SIRT1 Promotes M2 Microglia Polarization via Reducing ROS-Mediated NLRP3 Inflammasome Signaling After Subarachnoid Hemorrhage

Mounting evidence has suggested that modulating microglia polarization from pro-inflammatory M1 phenotype to anti-inflammatory M2 state might be a potential therapeutic approach in the treatment of subarachnoid hemorrhage (SAH) injury. Our previous study has indicated that sirtuin 1 (SIRT1) could ameliorate early brain injury (EBI) in SAH by reducing oxidative damage and neuroinflammation. However, the effects of SIRT1 on microglial polarization and the underlying molecular mechanisms after SAH have not been fully illustrated. In the present study, we first observed that EX527, a potent selective SIRT1 inhibitor, enhanced microglial M1 polarization and nod-like receptor pyrin domain-containing 3 (NLRP3) inflammasome activation in microglia after SAH. Administration of SRT1720, an agonist of SIRT1, significantly enhanced SIRT1 expression, improved functional recovery, and ameliorated brain edema and neuronal death after SAH. Moreover, SRT1720 modulated the microglia polarization shift from the M1 phenotype and skewed toward the M2 phenotype. Additionally, SRT1720 significantly decreased acetylation of forkhead box protein O1, inhibited the overproduction of reactive oxygen species (ROS) and suppressed NLRP3 inflammasome signaling. In contrast, EX527 abated the upregulation of SIRT1 and reversed the inhibitory effects of SRT1720 on ROS-NLRP3 inflammasome activation and EBI. Similarly, in vitro, SRT1720 suppressed inflammatory response, oxidative damage, and neuronal degeneration, and improved cell viability in neurons and microglia co-culture system. These effects were associated with the suppression of ROS-NLRP3 inflammasome and stimulation of SIRT1 signaling, which could be abated by EX527. Altogether, these findings indicate that SRT1720, an SIRT1 agonist, can ameliorate EBI after SAH by shifting the microglial phenotype toward M2 via modulation of ROS-mediated NLRP3 inflammasome signaling.


