S1PR3/RhoA signaling pathway in microglia mediates inflammatory activation in early brain injury after subarachnoid hemorrhage

S1PR3/RhoA signaling pathway in microglia mediates inflammatory activation in early brain injury after subarachnoid hemorrhage | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article S1PR3/RhoA signaling pathway in microglia mediates inflammatory activation in early brain injury after subarachnoid hemorrhage Lu Feng, Panxing Wu, Chao Ding, Xiuyou Yan, Xuanhao Zhu, Ming Lu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4374501/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Spontaneous subarachnoid hemorrhage (SAH) is a serious and common cerebrovascular disease with high mortality rate and poor prognosis. The immune response caused by abnormal activation of intracranial microglia is one of the main factors contributing to early brain injury after SAH. Sphingosine 1-phosphate (S1P) signaling pathway is widely involved in immune regulation, nerve cell differentiation and other processes. It has been reported that S1P expression is increased in cerebrospinal fluid after SAH, but its role in early brain injury and neuroinflammation induced by SAH remains unclear. In the rat model of SAH established by arterial puncture, low (0.5mg/kg) or high dose (5mg/kg) of the S1P receptor inhibitor FTY720 was administered immediately or at 24 hours after surgery. Improvement of behavioral scores and brain edema symptoms after SAH was observed in immediate treatment group at high dose. In addition, activation of cortical microglia near the perforation site was observed after SAH, and this activation was significantly inhibited after 5mg/kg FTY720 treatment immediately after surgery. Further studies showed that S1P could induce activation and M1 polarization of human microglia cells in vitro. This activation may be mediated through the S1PR3-Gα 12/13 -RhoA pathway. Therefore, our study highlights the important role of S1P signaling and microglia activation in SAH-induced early brain injury, and provides evidence for novel therapies targeting the neuroinflammatory process after SAH. Subarachnoid hemorrhage Sphingosine 1-phosphate S1P receptor inhibitor Microglia Neuroinflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Spontaneous subarachnoid hemorrhage (SAH) is the third leading cause of stroke, with high mortality and disability rate[ 1 ]. The pathogenesis of SAH is complex, of which 85% are caused by aneurysm rupture, called aneurysmal subarachnoid hemorrhage (aSAH) [ 2 ]. Clinically, about a quarter of patients with SAH die before receiving medical care, 33% die within 48h after the initial hemorrhage, and 50% die within 30 days [ 3 ]. Patients who survive early massive hemorrhage still suffer from a series of complications, including increased intracranial pressure and delayed cerebral ischemia (DCI). Historically, many studies mainly focused on the targeted treatment of cerebral vasospasm (CVS) after SAH, but the clinical outcomes have not been improved, and the incidence and mortality of DCI have not been reduced [ 4 ]. Recently, researchers have found that early brain injury (EBI) induced by SAH may be a key factor affecting patient outcome. EBI usually occurs within 72 hours before SAH and involves a series of pathophysiological events, such as brain edema, blood-brain barrier (BBB) destruction, oxidative stress, inflammation, excitotoxicity, and impaired ion homeostasis. Among them, the activation and amplification of harmful inflammatory response play a key role in nerve injury after SAH [ 5 ]. However, how EBI occurs and how it causes delayed cerebral ischemia and neurological damage, the underlying mechanisms are still unclear. Microglia, the resident macrophage in the central nervous system (CNS), plays an important role in the neuroinflammatory response induced by SAH[ 6 ]. Upon receiving risk-associated molecular pattern signals (DAMPs), microglia can rapidly activate, proliferate, and migrate to the site of injury, whereby they phagocytose cellular debris and harmful substances, and secrete various inflammatory cytokines and chemokines. Activated microglia also produce some anti-inflammatory cytokines and trophic factors to promote neuronal regeneration. Therefore, microglia can be classified into deleterious M1 subpopulation and beneficial M2 subpopulation according to their distinguishable phenotype. Usually, M1 microglia expressing MHCII, CD80 and CD11b secretes proinflammatory cytokines (including TNF-α, IL-1β, IL-6, COX-2, ROS and NO). In contrast, M2 microglia, which is positive for CD163 and CD206, mainly produce IL-10 and TGF-β, while phagocytosing cell debris and harmful substances. Early DAMPs signaling induce some microglia to polarize to an M2 phenotype; however, persistent brain injury may gradually replace these reparative M2 cells with a toxic M1 phenotype, resulting in prolonged inflammatory responses and subsequent impairment of neuronal regeneration[ 7 ]. Thus, the functional imbalance of activated microglia leading to an intensified immune response is one of the potential mechanisms of EBI. Sphingosine-1-phosphate (S1P) belongs to a class of lipids called sphingolipids [ 8 ], and its receptors are widely distributed in almost all tissues and organs of the human body. Studies have shown that S1P is widely involved in the regulation of vasculogenesis, angiogenesis, immune regulation, nerve cell differentiation, blood-brain barrier integrity construction and other processes by interacting with its receptor S1PRs (S1PR1-5) [ 9 ]. Its dysfunction may lead to autoimmune diseases, chronic inflammation, tumors and neurodegenerative diseases [ 10 , 11 ]. In 2010, the FDA approved Fingolimod (FTY720), an S1P receptor 1 (S1PR1) inhibitor, for the treatment of multiple sclerosis, an autoimmune disease, which has triggered extensive attention to the S1P signaling pathway among researchers [ 12 ]. In addition to S1PR1, the drug can also selectively inhibit the activation of S1PR3 and S1PR5 receptor signaling pathways, reduce the number of peripheral circulating lymphocytes, and significantly prolong the survival of experimental animal transplanted organs without impairing the immune response and immune memory function to pathogens, with low toxic side effects. A series of clinical studies support its use as a good immunosuppressive agent [ 13 ]. In recent years, increasing evidence has shown that the expression levels of S1P and its receptors undergo pathological changes in diseases related to CNS injury [ 14 ], suggesting that S1P pathway may also have the ability to regulate neuroinflammation after CNS injury. However, the detail mechanism of S1P-mediated neuroinflammation is still poorly understood. Therefore, further studies will help to clarify the mechanism of S1P in CNS injury-related diseases such as SAH, and provide theoretical basis for the application of immunomodulators targeting the S1P signaling pathway in CNS injury-related diseases. Materials and Methods 1. Cells and reagents HMC3 cell line (CRL-3304) were obtained from ATCC. Twelve-week-old rats were purchased from Charles River. FTY720 (CAS NO. 162359-56-0) were purchased from AdooQ Bioscience, IFN-γ was purchased from Peprotech (300-02), and S1P (S45337) from Shanghai yuanye Bio-Technology Co., Ltd.. C3 exoenzyme (Amylet Scientific) was used to block RhoA activation, and Y27632 (Sigma-Aldrich) was used to inhibit ROCK activation. 2. Cell culture and treatment HMC3 cells were maintained in RPMI1640 medium, containing 10% heat inactivated fetal bovine serum, 100 U/ml penicillin, 50U/ml streptomycin, 2mM glutamine, and 1mM sodium pyruvate at 37℃ in a 5% CO 2 humidified incubator. At a confluence of 70%, HMC3 cells were trypsinized and plated in 6 well plates. 48 hours later, IFN-γ or S1P was added to the culture medium at indicated concentrations. Cells were harvested for further analysis after a 6-hour treatment period. 3. siRNA vector construction and transfection Pre-designed shRNA oligos targeting S1P1, S1P2, S1P3 and G α12/13 were customized by Beijing Tsingke Biotech Co., Ltd (Online Resource Table S1). shRNA oligos were inserted into the FV055 vector containing the AmpR, GFP and puromycin sequences, then positive clones were selected and validated by sequencing. HMC3 cells were plated in 6 well plate, serum-starved for 18–24 h and transfected with plasmids expressing si-S1P1, si-S1P2, si-S1P3 and si-G α12/13 , respectively. Cells were collected for gene expression assay using QRT-PCR and western blotting. 