuron injury

Reduced infarct volume

Increased infarct volume

No significant effects

BCAO (40 min)

Significant reduction in neurological deficits Significant reduction in infarct volume, neurological deficits and increased survival rate

Joseph et al., 2012

Gaseous H2S

1h after reperfusion

70ppm (2h) + 50ppm (46h)

aged SD rat

tMCAO (90min occlusion)

Hypothermia and reduced infarct volume

Yin et al., 2013

NaHS

30min after occlusion (i.p.)

0. 2 or 4 µmol/kg

SD rat

global cerebral ischemia-

Decreased brain infarcted area 35

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Start of MCAO (i.p.)

NaHS

Reduced blood-brain-barrier disruption

ICR mice 50 mg/kg/day

25 µmol/kg

Reduced infarct volume and edema

tMCAO (60min) Reduced infarct volume, blood-brainbarrier disruption and neurological deficits after 2 days Reduced tPA-induced cerebral hemorrhage

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ACS48 and ADT are slow-releasing H2S donors; 2memantine is an NMDA antagonist; 3S-memantine is a H2S releasing NMDA antagonist with a molecular structure of ACS-48 linked to memantine

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1

3h after reperfusion (i.p.)

tMCAO (60min)

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ADT1

Wistar rat

25 µmol/kg/day

NaHS Wang et al., 2014

18-90 µmol/kg

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NaHS

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Gheibi et al., 2014

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reperfusion

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tMCAO (120min)

Reduced tPA-induced cerebral haemorrhage and BBB-disruption

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C57BL/6J mice

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ADT

Start of reperfusion (i.p.) 50 mg/kg

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Liu et al., 2016

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MAT

Met

Hcy

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SAHase

SAH

2

Cys

Ser

CBS

CBS 1

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H2 O Cystathionine

Ser

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H2 O 3

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4

Cys

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MT

MS

CSE

SAM

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CBS

Lan

H2S

SQR persulfide

e‐

ETHE1

Cys sulfite Fig. 1

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Ischemia

Oxidative  stress

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inflammation

−

−

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Anti‐inflammation

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H2S

+

↑ Arginase‐1 ↑ IL‐10

↑free radicals ↑MDA ↓Cu/Zn SOD ↓GSH peroxidase

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↑NF‐B ↑TNF‐ ↑Annexin‐1 ↑MMP‐9 ↑NOX ↑iNOS ↑IL‐1

+

−

Anti‐oxidation ↑ GSH

↑Nrf2 ↑ ‐GCS ↑Cys transport Fig. 2

BDNF/TrkB

PI3K

Bcl‐2

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p38MAPK

PTEN

Akt/PKB

SIRT‐1

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mTOR

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AIF

p53

HIF1

AC C

HSP70

Bax

PARP‐1

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Nrf2

caspases

Apoptosis Fig.3

ACCEPTED MANUSCRIPT Highlights  1. H2S is a signalling molecule implicated in stroke.  2. Using various stroke models, researchers have observed both deleterious and protective  effects following ischemic injuries. 

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3. This review discusses the observations for or against a deleterious or protective role of  H2S in stroke.  4. Current evidence suggests that H2S can be deleterious at high concentrations but  protective at low concentrations. 

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5. Therefore, either an enhancement or a reduction of its concentration may be of potential  use in future stroke therapy. 

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