INTRODUCTION
Subarachnoid hemorrhage (SAH) is a fatal cerebral vascular disease with high morbidity and mortality worldwide. Despite tremendous advances in diagnosis and neurosurgical care, it remains a difficult challenge to develop effective therapeutic interventions to improve SAH outcomes. The etiology of SAH is complicated. Accumulating evidence suggests that the robust post-SAH inflammatory response plays a crucial role in causing early brain injury (EBI) after SAH (1)(2)(3). With the occurrence of hemorrhage and ischemia, rapid influx of inflammatory cells, microglia activation, and an increase of proinflammatory cytokines production lead to neuronal cell death. In addition, inflammatory cells could induce the generation of reactive oxygen species (ROS) to further aggravate EBI. Therefore, diminishing cerebral inflammation is a promising method to reduce EBI and improve brain recovery after SAH.
As key innate immune cells, microglia play critical roles in the innate immune response and exert distinct tissue repair functions after SAH (4). Microglia can sense slight imbalances in immune homeostasis, and cytotoxic mediators and endogenous proteins released from damaged neural cells stimulate resting microglia activation (5). In response to acute brain injury, activated microglia can develop into either classically M1 (proinflammatory) or alternatively activated M2 (anti-inflammatory) phenotypes (6). For example, in a mouse model of spinal cord injury (SCI), both M1 and M2 marker levels were increased in the acute phase. But the flow cytometry results indicated that microglia and macrophages had mainly developed an M1 phenotype (7). In models of traumatic brain injury (TBI) and ischaemic stroke, a dynamic change of M2 to M1 phenotype was observed. In time, the M1 microglia/macrophages become dominant in the injury area and aggravate brain damage by exacerbating inflammatory response (8,9). In SAH area, numerous studies also reported that the M1 microglia/ macrophages was predominant in the early phase and subsequently transited to the M2 phenotype. Inhibition of microglia M1 polarization and promotion of a shift from M1 to M2 phenotype significantly ameliorated neuroinflammation and improved neurological outcomes after SAH (10)(11)(12). Thus, targeting the microglial phenotypic transformation might provide a promising means to reduce EBI after SAH. SIRT1, a member of the sirtuin family, plays a prominent role in regulating various biological functions, such as immune response, apoptosis, oxidative stress, and aging (3,13). The function of SIRT1 is increasingly recognized as especially important in the pathogenesis of neurological diseases. Accumulating studies have demonstrated that SIRT1 plays a protective role in various models of neuronal injury and neurodegeneration (14)(15)(16). For example, in a permanent focal ischemia mice model, SIRT1 plays a key role in neuroprotection against brain ischemia by inhibition of inflammatory and apoptotic pathways (17). In models of TBI, SIRT1 activation ameliorates oxidative damage and neuronal apoptosis by modulation of nuclear factor-erythroid 2-related factor 2 (Nrf2) signaling pathway (18). Meanwhile, emerging evidence has indicated that SIRT1 is involved in and plays a pivotal role in the pathophysiology of SAH. Vellimana et al. reported that SIRT1 inhibition blocked the protective effects of hypoxic preconditioning on vasospasm and neurological deficits after SAH. In contrast, SIRT1 activation by resveratrol pretreatment abrogated vasospasm and attenuated neurological deficits following SAH (19,20). Another two previous studies observed that SIRT1 inhibition by sirtinol aggravated brain edema, oxidative damage, neuroinflammation, and neuronal apoptosis after SAH (13,21). However, it is not clear whether SIRT1 activation modulates the phenotypes and functions of activated microglia after SAH as well as the potential mechanisms. Previous studies have reported that SIRT1 participates in regulating immune responses and inducing M2 microglia in a variety of neurological disorders (22,23). For example, Fu et al. showed that SIRT1 activation could modulate the microglia polarization shift from the M1 phenotype and skew toward the M2 phenotype in a cerebral ischemic/reperfusion injury model (24). Duan et al. indicated that activation of SIRT1 ameliorated chronic unpredictable mild stress-induced depressivelike behaviors via shifting microglial polarization toward the M2 phenotype (22). Therefore, we hypothesized that SIRT1 might modulate microglial phenotypic transformation to protect against EBI after SAH.
The nod-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is a key mediator of numerous vascular diseases through triggering a series of immune-inflammatory response (25). It has been demonstrated that the NLRP3 inflammasome is expressed in microglia and implicated in the modulation of inflammatory responses by switching microglia M1/M2 phenotypes (26,27). Intriguingly, emerging evidence indicates that SIRT1 could regulate NLRP3 inflammasome signaling in different research fields (3,28,29). However, the relationship between SIRT1 and NLRP3 inflammasome signaling in modulation of microglia polarization after SAH is still unknown. Based on these backgrounds, we sought to evaluate the effects of SIRT1 activation on microglia polarization and investigate the relationship between SIRT1 and NLRP3 inflammasome signaling in a SAH model.

METHODS AND MATERIALS
All procedures were approved by the Animal Care and Use Committee of Wannan Medical College and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Animals and SAH Model
Adult male Sprague-Dawley rats (250-300 g) were bought from the Nanjing Biomedical Research Institute of Nanjing University. A prechiasmatic cistern injection models was built according to our previous study (13). Briefly, after anesthetization with avertin (200 mg/kg), rats were fixed in a stereotactic apparatus. The skin overlaying the anterior skull was opened, and a hole was drilled in the midline 7.5 mm anterior to the bregma. Blood (0.35 mL) from the femoral artery was injected through the hole over 20 s. Sham animals received the same procedure with injection of 0.35 ml of physiologic saline instead of blood.

Primary Cell Culture
Primary neural cell culture was conducted from the cortex of neonatal rats according to previous studies (12,30). For primary neurons culture, neuronal cells were cultured onto poly-D-lysine -coated plates and suspended in neurobasal media supplemented with B27, glutamate, Hepes, penicillin and streptomycin. For primary microglia culture, cortical cells were suspended in serum-free DMEM-F12 culture medium. The purity of primary microglia was more than 90% in our study (Supplementary Figure 1). Regarding the neurons and microglia co-culture system, microglia were seeded in transwell upper chamber and the neurons were cultured in the plates. Co-culture medium was DMEM with 10% FBS. The co-culture system was harvested 24 h after indicated intervention.
For the in vitro SAH model, the co-culture system was stimulated with oxyhemoglobin (OxyHb, Bio Basic Inc., USA). The neuron-microglia co-cultures were divided into the following groups: control, OxyHb, OxyHb + SRT1720 (1, 5, and 10 mM), and OxyHb + SRT1720 + EX527. The culture medium and cells were collected for, ELISA, immunofluorescence staining, and cell viability analysis.