4. QRT-PCR Total mRNA of HMC3 cells was extracted using the RNeasy kit (Invitrogen) and the complement strand of DNA (cDNA) was synthesized using PrimeScript™ RT reagent Kit from Takara. Gene expression was determined by Real-time PCR using the TB Green® Premix Ex Taq™ II FAST qPCR kit. Primer sequences are shown in the Online Resource Table S2. Data were normalized to internal β-Actin, and fold change was determined as described previously[15] . Values for comparison for a single gene across multiple samples was determined using cycle threshold (Ct) data fitted to a standard curve. For comparison of multiple transcripts in a single sample, then the 2−ΔΔCt method was applied to the Ct value. 5. Western blot HMC3 cell samples were lysed with RIPA buffer (20 mm Tris, 250 mm NaCl, 3 mm EDTA, 3 mm EGTA, and 20 mm βglycerophosphate) supplemented with sodium vanadate, leupeptin, aprotinin, p-nitrophenyl phosphate, and phenyl methylsulfonyl fluoride. Protein concentration of samples were measured using Bradford Protein Assay kit (Beyotime). Equal amounts of protein (20 μg) were loaded onto 4–12% 10-well or 15-well SDS-PAGE gels. Gels were transferred to PVDF membranes, and the resulting blot was probed with specific antibodies. The COX-2 antibody (Affinity#AF7003) was used at 1:500 dilution, the β-actin antibody (Cell Signaling Technology #4970) was used at 1:1000 dilution. Rabbit secondary antibody was used at 1:4000 dilution. Fold changes were determined by chemiluminescence and normalized to β-actin. 6. Flow cytometry HMC3 cells were harvested immediately after S1P or IFN-γ treatment, and stained with FITC-conjugated CD11b antibody (Biolegend#301329), or CD80 antibody (Biolegend#375405)，or CD163 antibody (Biolegend#333617), or CD206 antibody (Biolegend#321103), or MHC-II antibody (Invitrogen#17-9956-42），or corresponding IgG isotypes controls without PMA fixation. The gates were established by fluorescence minus IgG isotype controls. 7. SAH model and treatment All animal experiments were approved by the ethics committee of Taizhou University, performed in accordance with the Guide for the Care and Use of Laboratory Animals by the National Institute of Health, China. SAH was performed by using an artery puncture method according to a previous study[16]. In brief, 12-week-old male rats were anesthetized with sodium pentobarbital (80 mg/kg body weight). The left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were exposed. A sharpened 4-0 monofilament nylon suture was advanced into ICA from ECA to perforate the artery at the bifurcation of the anterior and middle cerebral artery. And then the suture was immediately withdrawn to cause SAH. The procedure in the sham group was similar without the perforation. Rats were randomly divided into 5 groups, n=5 in each group. A: sham; B: SAH + vehicle ; C: SAH + LD (0.5 mg/kg FTY720); D: SAH +HD (5 mg/kg FTY720); E: SAH + HD24 (5 mg/kg FTY720 delayed intervention). FTY720 was formulated with physiological saline at a concentration of 0.15 mg/ml or 1.5 mg/ml. Then the rats in group C, D were given with FTY720 or vehicle (physiological saline) by intraperitoneal administration at 2 h after SAH injury. The FTY720 dose was determined according to previous studies[17]. Rats in group B received intraperitoneal administration with an equal volume of physiological saline at 2 h after SAH. Rats in group E received intraperitoneal administration with 5mg/kg FTY720 at 24 h after SAH. 8. Assessment of neurological score Neurological score was assessed at 24 h and 72 h after SAH according to previous studies by a blinded investigator[18]. In brief, animals’ neurological functions were evaluated by six tests, including symmetry in the movement of all limbs (0-3), spontaneous activity (0-3), forepaw outstretching (0-3), climbing (1-3), response to vibrissae touch (1-3) and body proprioception (1-3). The minimum neurological score was 3 (severe impairment) and the maximum was 18 (no neurological impairment). 9. Brain water content After the assessment of neurological score, rats were sacrificed by cervical dislocation. The brains were removed and weighed immediately to obtain the wet weight. The brain was then dried in an oven at 100°C for 72 h and weighed again to obtain the dry weight. The percentage of brain water content was calculated according to the formula: [(wet weight-dry weight)/wet weight] × 100%. 10. Immunohistochemical staining The brain tissues were fixed in 4% paraformaldehyde and embedded in paraffin before being cut into 5-μm thick sections. Cortex adjacent to the Perforated Site (CAPS), the motor cortex (M1 cortex), and hippocampus were selected for IBA-1 staining as previously described[19]. In brief, after a xylene/ethanol dewax-rehydration series, endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide. Brain sections were then incubated for 1 h with blocking buffer comprising 2.5% goat serum, 1% bovine serum albumin (BSA), and 0.1% Triton-100. The primary antibody of IBA-1 (Thermo Fisher# PA5-21274) was applied subsequently at 4 °C overnight. Horseradish peroxidase (HRP) conjugated secondary antibody was applied for 1 h at room temperature. Diaminobenzidine (DAB) was utilized for visualization of colorimetric reaction. Three random fields were examined on each brain area respectively of each animal under microscope (Leica) at × 40 magnification. 11. Statistical analysis IBM SPSS 23.0 software was used for statistical analysis of the data. All data were expressed as mean ± standard deviation (SD). Differences between two experimental groups were compared by the Student t-test. P<0.05 was considered to indicate a statistically significant difference. Results To test whether S1P blockage can ameliorate SAH-induced EBI, a rat SAH model was established using arterial puncture method. Compared with the sham-operated group, the SAH experimental group showed significant neurological deficits. Specifically, motor dysfunction in limb movement symmetry, forepaw outstretching, and climbing within 24 hours occurs immediately after SAH (Fig. 1 A). Motor dysfunction induced by SAH worsened after 72 hours (Fig. 1 B). A significant increase in brain water content, without alteration of the structural integrity of the brain (Fig. 1 C) or substantial change in body weight (Online Resource Fig. S1 ), was observed at 72 h after SAH in the experimental group, accompanied by an increase in the number of IBA1-positive microglia in the cortex adjacent to the perforated site (CAPS), hippocampus, and motor cortex, with a more amoeboid than bifurcating morphology (Fig. 2 A). These are direct evidence of early brain damage caused by the puncture-triggered arterial hemorrhage which spreads in the subarachnoid space and causes an acute inflammatory response. At 24 hours after surgery, all SAH groups, either with or without treatment, did not show significant difference for neurological scores. However, at 72 hours after SAH, in the groups that received 0.5mg/kg or 5mg/kg of FTY720 or the same volume of saline after surgery, the rats showed different responses in neurological scores. Compared with the control group (saline), 5mg/kg FTY720 administered immediately after surgery significantly alleviated neurological deficits after surgery, whereas 0.5mg/kg FTY720 administered immediately after surgery did not significantly improve neurological scores (Fig. 1 A-B, Table 1 ). Delayed administration of 5mg/kg FTY720 at 24 hours after surgery (before neurological scoring) did improve the neurological outcomes but the effect was modest compared with the immediate-treated group. Moreover, 5mg/kg of FTY720 immediately after surgery was the only group that achieved prolonged alleviation of neurological deficits within 72 hours after SAH. Likewise, brain edema was significantly alleviated in the 5mg/kg FTY720 immediate-treated group at 72 hours after surgery, but not in the other two groups (Fig. 1 C). These results confirmed that S1P receptor inhibitors could significantly improve the nerve injury and inhibit the inflammatory response caused by EBI after SAH. The blockage effects of S1P-dependent events are most likely to be maximized within 24 hours after SAH, and to be dose-dependent. Table 1 Neurological evaluation by six behavior tests for rats with indicated operation and treatments. Groups Sham SAH + vehicle SAH + LD SAH + HD SAH + HD24 Time point 24 hr 72 hr 24 hr 72 hr 24 hr 72 hr 24 hr 72 hr 24 hr 72 hr Neurological scores Total 18 18 10.4 ± 0.5 8.6 ± 0.5 10.8 ± 0.8 9.2 ± 0.8 11.8 ± 1.3 13.2 ± 1.5 10.8 ± 0.8 10.2 ± 1.3 Symmetry in the movement of all limbs 3 3 2.4 ± 0.5 1.6 ± 0.5 2.2 ± 0.4 1.8 ± 0.4 2.2 ± 0.4 2.6 ± 0.5 2.4 ± 0.5 2.2 ± 0.4 Spontaneous activity 3 3 1.4 ± 0.5 