cGAS-STING signaling regulates control of microglia polarization in cerebral ischemic stroke CURRENT STATUS: POSTED

Background Cerebral ischemic stroke is a highly debilitating disease, in which inflammation is well document to play a pivotal role in its pathophysiology. Microglia are the the major immuncompetent cells of the brain involved in different neuropathologies. Recent discovery of cyclic GMP-AMP synthase(cGAS) activation and its induction of the downstream signaling protein stimulator of interferon genes (STING) is increasingly recognized as a crucial determinant of neuropathophysiology. Although cGAS-STING pathway has been reported to play an important role in inflammatory response in myocardial infarction (MI), its mechanism in inflammatory response in ischemic stroke (IS) has remained to be fully explored. Methods In light of the above, this study sought to explore the roles of cGAS-STING pathway in inflammatory reaction in IS. It is hoped that the results would provide new insights for designing of therapeutic strategies targeting at IS. We used HT22 cells to establish an oxygen-glucose deprivation (OGD) cell model. The supernatant derived from this and which contained OGD-induced DAMPs(OIDs) was used to stimulate the BV2 microglia. Additionally, we used siRNA technology to interfere with cGAS gene expression to observe changes in downstream cytokines. Furthermore, we established middle cerebral artery occlusion (MCAO) mouse model and performed cGAS-siRNA lentivirus infection to further elucidate the mechanism of cGAS-STING pathway in vivo. Results We show here that OIDs strongly activated the cGAS-STING pathway and triggered accumulation of a plethora of proinflammatory factors in activated Microglia. Of note, the cascade reaction was successfully inhibited by cGAS-siRNA. Furthermore, we extended the study of cGAS-STING in a mouse MCAO model, which showed that inhibiting cGAS-STING pathway can effectively diminish MIDs(MCAO-induced DAMPs)-induced neuronal apoptosis and ultimately functional improvement. Conclusion The present results have shown GAS-STING signaling pathway controls the polarity transformation of microglia. The underlying mechanisms of cGAS-STING triggering microglial inflammatory response is now better clarified which made the pathway a potential therapeutic target of IS. of cGAS, IRF3, and IFNAR1 significantly improved early survival of animals in the MI model. Therefore, these findings suggest that components of cGAS–STING pathway represent promising targets for developing pharmacological intervention for MI and other pathway-related pathologies. We inhibited the expression of cGAS in microglia by siRNA technology and found that STING and p-IRF3 gene expression was inhibited. However, CD206-Arg1 labeled cell signaling was


Introduction
Ischemic stroke(IS) results from transient or permanent reduction in regional cerebral blood flow, and associated inflammation is recognized as a vital contributor to the pathophysiology [1]. IS causes necrosis of local cells and tissues in the brain, followed by accumulation of many substances called danger-associated molecular patterns(DAMPs) [2]. These DAMPs when combined with pattern recognition receptors (PRRs) result in production of inflammatory mediators [2]. Recent studies have identified different DAMPs and PRRs that may play a role in the inflammatory response after IS [3], but the downstream regulation of DAMPs-PRRs in activated microglial phenoxype polarity remains uncertain.
Microglia are the resident macrophages and acts as the first line of defense against injury in the central nervous system [4,5]. Activated microglia migrate swiftly towards the injury site and can aggravate strengthen tissue injury by producing an excess amount of inflammatory cytokines and cytotoxic substances after cerebral ischemic damage. However, activated microglia can exert dual functions, thus ,on the one hand, they participate tissue repair and remodeling by clearing up debris and producing anti-inflammatory cytokines and growth factors [6]; on the other hand, microglia activation and their polarization in phenotypes are sophisticated processes that can be regulated by a variety of cytokines and signal pathways. Activated microglia can generate a multitude of proinflammatory mediators including inducible nitric oxide synthase (iNOS, TNFa) [7] [8], and antiinflammatory cytokines (such as TGF-β) [9]. Microglia alter their cell polarity by regulating the expression level of these mediators to further exert their dual functions.
Cyclic GMP-AMP synthase (cGAS) has attracted the attention in recent years in search of proper therapeutic strategies to different neurological diseases. In this connection, discovery of cGAS as the mammalian cytosolic DNA sensor has promoted a better understanding of molecular mechanisms of neuroinflammation. To this end, cGAS-STING signal pathway has been described to play a crucial role in inflammatory response after MI [10,11]. It has been reported that cGAS can sense the MI-induced DAMPs as a novel PRRs in MI injury. Furthermore, cGAS governs macrophage polarity during the repair of damaged heart [10]. Very interesting, loss of function of cGAS promoted the transformation 4 of macrophage toward a reparative phenotype, and further inhibited pathological remodeling, promoted angiogenesis and early survival [10]. In other words, cGAS controlls the transition of macrophage polarity making it a potential target for MI treatment.
Exploring cGAS mechanism also had led to discovery of multifunctional and context-dependent role of cGAS-STING signaling in neurological diseases. Thus, crucial links between DNA damage and central nervous system (CNS) damages have been recognized, and DNA damage who found to activate the cGAS-STING pathway. It is noteworthy that cGAS mediates microglia-drived inflammation that triggered by genome instability and mislocalized DNA. On the one hand, cGAS can promote antineuroinflammation immunity; on the other hand, it acts as an important driving force of neuroinflammation. This bipolar roles of cGAS have brought out its great potential for the therapies of neuroinflammatory diseases. While there is the significant progress in cGAS-STING pathway making cGAS an exciting and potential therapeutic target in infection [12,13],immunology [14], and cancer [15], the dominant molecular mechanism of cGAS in IS-associated inflammation has remained largely unknown.
We speculate that the cGAS-STING pathway may play a similar role in post-IS inflammation as in post-MI inflammation. We surmised that regulation of cGAS-STING signaling and biased activation of downstream transcription factors may promote or prevent neuroinflammation. This study was aimed specifically to explore the role and mechanism of cGAS-STING in inflammatory process in CNS mediated by microglia through use of an OGD cell model and MCAO mouse model.