Drug Administration
For in vivo, SRT1720 (Selleck) was prepared in 1% dimethyl sulfoxide (DMSO) in physiologic saline before use. SRT1720 or vehicle was administered intraperitoneally at 2 h after surgery and then once a day until euthanasia. EX527 (Sigma-Aldrich, St. Louis, MO, USA) was prepared in 1% (DMSO). EX527 or vehicle was administered intraperitoneally for 3 days before SAH construction. In vitro, SRT1720 was dissolved in culture medium. EX527 was dissolved in 1% DMSO (in physiologic saline) and then added to culture medium to reach a final concentration of 20 mM. The doses of SRT1720 and EX527 were selected according to previous studies (31)(32)(33).

Detection of ROS Fluorescence
As previously reported (18), primary neurons were incubated with 2,7-dichlorodihydrofluorescein diacetate (DCFH, Sigma) for 10 min at 37°C. DCFH fluorescence was measured under an inverted fluorescence microscope. The mean relative fluorescence intensity for each group was measured with the Image-Pro Plus system.

Cell Viability Analysis
The lactate dehydrogenase (LDH) and Cell Counting Kit-8 (CCK-8) kits (Beyotime Biotechnology, China) were used to evaluate the viability of primary cultured neurons according to the manufacturer's instructions.

Biochemical Estimation
The levels of intracellular malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) were evaluated with commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) in accordance with the manufacturer's instructions.

Western Blotting
Western blotting was performed according to our previous study (18). After sample preparation, equal amounts of protein samples were separated by polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was then incubated with primary antibodies against SIRT1

Immunohistochemical Staining
Immunohistochemical staining was conducted as previously described (34). In brief, brain sections were fixed with 4% paraformaldehyde and embedded in paraffin. After being deparaffinized, the brain sections were incubated overnight at 4°C with primary antibodies against SIRT1 (1:50, Santa Cruz Biotechnology), NLRP3 (1:50, Santa Cruz Biotechnology), ASC (1:50, Santa Cruz Biotechnology), and caspase-1 p20 (1:50, Santa Cruz Biotechnology). After sections were washed with PBS, they were incubated with HRP-conjugated IgG for 60 min at room temperature. Slides were visualized by incubation with diaminobenzidine and hydrogen peroxide. Staining intensity was scored on a 0-to-4 scale system. In this system, 0 indicates no detectable positive cells and 4 indicates the highest density of positive cells.

Nissl Staining
Nissl staining was conducted as previously described (34). In brief, coronal sections were immersed in cresol violet at 37°C for 20 minutes, and then then mounted onto microscope slides with Permount. Staining was visualized under a light microscope and normal neurons with round and palely stained nuclei were counted.

Neurological Behavior
Neurologic functions were assessed using an 18-point scoring system reported by Sugawara et al (36). Rotarod test was used to assess motor deficits (30). The rotating speed was gradually increased from 4 to 40 rpm over a 5-min period. The latency to fall was recorded. The mean latency was calculated based on three consecutive trials.

Statistical Analysis
All data were performed with GraphPad Prism 8.02 (GraphPad Software, La Jolla, CA, USA), and expressed as mean ± SD. Differences among multiple groups were compared by one-way analysis of variance with Bonferroni post hoc test. Statistical significance was inferred at P < 0.05. Linear regression was performed for correlational analysis. Values of P < 0.05 was considered statistically significant.