1.2 ± 0.4 1.4 ± 0.5 1.4 ± 0.5 1.6 ± 0.5 1.8 ± 0.4 1.4 ± 0.5 1.0 ± 0.0 Forepaw outstretching 3 3 1.6 ± 0.5 1.2 ± 0.4 1.4 ± 0.5 1.0 ± 0.0 1.6 ± 0.5 2.2 ± 0.4 1.6 ± 0.5 1.4 ± 0.5 Climbing 3 3 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.4 ± 0.5 1.4 ± 0.5 1.0 ± 0.0 1.2 ± 0.4 Reaction to touch on either side of trunk 3 3 2.0 ± 0.0 1.8 ± 0.4 2.6 ± 0.5 1.8 ± 0.4 2.8 ± 0.4 3.0 ± 0.0 2.4 ± 0.5 2.4 ± 0.5 Response to vibrissae touch 3 3 2.0 ± 0.0 1.8 ± 0.4 2.2 ± 0.4 2 ± 0.0 2.2 ± 0.4 2.2 ± 0.4 2.0 ± 0.0 2.0 ± 0.0 Microglia play a pivotal role in the immune response after SAH. Activation of microglia, indicated by the IBA1 staining, is supposed to initiate at the CAPS, then spread to the distal motor cortex and to the hippocampus region deep in the brain. We showed that administration of FTY720 at 24 hours after SAH seemed to block only the remote activation of microglia while microglia located in the CAPS remained activated (Fig. 2 A-B). However, immediate-treatment of FTY720 globally inhibited the microglia activation, suggesting that FTY720 may only inhibit the early activation, but not reverse the activated status of microglia. To further explore the mechanism by which S1P regulates neuroinflammation, we first selected an immortalized human microglia cell line, HMC3, for in vitro studies. Similar to primary microglia, HMC3 can be induced and activated by IFN-γ to secrete inflammatory factors such as IL-6 and TGFβ, and upregulate M1 markers such as MHCII, CD80 and CD11b. If cultured in the presence of neuronal precursor cells or astrocytes, the expression of M2 markers (including CD163, CD206, etc.) is up-regulated [ 20 ]. We found that S1P activates HMC3 cells in a dose-dependent manner. At a concentration above 50nM, S1P induces induced an IFN-γ-induced phenotype in HMC3 and a marker of M1 polarization (Fig. 3 A, Online Resource Fig. S2). In contrast, S1P had no effect on the expression of several CD163 and CD206 associated with M2 polarization. HMC3 is derived from embryonic microglia, and the level of secreted TNF-α is very low even in the activated state, which is not easy to detect [ 21 ]. In contrast, the mRNA expression of several major inflammatory factors, including IL-6, TNF-α, and TGF-β1, was significantly upregulated in the activated state after receiving IFN-γ. Our results showed that S1P (50nM) induced an up-regulation of IL-6 and TNF-α expression at the mRNA level, but did not change the expression of anti-inflammatory factor TGF-β1 (Fig. 3 B). In addition, expression of the inflammation-related expression product cyclooxygenase-2 (COX2) was significantly increased after S1P induction (Fig. 4 A). All these results suggest that S1P induces HMC3 activation and M1 polarization, but not M2 polarization. S1P activates downstream signals through its receptors, including S1PR1-S1PR5. Among them, S1PR1-3 were the most studied. To further elucidate the mechanism by which S1P activates HMC3, siRNA vectors targeting S1pr1 , S1pr2 and S1pr3 were constructed. The results of QRT-PCR showed that the mRNA of S1pr1 , S1pr2 and S1pr3 were all detected in HMC3 cells. After 48 hours of transfection with si-S1P1, si-S1P2 or si-S1P3 vectors, the mRNA expression of S1pr1 , S1pr2 and S1pr3 in HMC3 cells was down-regulated by 62%, 63% and 58%, respectively (Online Resource Fig S3). Meanwhile, western blot showed that the expression of COX2 was still up-regulated after S1P induction in HMC3 cells transfected with si-S1P1 and si-S1P2. In contrast, the expression level of COX2 in HMC3 cells transfected with si-S1P3 was not responsive to S1P induction (Fig. 4 B). Thus, S1P acts primarily through S1PR3, but not S1PR1 and S1PR2, in HMC3 cells. When bound by S1P ligands, S1P receptors transmit signals downstream by activating their corresponding G-coupled protein receptors, where S1PR1 only couples G i/o , whereas S1PR2 and S1PR3 couple G i/o , G q or G 12/13 [ 22 ]. It has been reported that S1PR3 can activate downstream inflammatory signals and play a role in microglia-mediated inflammatory response [ 23 ], while S1P activates inflammatory astrocytes mainly through the S1PR3-G 12/13 -RhoA pathway [ 24 ]. To further demonstrate whether S1P activates microglia via G 12/13 -RhoA, we used selective inhibitors of RhoA/ROCK or knockdown of Gα 12/13 . The S1P-induced increase in COX2 expression was significantly inhibited by the addition of FTY720, the RhoA inhibitor C3, the ROCK inhibitor (Y27632) to the culture medium, or by transfection of HMC3 with siRNA targeting G α12/13 (Fig. 4 C). It should be noted that expression of G α12 and G α13 in HMC3 cells was reduced by 72% and 64% after transfection with vectors expressing si-GNA12/13 mixture, respectively (Online Resource Fig. S3). These results suggest that S1P can activate the pro-inflammatory phenotype of HMC3 by activating the S1PR3-G 12/13 -RhoA/ROCK signaling pathway. Discussion There is increasing evidence that neuroinflammation occurs rapidly after SAH and is an important factor in causing EBI [ 25 ]. S1P regulates a variety of cell functions through its receptors S1PR1-5 and is considered to be involved in the regulation of inflammatory process [ 22 ]. FTY720 is the first approved S1P receptor inhibitor. Reduction in circulating lymphocytes and limiting inflammatory cell migration into the CNS are considered to be its two most important pharmacological mechanisms [ 26 ]. However, unlike other immunosuppressants, FTY720 is lipophilic and therefore easily penetrates the blood-brain barrier, acting at the site of CNS injury through sphingosine kinase activation secreted by neuronal cells. Several preclinical and clinical studies have demonstrated the inhibitory effect of FTY720 on neuroinflammation caused by CNS injury [ 17 , 27 – 29 ]. Our findings are consistent with literature reports that FTY720 plays a protective role in the CNS injury caused by SAH. Meanwhile, our evidence further strengthens the notion that S1P signaling is an important molecular mechanism responsible for CNS neuroinflammation. However, several issues still require further discussion. First of all, the inflammatory response after SAH is complex and seems to get involved in both the acute phase and the post-CVS period[ 30 , 31 ]. Aneurysm rupture causes blood leakage throughout the subarachnoid space, followed by a sharp increase in intracranial pressure (ICP), a decrease in perfusion pressure (CPP), transient ischemia of brain tissue and death of nerve cells [ 25 ]. At this time, the blood-brain barrier rapidly breaks down, causing massive inflammatory cells (mainly neutrophils and macrophages) infiltration and microglia activation. After an acute response period, these inflammatory cells may remain trapped at the site of injury, undergo apoptosis, and cause a secondary and chronic inflammatory response [ 32 ]. Our study provides evidence that FTY720 directly inhibits the initial microglial activation within 24 hours after SAH, but prolonged observation of FTY720-treated SAH rats was absent due to the limited experiment animals in this study. Further studies are needed to elucidate whether and how the S1P pathway is involved in subsequent events of this complex process, including amplification of the inflammatory cascade and later neural repair. Therefore, the therapeutic strategy targeting S1P pathway still faces some challenges in CNS injury, including how to choose the intervention window, to optimize administration dose and frequency, and to develop personalized dosing regimens according to specific conditions of CNS injury. S1PR3 is widely expressed in heart, lung, kidney, spleen and other organs, and is involved in the maintenance of endothelial barrier, vascular tone and immune response. Different from S1PR1, which mainly functions through JAK2 and PI3K pathways [ 33 , 34 ], S1PR3 can extensively affect pathways including G α12/13 -RhoA-ROCK, VEGF, PI3K, STAT3 [ 35 ]. Among them, the G α12/13 -RhoA-ROCK pathway is considered to be closely related to the migration and M1 polarization of macrophages. In mouse peritoneal macrophages, the expression level of S1PR3 is lower than that of S1PR1 and S1PR2. However, LPS stimulation can up-regulate the expression of S1PR3 and stimulate S1PR3-mediated inflammatory activation, while the expression of S1PR1 is down-regulated [ 23 ]. Consistent with this, our in vitro studies showed that microglial activation in the CNS was dependent on S1PR3 but not S1PR1. However, this conclusion cannot be simply translated to in vivo studies because FTY720 is a broad-spectrum S1PR inhibitor with known substrates including S1PR1, S1PR3, and S1PR5 among others [ 26 ]. The