An Update on Antioxidative Stress Therapy Research for Early Brain Injury After Subarachnoid Hemorrhage

The main reasons for disability and death in aneurysmal subarachnoid hemorrhage (aSAH) may be early brain injury (EBI) and delayed cerebral ischemia (DCI). Despite studies reporting and progressing when DCI is well-treated clinically, the prognosis is not well-improved. According to the present situation, we regard EBI as the main target of future studies, and one of the key phenotype-oxidative stresses may be called for attention in EBI after laboratory subarachnoid hemorrhage (SAH). We summarized the research progress and updated the literature that has been published about the relationship between experimental and clinical SAH-induced EBI and oxidative stress (OS) in PubMed from January 2016 to June 2021. Many signaling pathways are related to the mechanism of OS in EBI after SAH. Several antioxidative stress drugs were studied and showed a protective response against EBI after SAH. The systematical study of antioxidative stress in EBI after laboratory and clinical SAH may supply us with new therapies about SAH.


INTRODUCTION
Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating disease, mainly induced by the rupture of an intracranial aneurysm and linked to high levels of morbidity and mortality (Bor et al., 2008;Connolly et al., 2012;Macdonald and Schweizer, 2017;Chao et al., 2021). Although we have progressed in treatment, 40% of aSAH survivors remain dependent as a consequence of neurological disability and behavioral and cognitive impairments (Brathwaite and Macdonald, 2014;Etminan and Macdonald, 2017). Clinical studies have shown that cerebral vasospasm (CVS) is not the single contributor to delayed cerebral ischemia (DCI) and poor prognosis in patients with aSAH (Naraoka et al., 2018;Mayer et al., 2019;Schupper et al., 2020;Post et al., 2021;Takeuchi et al., 2021). At present, amassing laboratory evidence demonstrates that early brain injury (EBI), which happens within 72 h after aSAH, causes subsequently pathophysiological changes and poor outcomes (Kusaka et al., 2004). The pathological changes and mechanisms of EBI collectively contain increased intracranial pressure (ICP), oxidative stress (OS), neuroinflammation, bloodbrain barrier (BBB) disruption, brain edema, and cell death. Among them, the OS responses include a wide variety of active and inactive substances, which play a substantial role in EBI after aSAH and may be associated with DCI and long-term outcomes (Cahill et al., 2006;Rowland et al., 2012;Sehba et al., 2012;Shao et al., 2020). Therefore, we should pay more attention to strategies targeting cerebral redox responses to some extent. In this review, we update the impact of OS in the occurrence and development of SAH and several major oxidative pathways and biomarkers related to EBI after SAH. Additionally, we also take an overlook for the potential therapeutic drugs.
Although widely accepted than other pathogenic mechanisms, DCI has not reached further improvement clinically. By the same token, the failure of clazosentan, despite mitigating moderate and severe vasospasm, has shifted the focus of preclinical and clinical research from DCI to EBI toward a more multifactorial etiology in recent times (Macdonald et al., 2008(Macdonald et al., , 2011Cahill and Zhang, 2009;Caner et al., 2012).
Typically, the elementary changes after SAH can be segmented into two periods: EBI and DCI. Pathological changes occurring in the initial stage of hemorrhage propagate and lead to inflammation, OS, and apoptosis. Studies showed that OS plays a key role in the pathogenesis of EBI after SAH.