Cell and animal
HT22 cells were supplied by the cell bank of Chinese Academy of Sciences while BV2 microglia cells were maintained in our laboratory for regulatory use.C57BL/6 mice purchased from Charles River.
HT22 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100U/mL penicillin and 100μg/mL streptomycin, and cultured in a 37 ° & 5% CO 2 cell culture incubator. When the cells grow to 80-90% confluency, the medium was removed and rinsed in PBS for 1-2 times. After this PBS was removed, 1ml trypsin was added for 1-3min; 3ml of complete medium were added and trypsin to stop digestion. The cells were transferred to a 15 ml centrifuge tube and centrifuged at 1000 rpm for 5 min. After the supernatant was discarded, 3 ml of medium was added to resuspend the cells, and then subcultured at a ratio of 1: 3.
Establishment of OGD cell model 5×10 6 HT22 cells were inoculated into a 10 cm culture dish, and after normalizing overnight, the medium was removed. The culture medium was then added a non-sugar DMEM medium (37°C, 95% N 2 , 5 % CO 2 ). After 4 hours of hypoxia culture, the sugar-free medium was removed, and complete DMEM medium was added. The HT22 cells were cultured in a normoxic incubator for 24 hours. The HT22 cell culture supernatant which was not deficient in sugar hypoxia was collected as the control.
The culture supernatant was transferred to a 15ml centrifuge tube, centrifuged at 1000 rpm for 10min, and the supernatant was collected. After this , the OGD medium was divided into two portions, one of the supernatants was collected, and after centrifugation at 100,000 g for 3 hours, the supernatant was removed and the mtDNA precipitate was collected, and then the mitochondrial DNA(mtDNA) was separated according to the kit instructions (biovision,K280-50). 1×10 6 BV2 cells were seeded into 6-well plates, cultured overnight, and the medium was removed.
After washing once with PBS, 2 ml of HT22 OGD medium or HT22 normal culture medium was added to each well (2 ml of OGD medium-isolated mtDNA was added to the mtDNA group). After culture for 24 hours, the medium was collected, washed twice with 2 ml of PBS, then PBS was removed. Ripa protein lysate was added to extract protein or trypsin was used to digest the cells for staining for flow detection.