SRT1720 Treatment Activates SIRT1 Signaling and Inhibits Oxidative Stress
After SAH SIRT1 is implicated in a variety of pathophysiological processes, including immune response and redox stress. We first evaluated the effects of SRT1720 on SIRT1 signaling and the subsequent oxidative damage after SAH. Immunohistochemistry showed that the immunoreactivity of SIRT1 was significantly increased after SAH (P = 0.0148), which was further enhanced after SRT1720 supplementation (P = 0.0388) (Figures 3A, B). In contrast, EX527 abrogated the enhanced immunoreactivity of SIRT1 by SRT1720 (P < 0.001) (Figures 3A, B). Consistent with the immunohistochemistry data, western blot analysis showed that SRT1720 treatment significantly increased expression of SIRT1 (P = 0.0021) and decreased expression of acetyl-FoxO1 (P = 0.0026). These changes were all abated by EX527 pretreatment (P < 0.05) (Figures 3C-E). We then evaluated the effects of SRT1720 on oxidative damage after SAH. Double immunofluorescent staining indicated that SRT1720 treatment significantly reduced the 8-OHdG immunity when compared with SAH + vehicle group (P < 0.001) (Figures 3F-H). In addition, SRT1720 administration significantly decreased oxidative insults (P = 0.0055) and restored the impairment antioxidant systems after SAH, including SOD and GSH-px levels (P = 0.0019, and P < 0.001) ( Figures 3I-K). However, EX527 pretreatment reversed the effects of SRT1720 on oxidative damage after SAH (P < 0.05) (Figures 3F-H).

SRT1720 Treatment Inhibits Inflammatory Response Following SAH
It has been demonstrated that SIRT1 is implicated in the modulation of immune response and could reduce neuroinflammation in a variety of neurological disorders. We then evaluated the effects of SRT1720 on inflammatory response after SAH. As shown, SAH significantly increased IL-1b, IL-6, TNF-a, IL-18, ICAM-1, and CCL-2 (P < 0.001) protein levels in the cortex when compared with the sham + vehicle group. SRT1720 treatment markedly reduced these proinflammatory cytokines after SAH (P = 0.0172, P < 0.001, P = 0.0239, P< 0.001, P = 0.0166, and P < 0.001, respectively) ( Figures 4A-F). However, all these changes were abrogated by EX527 pretreatment (P < 0.05) (Figures 4A-F).

SRT1720 Treatment Modulates M1/M2 Microglia Polarization After SAH
It has been shown that SIRT1 can modulate the microglia polarization shift from the M1 phenotype and skew toward the M2 phenotype in a variety of disorders. We next evaluated the effects of SIRT1 activation by SRT1720 on M1/M2 microglia polarization following SAH. As shown, the proportion of M1 microglia (IBA1 + /CD16/32 + ) was significantly increased after SAH as compared with sham group (P < 0.001), while treatment with SRT1720 decreased the amount of M1 microglia (P < 0.001). Additionally, SRT1720 markedly increased the quantity of M2 microglia (IBA1 + /CD206 + ) after SAH when compared with SAH + vehicle group (P < 0.001) (Figures 5A-F). However, EX527 pretreatment reversed the effects of SRT1720 on M1/M2 microglia polarization after SAH (P < 0.001) (Figures 5A-F).

SRT1720 Reduces Neuronal Cell Death and Neurological Deficits After SAH
Accumulating evidence indicated that both apoptosis and pyroptosis contribute to the EBI after SAH. Unlike apoptosis, pyroptosis is biochemically characterized as caspase-1 dependent and caspase-3 independent. Notably, suppression of NLPR3 inflammasome or caspase-1 reduces neuronal apoptosis and pyroptosis, and improves neurological outcomes in SAH models. We further explored the effects of SRT1720 on neuronal death and functional outcomes after SAH. As shown, the immunofluorescence staining results showed that there was a low level of caspase-1-and TUNEL-positive neurons in the sham group, which were dramatically increased after SAH (P = 0.001, and P < 0.001, respectively) ( Figures 7A-D). In contrast, SRT1720 administration could significantly reduce the number of caspase-1-and TUNEL-positive neurons after SAH (P = 0.0126, and P < 0.001, respectively) ( Figures 7A-D).