OXIDATIVE STRESS IN SUBARACHNOID HEMORRHAGE
Although progress is made in cell death, the actual pathogenesis of EBI after SAH is still rarely understood. Several pieces of research demonstrate that OS is one of the basic drivers of EBI (Zhang et al., 2016c;Fumoto et al., 2019).
Relying on the activity of the producer and removal systems, OS has its yin and yang faces, but those covered in this review are the result of the dysregulation of reduction-oxidation (redox) reactions. Sies and Jones (2020) also defined the elevated constitution of various reactive oxygen species (ROS) resulting in all classes of molecular damage as "oxidative distress." OS, a relative extra of ROS compared with antioxidants, has been associated with cardiovascular, neurodegenerative, and many other diseases. ROS is an overall term for derivatives of dioxygen not chemically precise, which serve as a normal character of aerobic life. Hydrogen peroxide (H 2 O 2 ) and the O 2 •− are pivotal agents produced by greater than 40 enzymes (Murphy, 2009;Go et al., 2015). Additionally, many other reactive categories are contained in redox signaling, for example, hydrogen sulfide and oxidized lipids (Fujii et al., 2010;Yu et al., 2014;Jarocka-Karpowicz et al., 2020).
After SAH, exhibiting aberrant redox hemostasis, the production of oxidants mainly comes from the disruption of mitochondria (Yan et al., 2015;Fan et al., 2021;, extravascular hemolyzed blood (Vecchione et al., 2009;Deng et al., 2018), and enzymatic sources of free radicals (Sies et al., 2017;Yang et al., 2017;Sies and Jones, 2020). Intrinsic antioxidant activity can be exhausted by excessive free radicals, resulting in lipid peroxidation, protein breakdown, and DNA damage. Mention must be made that beyond the biology of H 2 O 2 and O 2 •− , a significant area of ROS research is lipid-derived ROS. Oxidative DNA damage has also been widely distinguished by mutagenesis, DNA methylation, and chromatin structure. Although substantiation is also gathered about oxidative damage to RNA, the underlying functional effect has not yet been fully illustrated.

OXIDATIVE STRESS IN EARLY BRAIN INJURY
For decades, the treatment of CVS and DCI has always been the focus of clinical practice. However, mounting evidence showed that even angiographic vasospasm is reversed, and clinical outcomes remain frustrating (Macdonald et al., 2008;Gomis et al., 2010). So far, nimodipine remains the only medication proven to lessen DCI and unfavorable outcomes. Therefore, new therapeutic regimens are promising (Hänggi et al., 2015).
Previously, the aspecific erasure of ROS using antioxidant compounds was unsuccessful in offsetting SAH initiation. However, regulating specific ROS-mediated signaling pathways offers a viewpoint, mainly containing enzymatic defense systems like those regulated by the nuclear factor erythroid 2 (NF-E2)related factor 2 (Nrf2) (Zolnourian et al., 2019;Sies and Jones, 2020) and PI3K/Akt, the role of key molecules such as melatonin, sirtuins, and hydrogen sulfide, and the modifiable factors that are corporately thought as the exposome [by way of illustration, nutrition , lifestyle, and irradiation] (see overview in Table 1 and Figure 1). Discovering strategies for effectively detoxifying free radicals has become a theme of great interest from both practical and academic viewpoints.

KEAP1-NRF2-ARE Pathway
An important "sensors" protein that captures specific metabolic information after SAH and transforms it into an appropriate response is Kelch-like ECH-associated protein 1 (Keap1), which contains reactive cysteine residues that collectively respond to ROS resulting from heme. Covalent modification of Keap1 leads to reduce ubiquitination and accumulates Nrf2 (Bollong et al., 2018). It is combined with a given DNA site, the antioxidant response element, modulating the transcription of a series of antioxidant enzymes (Zolnourian et al., 2019). Lots of researches on stroke models have substantiated that Nrf2 levels lift soon after the stroke. It first appears within 3 h and peaks within 24 h post insult, with the peri-infarct area markedly increasing (Yang et al., 2009;Tanaka et al., 2011). The study indicated that Nrf2 expression is significantly activated in neurons, astrocytes, leukocytes, microglia, endothelin cells, smooth muscle cells, and adventitial cells after SAH-induced brain insult (Wang et al., 2010). Wang et al., found that the Nrf2-ARE pathway is upregulated in rat models with experimental SAH in the time course at 12, 24, and 48 h (Chen et al., 2011). Since then, lots of known activators of the Keap1-Nrf2-ARE pathway were overwhelmingly carried out in the study. Although controversial conclusions are sometimes achieved due to pleiotropic and primary mechanisms, more effective inducers with less crossing activation of other pathways are identified and reach favorable outcomes.
In addition to the agents summarized by former researchers, such as sulforaphane, astaxanthin, curcumin, lycopene, tetrabutyl hydroquinone, dimethyl fumarate, melatonin, and erythropoietin, there are also many other experimental studies conducted more recently. Efforts to target the Nrf2 signaling pathway therapeutically have largely focused on covalent small molecule agonists of Keap1. Mitoquinone (MitoQ), effective in the prevention of mitochondrial dysfunction, restrained OS-related neuronal death by stimulating mitophagy through Keap1/Nrf2/PHB2 pathway after SAH in rats . In the endovascular perforation mice SAH model, MitoQ treatment reduced OS, both short term and long term. Another activator of Nrf2, RTA 408, a new second-generation semisynthetic oleanane triterpenoid, manifested an antioxidant and anti-inflammatory phenotype (Reisman et al., 2014). After administrated intraperitoneally, vasospasm was reversed by RTA 408 through growth in Nrf2 and reduction in NF-κB (Tsai et al., 2020).
More recently, SS31 cell-membrane permeating mitochondria has been shown to exert potential neuroprotective effects (Petri et al., 2006). Via suppressing Bax translocation and cytochrome c release, SS31 ameliorated OS by inhibiting the mitochondrial pathway (Shen et al., 2020).

