The nuclear factor-erythroid 2-related factor 2 (Nrf2)–antioxidant response element (ARE) pathway was the key regulator in maintaining cellular homeostasis via antioxidant defense, making it a therapeutic candidate for SAH [3]. Nrf2 is a cap ‘n’ collar (CNC) transcription factor, which possesses a basic region leucine zipper structure [76]. In the latent state, Nrf2 is sequestered by Kelch-like erythroid cell-derived protein with CNC homology-associated protein 1 (Keap1)-dependent ubiquitination–proteasomal degradation in the cytoplasm. Modifying critical cysteine thiols of Keap1 and Nrf2 by some oxidants promotes Nrf2 dissociation from the Keap1/Nrf2 complex and translocation into nuclei. Subsequently, Nrf2 binds to ARE in the promoter of cytoprotective genes leading to upregulated expression of relevant proteins, such as heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1, and glutathione-S-transferase [34, 64].

Evidence from experimental SAH research indicates a protective role of the Nrf2/ARE pathway in EBI and CVS after SAH. Nrf2/ARE signaling was activated during the EBI period after SAH [66, 74]. Post-SAH treatment with melatonin and recombinant human erythropoietin reduced brain edema and improved neurobehavioral outcome via activating the Nrf2/ARE pathway and modulating oxidative stress after SAH, making these drugs promising for treatment. In addition, an elevated level of Nrf2 was detected in endothelial and smooth muscle cells in the basilar arterial walls [65]. The activation of Nrf2 increased in the arterial wall, parallel to the development of basilar artery vasospasm, in a double-injection SAH rabbit model. Because of the elevated expression of Nrf2, the Nrf2/ARE pathway was hypothesized to prevent CVS after SAH [78]. In an in vitro SAH model, sulforaphane, an agonist of the Nrf2–ARE pathway, can inhibit oxyhemoglobin (OxyHb)-induced inflammatory cytokine, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, release in vascular smooth muscle cells [77]. Oxyhemoglobin-induced inflammation was aggravated in astrocytes from Nrf2-knockout mice via nuclear factor (NF)-κB signaling [45]. Thus, the Nrf2/ARE pathway may exert anti-inflammatory and antioxidative effects that contribute to alleviation of EBI and CVS after SAH. However, additional in vivo experiments using Nrf2 agonists or antagonists are required to further investigate the role of Nrf2 on SAH-induced brain injury.

NF-κB signaling is involved in various CNS disorders because it regulates immune and inflammatory responses including infection, brain trauma, neurodegenerative diseases, and stroke [48, 54]. In mammalian cells, the NF-κB family of transcription factors consists of five members, Rel A (p65), c-Rel, Rel B, p50, and p52. Under inactive conditions, NF-κB is sequestered in the cytoplasm by binding with IκB family members. The IκB kinase (IKK) enzyme complex can phosphorylate IκB proteins to release active NF-κB, leading to translocation of NF-κB into the nucleus. Subsequently, NF-κB dimmers (p50–p65) are free to bind to the promoters of genes of inflammatory mediators and increase the release of those mediators [42]. An in vitro study demonstrated that the phosphorylation of IκB diminishes its association with NF-κB, leading to NF-κB translocation into the nucleus, where NF-κB binds to the promoter of nitric oxide synthase (NOS)-2 in endothelial cells [8].

NF-κB signaling in SAH has been explored and some trials are ongoing trials. Toll-like receptor (TLR)-4 is an important upstream receptor of NF-κB. At the acute stage of SAH, TLR4/NF-κB signaling is significantly activated, suggesting that this pathway may regulate the inflammatory response in experimental SAH [33]. Post-SAH administration of progesterone attenuated EBI via suppressing the activation of TLR4/NF-κB signaling in the cortex after SAH [68]. p65, a nuclear NF-κB subunit, was overexpressed in the basilar artery, which indicated that the NF-κB-mediated inflammatory response may also facilitate the development of CVS after SAH. Intracisternal administration of pyrrolidine dithiocarbamate, an inhibitor of NF-kB, reduced the levels of TNF-α, IL-1β, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 and alleviated CVS in a rat model of SAH [79]. In addition, Nle4, DPhe7-α-MSH (NDP-MSH) and trehalose exerted protective effects on CVS via inhibiting NF-κB signaling in the basilar artery [13, 15]. Similarly, 6-mercaptopurine increased the level of IκB, downregulating NF-κB activity. Thus, 6-mercaptopurine was capable of hindering the production of inflammatory cytokines (e.g., IL-1β, IL-6, and TNF-α) after SAH. The anti-inflammatory effect of 6-mercaptopurine contributed to its antivasospastic property in SAH animals [7]. Furthermore, because of the association of p65 with the estrogen receptor, 17β-estradiol blocked the binding of p65 to the gene target inducible NOS (iNOS). Therefore, this hormone drug reduced iNOS and showed a neuroprotective effect on CVS [57]. The activation of NF-κB was biphasic in a single injection rabbit SAH model. The peaks of NF-κB activity occurred around day 3 and day 10 after SAH. The first peak plays a prominent role in neuronal injury, but the exact role of the second peak requires additional investigation [72].

In conclusion, these data indicated an essential role of the NF-κB pathway in the pathogenesis of EBI and CVS after SAH.

HIF-1 is a critical regulator of cellular adaptation to hypoxic stress and is a heterodimeric DNA-binding complex composed of one α- and one β-subunit [30, 37]. Cytoplasmic HIF-1α is continuously degraded via ubiquitination in normoxic conditions. However, in hypoxia, the proteasomal degradation of HIF-1α is inhibited, leading to HIF-1α accumulation. Nondegraded HIF-1α recruits HIF-1β to form the functional HIF complex, which enters the nucleus. HIF-1 binds in the location of hypoxia response elements to induce the transcription activation of these genes (e.g., erythropoietin, vascular endothelial growth factor (VEGF), and heme oxygenase (HO)-1) [2].

During the EBI, the expression of HIF-1α, VEGF, and BNIP3 were increased in the hippocampus and cortex [44]. Hyperbaric oxygen reduced the expression of HIF-1α and its target genes (including VEGF and BNIP3), which resulted in fewer apoptotic cells [12]. A recent study demonstrated that HIF-1α may exert a deleterious effect in EBI by upregulating its downstream proteins BNIP3 and VEGF, resulting in cell apoptosis, blood brain barrier (BBB) disruption, and brain edema [69]. However, 3-(5ʹ-hydroxymethyl-2ʹ-furyl)-1-benzylinda-zole (YC-1), a HIF-1α inhibitor, increased cell apoptosis in the hippocampus and increased cognitive function damage in SAH rats, suggesting that HIF-1α might exert beneficial effects in SAH [10].

In the brainstem, an upregulated HIF-1α protein level, but not mRNA level, was detected in the acute (10 min) and chronic (7 days) phases of SAH. The difference in levels between protein and mRNA is not yet clear. Deferoxamine promoted the expression and activity of HIF-1α, which ameliorated basilar artery vasospasm [20]. Additionally, isoflurane significantly attenuated vasospasm by increasing endothelial HIF-1 and iNOS in SAH mice [38]. Conversely, HIF-1α was suggested as an important contributor in the development of CVS in other studies. 2-Methoxyestradiol might reduce CVS and improve neurological deficient via inhibiting HIF-1α/VEGF and HIF-1α/BNIP3 apoptotic pathways after SAH [70].

To date, both beneficial and detrimental effects of HIF-1α have been found in SAH. Therefore, the exact mechanism of HIF-1α in the pathogenesis of SAH is not yet fully elucidated.