SRT1720 Ameliorates Inflammatory Response, Oxidative Stress, and Neuronal Damage In Vitro
To confirm the proposed crosstalk between microglial and neuronal cells, a transwell co-culture system was established in vitro. We first evaluated whether SRT1720 had beneficial effects in vitro SAH model. As shown, primary neurons and microglia stimulated with OxyHb induced a marked decrease in neuronal viability (P < 0.05), and increase in ROS generation (P < 0.001) and proinflammatory cytokines including IL-1b (P < 0.001) and IL-6 (P < 0.001), all of which were reversed by SRT1720 treatment (P < 0.05) ( Figures 8A-F). In contrast, EX527 abrogated the beneficial effects of SRT1720 on oxidative stress, inflammatory response, and neuronal damage (P < 0.05) (Figures 8A-F). Consistent with the in vivo results, our findings indicated that SRT1720 could exert a cerebroprotective action against SAH damage in vitro.

SRT1720 Enhances SIRT1 Activation and Inhibits NLRP3 Inflammasome In Vitro
To further explore the mechanisms of action of SRT1720 in vitro SAH model, we examined the possible effects of SRT1720 on SIRT1 and NLRP3 expression in neuron-microglia co-cultures by using double immunofluorescence staining. At the cellular level, SIRT1 is known as a nuclear protein, which is predominantly expressed in neurons. NLRP3 inflammasome is mainly expressed in microglia and the basal level of the NLRP3 inflammasome is low under physiological conditions. Consistent with previous studies, our immunofluorescence staining data indicated that SIRT1 was weakly expressed in primary neurons and the immunoreactivity of NLRP3 was low in primary microglia in the control group. OxyHb stimulation induced the expression of SIRT1 (P = 0.0397) and NLRP3 (P < 0.001) in the co-culture system. SRT1720 treatment could significantly enhance the expression of SIRT1 (P < 0.001) in primary cortical neurons and decrease NLRP3 expression (P = 0.002) in primary microglia, which could be abrogated by EX527 administration (P < 0.05) (Figures 9A-D). These data consistent with the results in vivo suggesting that SIRT1 could modulate NLRP3 inflammasome activation and SRT1720 treatment was able to activate SIRT1 and inhibit NLRP3 inflammasome signaling.

SRT1720 Decreases Neuronal Pyroptosis and Apoptosis After SAH In Vitro
Both neuronal apoptosis and pyroptosis are involved in the pathophysiology of EBI after SAH. Since PI cannot pass intact plasma membrane and can only be present in DNA of cells where the plasma membrane has been injured. During pyroptosis, pores can be formed in the cell membrane and can be detected by PI staining [26]. Therefore, PI staining is widely used as a detection for neuronal pyroptosis. In our study, we performed PI and TUNEL staining to examine neuronal pyroptosis and apoptosis, respectively. Based on the data in vivo, we speculated that SIRT1 activation by SRT1720 could reduce neuronal apoptosis and pyroptosis in vitro. As shown, double immunofluorescence staining showed that PI-(P < 0.001) and TUNEL-positive (P < 0.001) neurons were significantly increased after OxyHb incubation, which could be decreased by SRT1720 administration (P = 0.0011, and P = 0.013, respectively) ( Figures 10A-D). These confirmed that SRT1720 was able to reduce neuronal pyroptosis and apoptosis after SAH in vitro. In contrast, EX527 abated the protective effects of SRT1720 on neuronal cell death in vitro (P < 0.05) (Figures 10A-D). These findings indirectly indicated that SIRT1 activation by SRT1720 probably inhibited NLRP3 inflammasome-mediated pyroptosis and apoptosis.