PI3K/Akt Pathway
The PI3K/Akt pathway is one of the important pathways that inhibit cell apoptosis and, therefore, plays a protective role against SAH. In recent years, antiapoptosis agents targeting this pathway generated the cross-talk with antioxidative effect . For example, Aggf1, also known as an angiogenic factor with G, patch, and FHA domain 1, in a recombinant human form, reduces BBB disruption and neuroinflammation through PI3K/Akt/NF-κB pathway after SAH in rats by significantly decreasing the level of myeloperoxidase (Zhu et al., 2018). For the first time, John H. Zhang et al., found that KISS1 siRNA knockdown (KD) aggravated neurological deficits and the brain expression of markers for OS in rats both 24 h and 28 days after SAH, suggesting that KP54 attenuated OS through activating GPR54/ARRB2/AKT/GSK3β pathway after SAH in rats (Mead et al., 2007;Huang et al., 2021). Another frontier hotspot involved in the PI3K/Akt pathway is the microglial polarizationmediated WMI (Xue et al., 2021). Additionally proposed by the John H. Zhang research group, low-density lipoprotein receptor-related protein-1 (LRP1), a scavenger receptor of apolipoprotein E (apoE), is validated for microglia polarization toward pro-antioxidative M2 phenotypes via Shc1/PI3K/Akt pathway after SAH in rats Rojo et al., 2018;Peng et al., 2019). Uniformly, apoE and apoE-mimetic peptides possess whole-brain protective effects that may reduce EBI after mice SAH via M1 microglial quiescence through the attenuation of the JAK2/STAT3/NOX2 signaling pathway axis (Pang et al., 2018). A shred of direct evidence presented by Kuanyu Li and his colleagues is that pAkt effectively inhibits iron accumulation, defense against OS, and ameliorates EBI in a model of experimental SAH in the temporal lobe (Jo et al., 2012;Hao et al., 2016;Zhang et al., 2016a). Interestingly, SC79 is an absorptive permeability without reported side effects, indicating a novel and promising delivery drug in patients with EBI after SAH.

More Recently Progressed Pathway
In addition to the Keap-Nrf2-ARE pathway and PI3K/Akt pathway, there are many other pathways that are oxidative related and proved to be effective. For instance, Scutellarin, a flavonoid from the Chinese herb Erigeron breviscapus, reduces vasospasm via the Erk5-KLF2-eNOS pathway after SAH . An agonist of the Shh co-receptor plays a part in neuroprotection against SAH-induced damage, mediated in part by antioxidant mechanisms, upregulating phospho-ERK levels, and mediating Shh signaling molecules in the prefrontal cortex . Benefitting from a broad distribution in neurons, astrocytes, and microglia, activation of TGR5 with INT-777 significantly attenuates OS through cAMP/PKCε/ALDH2 pathway after SAH in rats (Zuo G. et al., 2019). Recognized as a new component of the brain renin-angiotensin system, Mas is selectively target-activated by AVE 0991 and reduces OS through Mas/PKA/CREB/UCP-2 pathway . Moreover, 12/15-LOX is overexpressed in macrophages after SAH in mice, and restraint of the pathway attenuates brain injury and ameliorates unfavorable neurological outcomes. Progressing data support that various pathways may participate in the redox balance in EBI after SAH and needs to be added with a new insight of other underlying pathways.