widespread expression of S1PR is related to several adverse reactions such as slow heart rate and hypertension caused by FTY720 in clinical studies [ 28 ]. The pathologic consequences of systemic inhibition of the S1PR3 pathway are unclear. Therefore, detailed studies on different types of S1PR are necessary to develop more specific S1PR receptor inhibitors and optimize the benefit-risk balance of S1P pathway-based therapies. In summary, our study highlights the role of the S1P pathway in the inflammatory response after SAH, deepening our understanding of the complex inflammatory response process after SAH. In addition, our study also suggests that therapeutic strategies targeting S1P may play a protective role in attenuating CNS injury after SAH. Statements and Declarations Ethics Approval All animal experiments in this study was approved by the Ethics Committee of Taizhou University. Funding This study was supported by Taizhou social development science and technology Program project (Grant No. 21ywa31). Competing Interests The authors declare that they have no conflict of interest. References Claassen J, Park, S. 2022. Spontaneous subarachnoid haemorrhage. Lancet. https://doi.org/10.1016/S0140-6736(22)00938-2. Macdonald RL, Schweizer, T.A. 2017. Spontaneous subarachnoid haemorrhage. Lancet . https://doi.org/10.1016/S0140-6736(16)30668-7. Song HC, Yuan, S., Zhang, Z.W., Zhang, J.Y., Zhang, P., Cao, J., Li, H.Y., Li, X., Shen, H.T., Wang, Z., Chen, G. 2019. Sodium/Hydrogen exchanger 1 participates in early brain injury after subarachnoid hemorrhage both in vivo and in vitro via promoting neuronal apoptosis. Cell Transplant . https://doi.org/10.1177/0963689719834873. 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Dello Russo C, Cappoli N, Coletta I, Mezzogori D, Paciello F, Pozzoli G, Navarra P, Battaglia A. 2018. The human microglial HMC3 cell line: where do we stand? A systematic literature review. J Neuroinflammation . https://doi.org/10.1186/s12974-018-1288-0. Janabi N, Peudenier S, Heron B, Ng KH, Tardieu M. 1995. Establishment of human microglial cell lines after transfection of primary cultures of embryonic microglial cells with the SV40 large T antigen. Neurosci Lett . https://doi.org/10.1016/0304-3940(94)11792-h. Obinata H, Hla T. 2019. Sphingosine 1-phosphate and inflammation. Int Immunol . https://doi.org/10.1093/intimm/dxz037. Heo JY, Im DS. 2019. Pro-Inflammatory Role of S1P(3) in Macrophages. Biomol Ther (Seoul) . https://doi.org/10.4062/biomolther.2018.215. Dusaban SS, Chun J, Rosen H, Purcell NH, Brown JH. 2017. Sphingosine 1-phosphate receptor 3 and RhoA signaling mediate inflammatory gene expression in astrocytes. J Neuroinflammation. https://doi.org/10.1186/s12974-017-0882-x. Rass V, Helbok R. 2019. Early Brain Injury After Poor-Grade Subarachnoid Hemorrhage. Curr Neurol Neurosci Rep . https://doi.org/10.1007/s11910-019-0990-3. Zhang L, Wang H. 2020. FTY720 in CNS injuries: Molecular mechanisms and therapeutic potential. Brain Res Bull . https://doi.org/10.1016/j.brainresbull.2020.08.013. Miron VE, Schubart A, Antel JP. 2008. Central nervous system-directed effects of FTY720 (fingolimod). J Neurol Sci . https://doi.org/10.1016/j.jns.2008.06.031. Fu Y, Hao J, Zhang N, Ren L, Sun N, Li YJ, Yan Y, Huang D, Yu C, Shi FD. 2014. Fingolimod for the treatment of intracerebral hemorrhage: a 2-arm proof-of-concept study. JAMA Neurol . https://doi.org/10.1001/jamaneurol.2014.1065. Zhu Z, Fu Y, Tian D, Sun N, Han W, Chang G, Dong Y, Xu X, Liu Q, Huang D, Shi FD. 2015. Combination of the Immune Modulator Fingolimod With Alteplase in Acute Ischemic Stroke: A Pilot Trial. Circulation . https://doi.org/10.1161/CIRCULATIONAHA.115.016371. de Oliveira Manoel AL, Macdonald RL. 2018. Neuroinflammation as a Target for Intervention in Subarachnoid Hemorrhage. Front Neurol . https://doi.org/10.3389/fneur.2018.00292. Roa JA, Sarkar D, Zanaty M, Ishii D, Lu Y, Karandikar NJ, Hasan DM, Ortega SB, Samaniego EA. 2020. Preliminary results in the analysis of the immune response after aneurysmal subarachnoid hemorrhage. Sci Rep . https://doi.org/10.1038/s41598-020-68861-y. Pradilla G, Chaichana KL, Hoang S, Huang J, Tamargo RJ. 2010. Inflammation and cerebral vasospasm after subarachnoid hemorrhage. Neurosurg Clin N Am . https://doi.org/10.1016/j.nec.2009.10.008. Zhao S, Adebiyi MG, Zhang Y, Couturier JP, Fan X, Zhang H, Kellems RE, Lewis DE, Xia Y. 2018. Sphingosine-1-phosphate receptor 1 mediates elevated IL-6 signaling to promote chronic inflammation and multitissue damage in sickle cell disease. FASEB J . https://doi.org/10.1096/fj.201600788RR. Cianciulli A, Porro C, Calvello R, Trotta T, Lofrumento DD, Panaro MA. 2020. Microglia Mediated Neuroinflammation: Focus on PI3K Modulation. Biomolecules . https://doi.org/10.3390/biom10010137. Li Q, Li Y, Lei C, Tan Y, Yi G. 2021. Sphingosine-1-phosphate receptor 3 signaling. Clin Chim Acta . https://doi.org/10.1016/j.cca.2021.03.025. Additional Declarations No competing interests reported. Supplementary Files Onlineresource.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4374501","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":301534870,"identity":"94111070-7355-49c2-acba-178e6ba3bfe4","order_by":0,"name":"Lu Feng","email":"","orcid":"","institution":"Taizhou Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Feng","suffix":""},{"id":301534871,"identity":"aeae6e59-eeb7-4ca5-9293-47aa1fbb01ba","order_by":1,"name":"Panxing Wu","email":"","orcid":"","institution":"Taizhou Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Panxing","middleName":"","lastName":"Wu","suffix":""},{"id":301534872,"identity":"867f6292-d569-433b-b3ea-5217d1213729","order_by":2,"name":"Chao Ding","email":"","orcid":"","institution":"Taizhou Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Ding","suffix":""},{"id":301534873,"identity":"e92ddf3f-ac23-480c-beb8-e56599044fb1","order_by":3,"name":"Xiuyou Yan","email":"","orcid":"","institution":"Taizhou Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiuyou","middleName":"","lastName":"Yan","suffix":""},{"id":301534874,"identity":"61c77449-85c8-4984-9c93-6597b6c8e24a","order_by":4,"name":"Xuanhao Zhu","email":"","orcid":"","institution":"Taizhou Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xuanhao","middleName":"","lastName":"Zhu","suffix":""},{"id":301534875,"identity":"dcc41412-b46b-439c-9041-00967f55de52","order_by":5,"name":"Ming Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYFCCA2xAwkKOjb39AElaJIz5eM4kEG0NWEviPAkHA+LUyzcev/aYN0civU2CIYHhR8U2wloMDpwpN5y5TSK3TbrxAGPPmdtEaGE4kybxEaRF5kACM2MbEVrkG4BaErdJpLNJJBgQp4XhwPFjIFsSiNcC9AubJNAvhm3AQD5IlF/kZxx/Js27zUZevr394IMfFcQ4TOIMIjoOEKEeCPjbHxCncBSMglEwCkYuAAC3fD0SepuqFAAAAABJRU5ErkJggg==","orcid":"","institution":"The first people's Hospital of Xiaoshan District of Hangzhou City","correspondingAuthor":true,"prefix":"","firstName":"Ming","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2024-05-06 06:31:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4374501/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4374501/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56542989,"identity":"5bc9a806-a56c-4794-a91f-b5e623e8b117","added_by":"auto","created_at":"2024-05-15 14:34:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":340672,"visible":true,"origin":"","legend":"\u003cp\u003eS1P receptor inhibitor FTY720 ameliorates behavior scores in a rat SAH model. Neurological scores at 24 hours (a) or 72 hours (b) after SAH was indicated. c, brain water contents at 72 hours after SAH. ****, p\u0026lt;0.0001, ***, p\u0026lt;0.001, **, p\u0026lt;0.01. d, morphological overview of rat brains at the time of sacrificed. Hemorrhagic spots were indicated by red arrows. Scale bar, 1cm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4374501/v1/3fa10eb5cc5124e10301fd0c.png"},{"id":56542991,"identity":"f65927b8-ccce-4154-ab4a-2bd980d3486c","added_by":"auto","created_at":"2024-05-15 14:34:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":473163,"visible":true,"origin":"","legend":"\u003cp\u003eMicroglia activation in specific brain regions at 72 hours after SAH. Immunohistochemical staining of IBA1 at the Cortex adjacent to the Perforated Site (CAPS), the Motor cortex (M1 cortex), and hippocampus of rat brains was performed and results were shown ina. Scale bar, 20μm. Statistical summary of IBA1 positive cells in each field at the magnification of 400× was shown in b. ****, p\u0026lt;0.0001, ***, p\u0026lt;0.001, **, p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4374501/v1/04869479ed3232ed92118780.png"},{"id":56542993,"identity":"877969d7-625f-4d88-bfe0-d34eefc10274","added_by":"auto","created_at":"2024-05-15 