DISCUSSION
In this study, we showed that EX527 significantly inhibited SIRT1 expression, aggravated neurological outcomes and motor functions after SAH. In the meantime, EX527 pretreatment induced M1 microglia polarization and NLRP3 inflammasome activation after SAH. SIRT1 activation by SRT1720 exerted a neuroprotective effect after SAH. The application of SRT1720 significantly ameliorated neurological deficits, brain edema, oxidative damage, neuroinflammation, and neural cell death after SAH. In addition, SIRT1 activation by SRT1720 decreased the number of M1 microglia and upregulated the expression of M2 microglia, leading to improved SAH recovery. Furthermore, EX527 abated the beneficial effects of SRT1720 against SAH both in vivo and in vitro. Meanwhile, we confirmed that ROS-mediated NLRP3 inflammasome signaling might be involved in the beneficial effects of SRT1720 on microglia polarization after SAH. Cerebral inflammation is a vital component of the pathological cascade in the EBI after SAH (12). Microglia, as the resident immunocytes of the central nervous system (CNS), react to various brain injury and can be activated by blood components, including red cells, and heme (37). It has been demonstrated that microglia respond to acute brain injury by becoming activated and developing classic M1-like or alternative M2-like phenotypes. M1 microglia are the primary source of cytokines, chemokines, ROS, and other immunomodulatory molecules, which further increase brain damage. In contrast, M2 microglia are considered as nerve repair cells, as they produce anti-inflammatory factors and upregulate neuroprotective factors in neurological disorders (6,37). Though the polarization of microglia is controversial, the dichotomy between M1 and M2 phenotypes classification remains useful for clarifying the character of microglia in various brain disorders (4,38). In models of TBI, spinal cord injury, ischemic stroke, and intracerebral hemorrhage, an M2 to M1 shift was observed in the early period (6,8,37). In time, the M1 microglia/ macrophages become dominant in the injury area and aggravate brain damage by exacerbating inflammatory response. Similarly, in SAH area, the phenomenon of microglia polarization has also been reported. Mounting evidence has indicated that inhibition of microglia M1 polarization and promotion of a shift from M1 to M2 phenotype could ameliorate EBI and improve neurological functions after SAH (10)(11)(12). SIRT1 is a member of NAD + -dependent protein deacetylases implicated in a variety of pathophysiological processes, including immune response and redox stress (13,15). Multiple studies have demonstrated that SIRT1 exerts a protective role in various models of neuronal injury and neurodegeneration. For example, Vellimana et al. reported that SIRT1 activation by resveratrolconditioning provided cerebroprotection effects after cerebral ischemia at both early and delayed time points (20). Hattori et al. demonstrated that SIRT1 overexpression upregulates the nitric oxide system and counters cerebral hypoperfusion injury (39). In SAH area, accumulating studies also indicate that SIRT1 activation could attenuate EBI and delayed cerebral vasospasm, and inhibition of SIRT1 could aggravate SAH-induced vascular dysfunction and functional deficits (3,20,31,40). These suggest that SIRT1 might represent a potential therapeutic target for treating SAH. However, to data, little is known about the potential effects of SIRT1 on microglia polarization and the underlying molecular mechanisms after SAH.
According to current literature, SIRT1 can modulate the microglia polarization shift from the M1 phenotype and skew toward the M2 phenotype in a variety of disorders (22,24,33,41). A previous study by Chen et al. reported that SIRT1 activation by SRT1720 significantly improved functional recovery after SCI by reducing the levels of proinflammatory cytokines, and the number of M1 macrophages (33). Fu et al. indicated that SIRT1 activation could modulate the microglia polarization shift from the M1 phenotype and skew toward the M2 phenotype in a cerebral ischemic/reperfusion injury model (24). Thus, we speculated that SIRT1 might modulate microglial phenotypic transformation to protect against EBI after SAH. In our study, we first employed EX527, a potent and selective SIRT1 inhibitor, to suppress SIRT1 to mimic SIRT1 knockout. We observed that EX527 significantly inhibited SIRT1 activation and promoted microglia phenotype toward M1 after SAH. To further confirm whether SIRT1 modulates microglia polarization, a specific activator of SIRT1 SRT1720 was used. SRT1720 is reported as a potent SIRT1 activator with 1000-fold efficacy compared to resveratrol. Multiple studies have demonstrated that SRT1720 can effectively activate SIRT1 expression and activation in a variety of diseases models (33,42). In the CNS diseases, SRT1720 has been proved to ameliorate neuronal injury and improve functional recovery in