Melatonin
The number of data accumulated till now concerning the protective action of melatonin against OS is preponderant (Galano et al., 2011;Wu H. J. et al., 2017;Luo et al., 2019;Shao et al., 2020). Melatonin, a lipophilic amino acid that originated from tryptophan, N-acetyl-5-methoxytryptamine, is synthesized in the pineal gland and other organs and exhibits both direct and indirect antioxidant effects. Melatonin first reported the antioxidative function in preventing focal regions of injury via inducing HO-1 expression following a rat SAH model in 2002 (Martinez-Cruz et al., 2002). Before the postclazosentan era, studies regarding the EBI experiments were still broadly focused on delayed brain injury, such as setting the assessment point at day 5 (Fang et al., 2009). Although melatonin shows no improvement in neurologic scores, the phenomenon is settled by large doses with immensely lessened mortality (Ayer et al., 2008a,b). However, the latest study shows that patients with higher serum melatonin concentrations are more likely to have a poor prognosis (Zhan et al., 2021). The increased concentrations of serum melatonin correlate with admission WFNS scores and mFS and serum melatonin appears as an independent predictor for poor 6-month prognosis after aSAH, with a high discriminatory ability for the risk of the poor outcome under the ROC curve, indicating that serum melatonin might serve as a promising prognostic biomarker for aSAH (Zhan et al., 2021). In vivo experiments exhibit that melatonin supplied protection from the effects of EBI after SAH by adjusting the expression of murine SIRT3 (one of the members of the sirtuin family) . Another two recent studies also denoted that melatonin plays a neuroprotective role by increasing the expression of NRF2-mitophagy and ER stress via inducing antioxidant, LC3-II/LC3-I, and Atg 5-mediated autophagy, NLRP3 inflammasome-mediated anti-inflammatory effects, and detoxifying enzymes post-SAH Dong et al., 2016;Wu H. J. et al., 2017;Sun et al., 2018). Melatonin may reduce neurobehavioral dysfunction in the SAH model through the TRL4 pathway (Wang et al., 2013). Furthermore, melatonin reduces the EBI by influencing NLRP3 inflammasomeassociated apoptosis (Dong et al., 2016) and inhibiting NF-κB activation and attenuating HO-1, NQO-1, and c-GCLC expressions (Jumnongprakhon et al., 2015). The mechanism of these antiapoptosis effects was linked to the improvement of autophagy through a mitochondrial pathway (Chen et al., 2014b). Melatonin inhibits the disruption of tight junction proteins possibly linked to the adjustment of proinflammatory cytokines (Chen et al., 2014a). Taken together, these results demonstrate that regulation of melatonin attenuates symptomatic dysfunction .