14:34:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":137896,"visible":true,"origin":"","legend":"\u003cp\u003eImmunological phenotypes of HMC3 treated with S1P or IFN-γ. a, Expression of surface markers of HMC3 cells after a 6-hour treatment period as indicated. Normal saline was used as negative control. **, p\u0026lt;0.01 versus control. ##, p\u0026lt;0.01 and #, p\u0026lt;0.05 versus the S1P (10nM) group, ττττ, p\u0026lt;0.0001 versus the S1P (50nM) group. b, QRT-PCR analysis of gene expression of IL-6, TNF-α and TGF-β of HMC3 cells with indicated treatment. ****, p\u0026lt;0.0001, ***, p\u0026lt;0.001, **, p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4374501/v1/86e21278bee362989c80193a.png"},{"id":56543902,"identity":"2804ebaf-ba4e-40b2-aca7-85448d6d519f","added_by":"auto","created_at":"2024-05-15 14:42:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":93100,"visible":true,"origin":"","legend":"\u003cp\u003eS1P may induce HMC3 activation via S1PR3-G\u003csub\u003eα12/13\u003c/sub\u003e-RhoA/ROCK pathway. a, S1P induces HMC3 activation indicated by COX2 expression at a dose-dependent manner; b, S1P induces HMC3 activation through S1PR3, but not S1PR2 and S1PR3. c, S1P did not activate COX2 expression in the presence of either C3 exoenzyme (RhoA inhibitor, 0.5μg/ml), Y27632 (ROCK inhibitor, 2.5μM), or G\u003csub\u003eα12/13\u003c/sub\u003e targeting shRNA.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4374501/v1/91c57d70060bd982c40e8680.png"},{"id":57041656,"identity":"a64ffe3b-f516-4356-9946-2c2d3e152ace","added_by":"auto","created_at":"2024-05-23 21:46:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1308635,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4374501/v1/0191da95-e9ae-4931-bcc5-4d28156f0a42.pdf"},{"id":56542992,"identity":"0d6ae3f6-0b78-4a92-8efb-fd77dc11e3bc","added_by":"auto","created_at":"2024-05-15 14:34:55","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":299556,"visible":true,"origin":"","legend":"","description":"","filename":"Onlineresource.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4374501/v1/6e62c32b044865fb9b0265f0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"S1PR3/RhoA signaling pathway in microglia mediates inflammatory activation in early brain injury after subarachnoid hemorrhage","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSpontaneous subarachnoid hemorrhage (SAH) is the third leading cause of stroke, with high mortality and disability rate[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The pathogenesis of SAH is complex, of which 85% are caused by aneurysm rupture, called aneurysmal subarachnoid hemorrhage (aSAH) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Clinically, about a quarter of patients with SAH die before receiving medical care, 33% die within 48h after the initial hemorrhage, and 50% die within 30 days [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Patients who survive early massive hemorrhage still suffer from a series of complications, including increased intracranial pressure and delayed cerebral ischemia (DCI). Historically, many studies mainly focused on the targeted treatment of cerebral vasospasm (CVS) after SAH, but the clinical outcomes have not been improved, and the incidence and mortality of DCI have not been reduced [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Recently, researchers have found that early brain injury (EBI) induced by SAH may be a key factor affecting patient outcome. EBI usually occurs within 72 hours before SAH and involves a series of pathophysiological events, such as brain edema, blood-brain barrier (BBB) destruction, oxidative stress, inflammation, excitotoxicity, and impaired ion homeostasis. Among them, the activation and amplification of harmful inflammatory response play a key role in nerve injury after SAH [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, how EBI occurs and how it causes delayed cerebral ischemia and neurological damage, the underlying mechanisms are still unclear.\u003c/p\u003e \u003cp\u003eMicroglia, the resident macrophage in the central nervous system (CNS), plays an important role in the neuroinflammatory response induced by SAH[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Upon receiving risk-associated molecular pattern signals (DAMPs), microglia can rapidly activate, proliferate, and migrate to the site of injury, whereby they phagocytose cellular debris and harmful substances, and secrete various inflammatory cytokines and chemokines. Activated microglia also produce some anti-inflammatory cytokines and trophic factors to promote neuronal regeneration. Therefore, microglia can be classified into deleterious M1 subpopulation and beneficial M2 subpopulation according to their distinguishable phenotype. Usually, M1 microglia expressing MHCII, CD80 and CD11b secretes proinflammatory cytokines (including TNF-α, IL-1β, IL-6, COX-2, ROS and NO). In contrast, M2 microglia, which is positive for CD163 and CD206, mainly produce IL-10 and TGF-β, while phagocytosing cell debris and harmful substances. Early DAMPs signaling induce some microglia to polarize to an M2 phenotype; however, persistent brain injury may gradually replace these reparative M2 cells with a toxic M1 phenotype, resulting in prolonged inflammatory responses and subsequent impairment of neuronal regeneration[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Thus, the functional imbalance of activated microglia leading to an intensified immune response is one of the potential mechanisms of EBI.\u003c/p\u003e \u003cp\u003eSphingosine-1-phosphate (S1P) belongs to a class of lipids called sphingolipids [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and its receptors are widely distributed in almost all tissues and organs of the human body. Studies have shown that S1P is widely involved in the regulation of vasculogenesis, angiogenesis, immune regulation, nerve cell differentiation, blood-brain barrier integrity construction and other processes by interacting with its receptor S1PRs (S1PR1-5) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Its dysfunction may lead to autoimmune diseases, chronic inflammation, tumors and neurodegenerative diseases [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In 2010, the FDA approved Fingolimod (FTY720), an S1P receptor 1 (S1PR1) inhibitor, for the treatment of multiple sclerosis, an autoimmune disease, which has triggered extensive attention to the S1P signaling pathway among researchers [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In addition to S1PR1, the drug can also selectively inhibit the activation of S1PR3 and S1PR5 receptor signaling pathways, reduce the number of peripheral circulating lymphocytes, and significantly prolong the survival of experimental animal transplanted organs without impairing the immune response and immune memory function to pathogens, with low toxic side effects. A series of clinical studies support its use as a good immunosuppressive agent [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In recent years, increasing evidence has shown that the expression levels of S1P and its receptors undergo pathological changes in diseases related to CNS injury [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], suggesting that S1P pathway may also have the ability to regulate neuroinflammation after CNS injury. However, the detail mechanism of S1P-mediated neuroinflammation is still poorly understood. Therefore, further studies will help to clarify the mechanism of S1P in CNS injury-related diseases such as SAH, and provide theoretical basis for the application of immunomodulators targeting the S1P signaling pathway in CNS injury-related diseases.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e1. Cells and reagents\u003c/p\u003e\n\u003cp\u003eHMC3 cell line (CRL-3304) were obtained from ATCC. Twelve-week-old rats were purchased from Charles River. FTY720 (CAS NO. 162359-56-0) were purchased from AdooQ Bioscience, IFN-γ\u0026nbsp;was purchased from Peprotech (300-02), and S1P (S45337) from Shanghai yuanye Bio-Technology Co., Ltd.. C3 exoenzyme (Amylet Scientific) was used to block RhoA activation, and Y27632 (Sigma-Aldrich) was used to inhibit ROCK activation.\u003c/p\u003e\n\u003cp\u003e2. Cell culture and treatment\u003c/p\u003e\n\u003cp\u003eHMC3 cells were maintained in RPMI1640 medium, containing 10% heat inactivated fetal bovine serum, 100 U/ml penicillin, 50U/ml streptomycin, 2mM glutamine, and 1mM sodium pyruvate at 37℃\u0026nbsp;in a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified incubator. At a confluence of 70%, HMC3 cells were trypsinized and plated in 6 well plates. 