many neurological disorders (43,44). Moreover, SRT1720 can cross the blood-brain barrier and act directly on the CNS (33). In our study, we confirmed that SRT1720 significantly increased SIRT1 activation and improved locomotor recovery after SAH. Additionally, we observed that the number of M1 microglia was significantly decreased after SRT1720 treatment, while the percentage of M2 microglia was increased. Our data suggested that SIRT1 activation by SRT1720 is able to regulate microglia M1/M2 polarization after SAH, which may explain the observed locomotor recovery after injury. However, the mechanisms of microglial M1-to-M2 phenotype transition by SRT1 activation remain unknown. The NLRP3 inflammasome is a cytoplasmic multiprotein complex of the innate immune system that can positively trigger a series of proinflammatory pathways. Mounting evidence has demonstrated that NLRP3 inflammasome is involved in   (29,50). ROS can elicit a plethora of detrimental effects on cellular functions by causing damages to proteins, lipids and nucleic acids (51). Neurons can generate elevated amounts of ROS compared to other organs and are particularly vulnerable to ROS (52). It is known that both ROS overproduction and neuroinflammation are considered crucial elements of EBI after SAH and each of them promotes and amplifies the other one (3,53). Additionally, ROS production is one of the major signals that trigger the NLRP3 inflammasome activation (54). According to previous studies, SIRT1 is predominantly expressed in neurons and the elevated SIRT1 activation could significantly reduce oxidative damage by deacetylating FoxOs (13,31). As transcription factors, FoxOs play a critical role in the maintenance of intracellular ROS homeostasis.   (55). As an upstream mediator of FoxO1, SIRT1 could deacetylate FoxO1 to enhance FoxO1 DNA binding and induce the expression of FoxO1 target genes to mitigate oxidative stress in a variety of disorders (31,55). Consistent with previous studies, we observed that SIRT1 activation by SRT1720 significantly increased deacetylation of FoxO1, ameliorated the overproduction of ROS and inhibited NLRP3 inflammasome activation after SAH. Concomitant with the suppression of ROS-NLRP3 inflammasome pathway, SRT1720 ameliorated SAHinduced functional deficits and neuronal apoptosis and pyroptosis. In contrast, EX527 pretreatment abated the inhibitory effects of SRT1720 on ROS-NLRP3 inflammasome signaling and the neuroprotective effect in SAH. Similarly, in vitro, SIRT1 activation by SRT1720 ameliorated neuroinflammation, oxidative damage, and neuronal damage in neurons and microglia co-culture system stimulated with OxyHb, which were associated with ROS-NLRP3 inflammasome suppression, and were counteracted by EX527. Taken together, SIRT1 inhibits neuroinflammation by reduction in M1 microglia polarization and driving the microglial phenotype toward M2, which might be mediated by modulation of the ROS-NLRP3 inflammasome signaling pathway. Driving microglia M2 polarization and maintaining the balance of M1/M2 should be of great significance for the treatment of acute brain injuries. However, most of compounds discovered can only suppress inflammation by decreasing M1 phenotype microglia, and only a few compounds can promote microglia M2 polarization. This study showed that SIRT1 activation can significantly decrease the M1 phenotype microglia and promote the M2 polarization of microglia, which might explain the observed neuroprotection. However, we have to admit that there are several limitations in our study. Firstly, EX527 may not act as a highly specific inhibitor of SIRT1. EX527 can also slightly affect SIRT2 and SIRT3 pathway. Additionally, SRT1720 can affect other SIRTs members including SIRT3. Thus, SIRT1 gene knockout mice and SIRT1 overexpression by transfection of adenoassociated virus should be performed in the future to confirm our investigations. Secondly, the long-term inflammation also plays an important role in the pathophysiology after SAH (56). We did not evaluate the potential effects of SIRT1 on the long-term neuroinflammation. In our future studies, the endovascular puncture SAH model with heavier brain damage will be further employed. Lastly, it has reported that M2 microglia could divide into multiple phenotypes, including M2a, M2b, and M2c (6). In our study, we did not explore the functionality of the various M2 subtypes after SRT1720 treatment. Therefore, more pre-clinical studies are required to decipher these issues.

CONCLUSION
In summary, we provide the first preclinical evidence that SIRT1 activation by SRT1720 might have dramatic effects on inflammation and microglial polarization after SAH. These effects were at least in part through inhibiting ROS-mediated NLRP3 inflammasome signaling. Thus, SIRT1 might be a promising drug target for SAH treatment and the SIRT1 agonist SRT1720 may have great potential for SAH.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.