Sirtuins
Sirtuins (SIRTs), including the seven SIRTs identified, are a family of deacetylases with homology. Lines of studies showed that SIRTs could modulate diverse biological functions, Sirtuin 1 (SIRT1) with antioxidative properties particularly. Demonstrating that sequential inhibition of forkhead transcription factors of the O class-, NF-κ B-, and p53-induced oxidative pathways, SIRT1 enhanced the neuroprotective role against EBI in rats . A well-recognized antioxidant, astaxanthin, mitigates SAH-induced EBI by increasing SIRT1 and suppressing the TLR4 signaling pathway Zhang et al., 2014;. Interestingly, derived from seaweeds, fucoxanthin (Fx) mitigates SAH-induced oxidative damage via the SIRT1-dependent pathway . The activation of melanocortin 1 receptor with BMS-470539 immensely reduced EBI after SAH by restraining OS, apoptosis, and mitochondrial fission via the AMPK/SIRT1/PGC-1α signaling pathway . Modulated by SIRT1 activation, salvianolic acid B protects against SAH-triggered oxidative damage by upregulating the Nrf2 antioxidant signaling pathway . Salvianolic acid homolog A also presented antioxidative, antiapoptotic, and anti-inflammatory properties (Gu et al., 2017;Zhang X. et al., 2018). Other members of the SIRT family are increasingly studied recently. SIRT3, a type of NAD-dependent deacetylase, remarkedly activated in vivo and in vitro following SAH, is involved in the PGC-1a/SIRT3 pathway attenuating OS (Zhang K. et al., 2020). Drawing on the successful experience of the SIRT6 protective role of the heart from I/R injury via upregulating antioxidants and suppressing OS, the activation of RXR ameliorated neurological deficits after SAH at least partially via adjusting the PPARγ/SIRT6/FoxO3a pathway .

Hydrogen Sulfide
Hydrogen sulfide (H 2 S), a neuromodulator, which can be generated in the CNS from L-cysteine by cystathionineβ-synthase (CBS), may prove protective effects in experimental SAH (Xiong et al., 2020). The hypothesis that signaling through hydrogen sulfide may mediate protection from DCI clinically in patients with SAH was proposed in 2011 (Grobelny et al., 2011) and further demonstrated by Yu et al. (2014). Afterward, hydrogen sulfide attenuated brain edema formation and promoted the secretion of inflammatory cytokines (Cui et al., 2016). Soon after the proinflammation demonstrated in EBI after SAH, exogenous hydrogen sulfide functioning as an antioxidant and antiapoptotic mediator, donated by NaSH and L-cysteine, could significantly reduce EBI (Cui et al., 2016;Xiong et al., 2020). Inspired by inhaled hydrogen gas markedly decreasing OS on ischemia/reperfusion injury and stroke in rats (Ohsawa et al., 2007), hydrogen gas therapy was conducted, and the rate of survival and neurological deficits were improved in a pilot study as expected (Camara et al., 2019). Mechanistically, the above advantageous effects might be linked to the suppression of the ROS/NLRP3 axis (Zhuang et al., 2019). Similarly, hydrogen-rich saline exhibited the satisfying outcome of alleviating EBI through alleviating OS following experimental SAH in both rabbit and rat models (Zhuang et al., 2012;Shao et al., 2016). Furthermore, sodium/hydrogen exchanger 1 participates in EBI activated by SAH via mediating OS (Song et al., 2019). As abovementioned earlier, a recent study shows that postconditioning with hydrogen gas ameliorated SAHinduced neuronal pyroptosis (Zhang C. S. et al., 2021). Produced endogenously through HO, carbon oxide (CO) minimizes neurobehavioral deficits, indicating that posttreatment with CO gas or CO-donors can be further tested as a potential therapy against SAH (Kamat et al., 2019).

Modifiable Factors
Peaking onset age between 50 and 60 years, many patients with aSAH have modifiable hypertension, dyslipidemia, diabetes mellitus, cardiovascular diseases, and so on (de Rooij et al., 2007;Zhang L. et al., 2017;Macdonald and Schweizer, 2017). To control the clinical status, they are recommended with antihypertensive drugs, statins, and so on, part of whom demonstrate the antioxidative effect in the laboratory, for example, telmisartan, ameliorates OS, and SAH-induced CVS (Erdi et al., 2016). One of the L-type calcium channel blockers, verapamil, can inhibit vasospasm, OS, and apoptosis following experimental SAH (Akkaya et al., 2019). Rosuvastatin, commonly used clinically, ameliorates EBI after SAH through restraining SOD formation and NF-κB activation in rats. Moreover, 3,4-dihydroxyphenylethanol may be a powerful agent in the treatment of EBI after SAH because of its free radical scavenging capacity and modulating the Akt and NF-κB signaling pathway (Zhong et al., 2016;Fu and Hu, 2016). In addition to the intervening abovementioned diseases, other drugs commonly used clinically, such as heparin (Hayman et al., 2017), albumin , and propofol , all projected an antioxidative role. Some authors believe that preconditioning would provide the greatest chance of benefit but is obviously not effective (Mayor et al., 2013;Zolnourian et al., 2019;Ruan et al., 2020;Otsuka et al., 2021). Given the sophistication of brain damage after aSAH, therapeutic multimodality is promising. Supported by the evidence shown above, we suppose that the regular drug taken with high adherence may benefit favorable outcomes after aneurysm rupture than those who do not.