48 hours later, IFN-γ\u0026nbsp;or S1P was added to the culture medium at indicated concentrations. Cells were harvested for further analysis after a 6-hour treatment period.\u003c/p\u003e\n\u003cp\u003e3. siRNA vector construction and transfection\u003c/p\u003e\n\u003cp\u003ePre-designed shRNA oligos targeting S1P1, S1P2, S1P3 and G\u003csub\u003eα12/13\u003c/sub\u003e were customized by Beijing Tsingke Biotech Co., Ltd (Online Resource Table S1). shRNA oligos were inserted into the FV055 vector containing the AmpR, GFP and puromycin sequences, then positive clones were selected and validated by sequencing. HMC3 cells were plated in 6 well plate, serum-starved for 18–24 h and transfected with plasmids expressing si-S1P1, si-S1P2, si-S1P3 and si-G\u003csub\u003eα12/13\u003c/sub\u003e, respectively. Cells were collected for gene expression assay using QRT-PCR and western blotting.\u003c/p\u003e\n\u003cp\u003e4. QRT-PCR\u003c/p\u003e\n\u003cp\u003eTotal mRNA of HMC3 cells was extracted using the RNeasy kit (Invitrogen) and the complement strand of DNA (cDNA) was synthesized using PrimeScript™ RT reagent Kit from Takara. Gene expression was determined by Real-time PCR using the TB Green® Premix Ex Taq™ II FAST qPCR kit. Primer sequences are shown in the Online Resource Table S2. Data were normalized to internal\u0026nbsp;β-Actin, and fold change was determined as described previously[15] . Values for comparison for a single gene across multiple samples was determined using cycle threshold (Ct) data fitted to a standard curve. For comparison of multiple transcripts in a single sample, then the 2−ΔΔCt method was applied to the Ct value.\u003c/p\u003e\n\u003cp\u003e5. Western blot\u003c/p\u003e\n\u003cp\u003eHMC3 cell samples were lysed with RIPA buffer (20 mm Tris, 250 mm NaCl, 3 mm EDTA, 3 mm EGTA, and 20 mm βglycerophosphate) supplemented with sodium vanadate, leupeptin, aprotinin, p-nitrophenyl phosphate, and phenyl methylsulfonyl fluoride. Protein concentration of samples were measured using Bradford Protein Assay kit (Beyotime). Equal amounts of protein (20 μg) were loaded onto 4–12% 10-well or 15-well SDS-PAGE gels. Gels were transferred to PVDF membranes, and the resulting blot was probed with specific antibodies. The COX-2 antibody (Affinity#AF7003) was used at 1:500 dilution, the\u0026nbsp;β-actin antibody (Cell Signaling Technology #4970) was used at 1:1000 dilution. Rabbit secondary antibody was used at 1:4000 dilution. Fold changes were determined by chemiluminescence and normalized to\u0026nbsp;β-actin.\u003c/p\u003e\n\u003cp\u003e6. Flow cytometry\u003c/p\u003e\n\u003cp\u003eHMC3 cells were harvested immediately after S1P or IFN-γ\u0026nbsp;treatment, and stained with FITC-conjugated CD11b antibody (Biolegend#301329), or CD80 antibody (Biolegend#375405)，or CD163 antibody (Biolegend#333617), or CD206 antibody (Biolegend#321103), or MHC-II antibody (Invitrogen#17-9956-42），or corresponding IgG isotypes controls without PMA fixation. The gates were established by fluorescence minus IgG isotype controls.\u003c/p\u003e\n\u003cp\u003e7. SAH model and treatment\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the ethics committee of Taizhou University, performed in accordance with the Guide for the Care and Use of Laboratory Animals by the National Institute of Health, China. SAH was performed by using an artery puncture method according to a previous study[16]. In brief, 12-week-old male rats were anesthetized with sodium pentobarbital (80 mg/kg body weight). The left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were exposed. A sharpened 4-0 monofilament nylon suture was advanced into ICA from ECA to perforate the artery at the bifurcation of the anterior and middle cerebral artery. And then the suture was immediately withdrawn to cause SAH. The procedure in the sham group was similar without the perforation.\u003c/p\u003e\n\u003cp\u003eRats were randomly divided into 5 groups, n=5 in each group. A: sham; B: SAH + vehicle ; C: SAH + LD (0.5 mg/kg FTY720); D: SAH +HD (5 mg/kg FTY720); E: SAH + HD24 (5 mg/kg FTY720 delayed intervention). FTY720 was formulated with physiological saline at a concentration of 0.15 mg/ml or 1.5 mg/ml. Then the rats in group C, D were given with FTY720 or vehicle (physiological saline) by intraperitoneal administration at 2 h after SAH injury. The FTY720 dose was determined according to previous studies[17]. Rats in group B received intraperitoneal administration with an equal volume of physiological saline at 2 h after SAH. Rats in group E received intraperitoneal administration with 5mg/kg FTY720 at 24 h after SAH.\u003c/p\u003e\n\u003cp\u003e8. Assessment of neurological score\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNeurological score was assessed at 24 h and 72 h after SAH according to previous studies by a blinded investigator[18]. In brief, animals’ neurological functions were evaluated by six tests, including symmetry\u0026nbsp;in the movement of all limbs (0-3), spontaneous activity (0-3), forepaw outstretching (0-3), climbing (1-3), response to vibrissae touch (1-3) and body proprioception (1-3). The minimum neurological score was 3 (severe impairment) and the maximum was 18 (no neurological impairment).\u003c/p\u003e\n\u003cp\u003e9. Brain water content\u003c/p\u003e\n\u003cp\u003eAfter the assessment of neurological score, rats were sacrificed by cervical dislocation. The brains were removed and weighed immediately to obtain the wet weight. The brain was then dried in an oven at 100°C for 72 h and weighed again to obtain the dry weight. The percentage of brain water content was calculated according to the formula: [(wet weight-dry weight)/wet weight] × 100%.\u003c/p\u003e\n\u003cp\u003e10. Immunohistochemical staining\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe brain tissues were fixed in 4% paraformaldehyde and embedded in paraffin before being cut into 5-μm thick sections. Cortex adjacent to the Perforated Site (CAPS), the motor cortex (M1 cortex), and hippocampus were selected for IBA-1 staining as previously described[19]. In brief, after a xylene/ethanol dewax-rehydration series, endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide. Brain sections were then incubated for 1 h with blocking buffer comprising 2.5% goat serum, 1% bovine serum albumin (BSA), and 0.1% Triton-100. The primary antibody of IBA-1 (Thermo Fisher#\u0026nbsp;PA5-21274) was applied subsequently at 4 °C overnight. Horseradish peroxidase (HRP) conjugated secondary antibody was applied for 1 h at room temperature. Diaminobenzidine (DAB) was utilized for visualization of colorimetric reaction. Three random fields were examined on each brain area respectively of each animal under microscope (Leica) at × 40 magnification.\u003c/p\u003e\n\u003cp\u003e11. Statistical analysis\u003c/p\u003e\n\u003cp\u003eIBM SPSS 23.0 software was used for statistical analysis of the data. All data were expressed as mean ± standard deviation (SD). Differences between two experimental groups were compared by the Student t-test. P\u0026lt;0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTo test whether S1P blockage can ameliorate SAH-induced EBI, a rat SAH model was established using arterial puncture method. Compared with the sham-operated group, the SAH experimental group showed significant neurological deficits. Specifically, motor dysfunction in limb movement symmetry, forepaw outstretching, and climbing within 24 hours occurs immediately after SAH (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Motor dysfunction induced by SAH worsened after 72 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). A significant increase in brain water content, without alteration of the structural integrity of the brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) or substantial change in body weight (Online Resource Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), was observed at 72 h after SAH in the experimental group, accompanied by an increase in the number of IBA1-positive microglia in the cortex adjacent to the perforated site (CAPS), hippocampus, and motor cortex, with a more amoeboid than bifurcating morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These are direct evidence of early brain damage caused by the puncture-triggered arterial hemorrhage which spreads in the subarachnoid space and causes an acute inflammatory response. At 24 hours after surgery, all SAH groups, either with or without treatment, did not show significant difference for neurological scores. However, at 72 hours after SAH, in the groups that received 0.5mg/kg or 5mg/kg of FTY720 or the same volume of saline after surgery, the rats showed different responses in neurological scores. Compared with the control group (saline), 5mg/kg FTY720 administered immediately after surgery significantly alleviated neurological deficits after surgery, whereas 0.5mg/kg FTY720 administered immediately after surgery did not significantly improve neurological scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Delayed administration of 5mg/kg FTY720 at 24 hours after surgery (before neurological scoring) did improve the neurological outcomes but the effect was modest compared with the immediate-treated group. Moreover, 5mg/kg of FTY720 immediately after surgery was the only group that achieved prolonged alleviation of neurological deficits within 72 hours after SAH. Likewise, brain edema was significantly alleviated in the 5mg/kg FTY720 immediate-treated group at 72 hours after surgery, but not in the other two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). These results confirmed that S1P receptor inhibitors could significantly improve the nerve injury and inhibit the inflammatory response caused by EBI after SAH. The blockage effects of S1P-dependent events are most likely to be maximized within 24 hours after SAH, and to be dose-dependent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNeurological evaluation by six behavior tests for rats with indicated operation and treatments.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eSham\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eSAH\u0026thinsp;+\u0026thinsp;vehicle\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eSAH\u0026thinsp;+\u0026thinsp;LD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eSAH\u0026thinsp;+\u0026thinsp;HD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003eSAH\u0026thinsp;+\u0026thinsp;HD24\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime point\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24 hr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72 hr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24 hr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e72 hr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24 hr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e72 hr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e24 hr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e72 hr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e24 hr\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e72 hr\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeurological scores\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e11.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e13.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e10.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSymmetry in the movement of all limbs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpontaneous activity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eForepaw outstretching\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClimbing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReaction to touch\u003c/p\u003e \u003cp\u003eon either side of\u003c/p\u003e \u003cp\u003etrunk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResponse to vibrissae touch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMicroglia play a pivotal role in the immune response after SAH. Activation of microglia, indicated by the IBA1 staining, is supposed to initiate at the CAPS, then spread to the distal motor cortex and to the hippocampus region deep in the brain. We showed that administration of FTY720 at 24 hours after SAH seemed to block only the remote activation of microglia while microglia located in the CAPS remained activated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). However, immediate-treatment of FTY720 globally inhibited the microglia activation, suggesting that FTY720 may only inhibit the early activation, but not reverse the activated status of microglia.\u003c/p\u003e \u003cp\u003eTo further explore the mechanism by which S1P regulates neuroinflammation, we first selected an immortalized human microglia cell line, HMC3, for in vitro studies. Similar to primary microglia, HMC3 can be induced and activated by IFN-γ to secrete inflammatory factors such as IL-6 and TGFβ, and upregulate M1 markers such as MHCII, CD80 and CD11b. If cultured in the presence of neuronal precursor cells or astrocytes, the expression of M2 markers (including CD163, CD206, etc.) is up-regulated [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We found that S1P activates HMC3 cells in a dose-dependent manner. At a concentration above 50nM, S1P induces induced an IFN-γ-induced phenotype in HMC3 and a marker of M1 polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Online Resource Fig. S2). In contrast, S1P had no effect on the expression of several CD163 and CD206 associated with M2 polarization. HMC3 is derived from embryonic microglia, and the level of secreted TNF-α is very low even in the activated state, which is not easy to detect [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In contrast, the mRNA expression of several major inflammatory factors, including IL-6, TNF-α, and TGF-β1, was significantly upregulated in the activated state after receiving IFN-γ. Our results showed that S1P (50nM) induced an up-regulation of IL-6 and TNF-α expression at the mRNA level, but did not change the expression of anti-inflammatory factor TGF-β1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In addition, expression of the inflammation-related expression product cyclooxygenase-2 (COX2) was significantly increased after S1P induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). All these results suggest that S1P induces HMC3 activation and M1 polarization, but not M2 polarization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eS1P activates downstream signals through its receptors, including S1PR1-S1PR5. Among them, S1PR1-3 were the most studied. To further elucidate the mechanism by which S1P activates HMC3, siRNA vectors targeting \u003cem\u003eS1pr1\u003c/em\u003e, \u003cem\u003eS1pr2\u003c/em\u003e and \u003cem\u003eS1pr3\u003c/em\u003e were constructed. The results of QRT-PCR showed that the mRNA of \u003cem\u003eS1pr1\u003c/em\u003e, \u003cem\u003eS1pr2\u003c/em\u003e and \u003cem\u003eS1pr3\u003c/em\u003e were all detected in HMC3 cells. After 48 hours of transfection with si-S1P1, si-S1P2 or si-S1P3 vectors, the mRNA expression of \u003cem\u003eS1pr1\u003c/em\u003e, \u003cem\u003eS1pr2\u003c/em\u003e and \u003cem\u003eS1pr3\u003c/em\u003e in HMC3 cells was down-regulated by 62%, 63% and 58%, respectively (Online Resource Fig S3). Meanwhile, western blot showed that the expression of COX2 was still up-regulated after S1P induction in HMC3 cells transfected with si-S1P1 and si-S1P2. In contrast, the expression level of COX2 in HMC3 cells transfected with si-S1P3 was not responsive to S1P induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Thus, S1P acts primarily through S1PR3, but not S1PR1 and S1PR2, in HMC3 cells.\u003c/p\u003e \u003cp\u003eWhen bound by S1P ligands, S1P receptors transmit signals downstream by activating their corresponding G-coupled protein receptors, where S1PR1 only couples G\u003csub\u003ei/o\u003c/sub\u003e, whereas S1PR2 and S1PR3 couple G\u003csub\u003ei/o\u003c/sub\u003e, G\u003csub\u003eq\u003c/sub\u003e or G\u003csub\u003e12/13\u003c/sub\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. It has been reported that S1PR3 can activate downstream inflammatory signals and play a role in microglia-mediated inflammatory response [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], while S1P activates inflammatory astrocytes mainly through the S1PR3-G\u003csub\u003e12/13\u003c/sub\u003e-RhoA pathway [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. To further demonstrate whether S1P activates microglia via G\u003csub\u003e12/13\u003c/sub\u003e-RhoA, we used selective inhibitors of RhoA/ROCK or knockdown of Gα\u003csub\u003e12/13\u003c/sub\u003e. The S1P-induced increase in COX2 expression was significantly inhibited by the addition of FTY720, the RhoA inhibitor C3, the ROCK inhibitor (Y27632) to the culture medium, or by transfection of HMC3 with siRNA targeting G\u003csub\u003eα12/13\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). It should be noted that expression of G\u003csub\u003eα12\u003c/sub\u003e and G\u003csub\u003eα13\u003c/sub\u003e in HMC3 cells was reduced by 72% and 64% after transfection with vectors expressing si-GNA12/13 mixture, respectively (Online Resource Fig. S3). These results suggest that S1P can activate the pro-inflammatory phenotype of HMC3 by activating the S1PR3-G\u003csub\u003e12/13\u003c/sub\u003e-RhoA/ROCK signaling pathway.