PERSPECTIVES AND LIMITATIONS
Shifted from phenotype research on OS in EBI to pathwayrelated research, the mechanisms become evident than at any time in the past Ye et al., 2018;Pan et al., 2021). However, since the term EBI was coined from an angle of preclinical mechanism insight, there is still a long way to apply clinically. Preclinical and clinical studies should proceed hand in hand, and none of the multi-omics and clinical trials should stop Xu W. et al., 2018).
In addition to the traditional markers, such as malondialdehyde (MDA), SOD, reduced/oxidized glutathione (GSH/GSSG) ratio, and myeloperoxidase (MPO), new approaches that measure energetics and metabolomics of cells should be explored, such as the bioenergetic health index (BHI) (Chacko et al., 2016), to further guide the development of therapies. A lot of conducted studies showed that drugs introduced into the area of EBI after aSAH previously drew on the strength of the fields of ischemic stroke or traumatic brain injury. Furthermore, a close tracing of novel antioxidants is necessary, even in other disciplines with mutual adoption, promotion, and advancement, especially traditional Chinese medicine and nutrition Zhang et al., 2015;Li Q. et al., 2016;Han et al., 2017b;Liu et al., 2017Liu et al., , 2020Shao et al., 2019;Du et al., 2020;Wang T. et al., 2020). As the OS-related mechanisms and pathways have surfaced and matured, more modern technologies should emphasize their parts, such as designing a metabolite-derived protein alteration integrating glycolysis with Keap1-Nrf2 signaling directly (Bollong et al., 2018) or recombinant human drugs Sun C. M. et al., 2019;Wang J. et al., 2020;Wu et al., 2020;Tu et al., 2021). Effective drugs validated in a laboratory should be carefully compared and put into clinical use properly. Typical drug pharmaceutical effects combined with evolving drug-loaded methods may be promising in future exploration. For example, the conventional antioxidant agent, curcumin, loaded into a nanosized PLGA-encapsulated the therapeutic potential was enhanced for precision medicine in downregulating the NF-κB pathway and preventing OS in EBI Zhang et al., 2016b;Cai et al., 2017;Zhang Z. Y. et al., 2017).
There are several limitations in this study. First, at present, there remains a translational cleft between experimental SAH and clinical SAH, especially aSAH. Although injection and endovascular perforation models are well established and invaluable, no model, even the in vivo cerebral aneurysmal models, could perfectly replicate the actual rupture of an aneurysm in human beings. Second, the unequivocal definition of EBI could not be circumvented in animal models with the variation assessment points in 3, 12, 24, or 72 h. Clear evaluation time is still up for debate. Third, based on a multifactorial pathophysiology after EBI, OS plays a pivotal part but is still only one of the considerable phenotypes with promising therapeutic strategies covering as many pathways as possible.

CONCLUSION
Management of EBI after aSAH remains not only a challenge but also an opportunity. With in-depth understanding of oxidative pathophysiology of EBI, the way ahead becomes gradually clearer. Despite initial experimental studies demonstrating the effectiveness of the abovementioned antioxidants in EBI, these studies are comparatively rudimentary, with further translational medicine demanded to prove the utility of all of them clinically.

AUTHOR CONTRIBUTIONS
FL, RL, W-JT, KW, and YC contributed to the search and assessment of the available literature. FL and RL wrote the manuscript. XC and JZ helped to revise the manuscript to the final form. All authors contributed to the article and approved the submitted version.