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThere is increasing evidence that neuroinflammation occurs rapidly after SAH and is an important factor in causing EBI [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. S1P regulates a variety of cell functions through its receptors S1PR1-5 and is considered to be involved in the regulation of inflammatory process [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. FTY720 is the first approved S1P receptor inhibitor. Reduction in circulating lymphocytes and limiting inflammatory cell migration into the CNS are considered to be its two most important pharmacological mechanisms [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, unlike other immunosuppressants, FTY720 is lipophilic and therefore easily penetrates the blood-brain barrier, acting at the site of CNS injury through sphingosine kinase activation secreted by neuronal cells. Several preclinical and clinical studies have demonstrated the inhibitory effect of FTY720 on neuroinflammation caused by CNS injury [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Our findings are consistent with literature reports that FTY720 plays a protective role in the CNS injury caused by SAH. Meanwhile, our evidence further strengthens the notion that S1P signaling is an important molecular mechanism responsible for CNS neuroinflammation.\u003c/p\u003e \u003cp\u003eHowever, several issues still require further discussion. First of all, the inflammatory response after SAH is complex and seems to get involved in both the acute phase and the post-CVS period[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Aneurysm rupture causes blood leakage throughout the subarachnoid space, followed by a sharp increase in intracranial pressure (ICP), a decrease in perfusion pressure (CPP), transient ischemia of brain tissue and death of nerve cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. At this time, the blood-brain barrier rapidly breaks down, causing massive inflammatory cells (mainly neutrophils and macrophages) infiltration and microglia activation. After an acute response period, these inflammatory cells may remain trapped at the site of injury, undergo apoptosis, and cause a secondary and chronic inflammatory response [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Our study provides evidence that FTY720 directly inhibits the initial microglial activation within 24 hours after SAH, but prolonged observation of FTY720-treated SAH rats was absent due to the limited experiment animals in this study. Further studies are needed to elucidate whether and how the S1P pathway is involved in subsequent events of this complex process, including amplification of the inflammatory cascade and later neural repair. Therefore, the therapeutic strategy targeting S1P pathway still faces some challenges in CNS injury, including how to choose the intervention window, to optimize administration dose and frequency, and to develop personalized dosing regimens according to specific conditions of CNS injury.\u003c/p\u003e \u003cp\u003eS1PR3 is widely expressed in heart, lung, kidney, spleen and other organs, and is involved in the maintenance of endothelial barrier, vascular tone and immune response. Different from S1PR1, which mainly functions through JAK2 and PI3K pathways [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], S1PR3 can extensively affect pathways including G\u003csub\u003eα12/13\u003c/sub\u003e-RhoA-ROCK, VEGF, PI3K, STAT3 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Among them, the G\u003csub\u003eα12/13\u003c/sub\u003e-RhoA-ROCK pathway is considered to be closely related to the migration and M1 polarization of macrophages. In mouse peritoneal macrophages, the expression level of S1PR3 is lower than that of S1PR1 and S1PR2. However, LPS stimulation can up-regulate the expression of S1PR3 and stimulate S1PR3-mediated inflammatory activation, while the expression of S1PR1 is down-regulated [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Consistent with this, our in vitro studies showed that microglial activation in the CNS was dependent on S1PR3 but not S1PR1. However, this conclusion cannot be simply translated to in vivo studies because FTY720 is a broad-spectrum S1PR inhibitor with known substrates including S1PR1, S1PR3, and S1PR5 among others [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The widespread expression of S1PR is related to several adverse reactions such as slow heart rate and hypertension caused by FTY720 in clinical studies [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The pathologic consequences of systemic inhibition of the S1PR3 pathway are unclear. Therefore, detailed studies on different types of S1PR are necessary to develop more specific S1PR receptor inhibitors and optimize the benefit-risk balance of S1P pathway-based therapies.\u003c/p\u003e \u003cp\u003eIn summary, our study highlights the role of the S1P pathway in the inflammatory response after SAH, deepening our understanding of the complex inflammatory response process after SAH. In addition, our study also suggests that therapeutic strategies targeting S1P may play a protective role in attenuating CNS injury after SAH.\u003c/p\u003e"},{"header":"Statements and Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments in this study was approved by the Ethics Committee of Taizhou University.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was supported by Taizhou social development science and technology Program project (Grant No. 21ywa31).\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eClaassen J, Park, S. 2022. 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Sphingosine-1-phosphate receptor 3 signaling. \u003cem\u003eClin Chim Acta\u003c/em\u003e. https://doi.org/10.1016/j.cca.2021.03.025.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Subarachnoid hemorrhage, Sphingosine 1-phosphate, S1P receptor inhibitor, Microglia, Neuroinflammation","lastPublishedDoi":"10.21203/rs.3.rs-4374501/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4374501/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSpontaneous subarachnoid hemorrhage (SAH) is a serious and common cerebrovascular disease with high mortality rate and poor prognosis. The immune response caused by abnormal activation of intracranial microglia is one of the main factors contributing to early brain injury after SAH. Sphingosine 1-phosphate (S1P) signaling pathway is widely involved in immune regulation, nerve cell differentiation and other processes. It has been reported that S1P expression is increased in cerebrospinal fluid after SAH, but its role in early brain injury and neuroinflammation induced by SAH remains unclear. In the rat model of SAH established by arterial puncture, low (0.5mg/kg) or high dose (5mg/kg) of the S1P receptor inhibitor FTY720 was administered immediately or at 24 hours after surgery. Improvement of behavioral scores and brain edema symptoms after SAH was observed in immediate treatment group at high dose. In addition, activation of cortical microglia near the perforation site was observed after SAH, and this activation was significantly inhibited after 5mg/kg FTY720 treatment immediately after surgery. Further studies showed that S1P could induce activation and M1 polarization of human microglia cells in vitro. This activation may be mediated through the S1PR3-Gα\u003csub\u003e12/13\u003c/sub\u003e-RhoA pathway. Therefore, our study highlights the important role of S1P signaling and microglia activation in SAH-induced early brain injury, and provides evidence for novel therapies targeting the neuroinflammatory process after SAH.\u003c/p\u003e","manuscriptTitle":"S1PR3/RhoA signaling pathway in microglia mediates inflammatory activation in early brain injury after subarachnoid hemorrhage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-15 14:34:50","doi":"10.21203/rs.3.rs-4374501/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b7fbba1e-0402-45cf-8650-216f60180e80","owner":[],"postedDate":"May 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-12T16:38:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-15 14:34:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4374501","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4374501","identity":"rs-4374501","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}