Lentivirus injection
Eight weeks old C57 mice were acclimated for one week, and intraperitoneally injected with 4% chloral hydrate (dosage of 1ml/100g). The anesthetized mice were fixed on a brain stereotactic 6 device, such that the incisor hook plane was lower than the external auditory canal (ear). The center of the rod tip is about 2.4mm; cut off the hair on the top of the mouse head, wipe the top of the head with iodine; use the scalpel to cut the cranial skin and fascia along the midline of the skull, and wipe the skull periosteum with 3% hydrogen peroxide. Before and after exposure, adjust the incisor hook plane so that the anterior and posterior iliac crest are on the same level; before the Bregma point is the origin, the si-cGAS lentivirus is injected into the right ventricle of the mouse (center line right 0.9mm) ; 0.1mm before the front; 3.1mm deep from the front). The micro-syringe was pipetted with si-cGAS lentivirus. First, si-cGAS lentivirus was injected at the coordinate site, the injection dose was 2ul (1*109/ml), the injection speed was 0.5ul/min, the needle was left for 5min after the injection, and then the needle was slowly withdrawn. The previous point is the origin, and the second injection site is repositioned. After the last withdrawal, the blood is stopped, disinfected, and the needle is sutured.
The mouse is placed in the cage, and the temperature is kept warm.

MCAO mouse model
MCAO was performed 1 week after brain stereotaxic injection. 10% chloral hydrate was intraperitoneally injected at a dose of 400 mg/kg. After anesthesia for 3-5 minutes, the neck was sheared, iodophor was prepared, and the oral secretions were wiped with a sterile dry cotton ball to prevent dyspnea during surgery. A mid-line incision was made at the neck skin and muscles retracted in turn. The right common carotid artery was exposed till its bifurcation into the internal and the external carotid arteries bifurcation. The common carotid artery was separated and clamped. The external and external carotid arteries were pulled out with 4-0 sutures followed by ligation of the external carotid artery. A small opening was cut in the external carotid artery. A pre-prepared wire plug was inserted into the internal carotid artery distally with the length of about 9.5±0.05mm. After 1 h of ischemia, the plug was pulled out and the external carotid artery was ligated and the skin was sutured.

Western blot analysis
After the cells were cultured to the specified point, total protein was extracted. The protein concentration was determined according to the Biyuntian BCA Assay Kit, and the separation gel was 7 configured according to the protein concentration. Electrophoresis was then carried out, and the voltage used for the gel layer was at 60 V for 30 min. The protein samples in the separation gel, it were electrophoresed for 90 min with 80V voltage. After the electrophoresis is finished, the electrophoresis can be stopped immediately after the bromophenol blue is run, and the membrane is transferred. After the mold is transferred, the film is dried for use. Place the membrane in a Petri dish containing the blocking solution and shake for 1 h at room temperature with a destaining shaker.
They were then incubated with primary and secondary antibodies, respectively, and finally chemiluminescent reaction was performed, and Tanon ECL was used for chemiluminescence. Take a picture with a Tanon gel imager to get a picture.

Flow cytometric analysis
BV2 cell culture supernatant was removed. The adherent cells were trypsinized, centrifuged at 1000 rpm for 5min, and the supernatant was discarded. The cells were washed three times with pre-cooled PBS, centrifuged at 1000rpm for 5min at 4°C, and the supernatant was discarded. The cells were resuspend by adding 100ul PBS. CD16 (1:100 dilution) was added and mixed gently. Incubation was at room temperature for 30min in the dark for CD206 and ARG1 co-staining, the cells were fixed in 4% paraformaldehyde for 10 min at room temperature. After rinsing 3 times with 1ml PBS, PBS-Tritonx100 (0.25%) was added to rupture the membrane at 4°C for 10min. Following washing 3 times with PBS, 100ul PBS was added. The cells in suspension were incubated with CD206 and ARG1 antibodies at room temperature for 30min. After centrifugation the supernatant was discarded. The cells were resuspended in 1 ml of PBS, centrifuged at 1000rpm for 5min at 4°C, and the supernatant was discarded (repeated 2 times). 400μl PBS was then added and mixed well before flow cytometry.

ELISA
We used the ELISA kits for detection of IL-10, iNOS, TGF-β, and TNFα, respectively. The supernatants of the different treated cells were collected according to the experimental time points. Biotin-labeled antibody, HRP-labeled avidin, wash buffer, and standard protein were diluted according to the kit instructions. The standard and culture supernatant samples were added into 96-well coated plates.
Each sample in triplicate was incubated in a 37°C incubator. After 2 hours of incubation, the solution was removed. 100μL of biotin-labeled antibody was added to each well and incubated at 37°C. After 1 h of incubation, the solution was removed and washed twice with 200μL of washing buffer for 2min each. Then, 100μL of HRP-labeled avidin was added to each well and then incubated in a 37°C for 1 hour, after which the solution was removed, and 200μL of the washing buffer was added 5 times over 2 minutes each 90μL of TMB reaction substrate was added to each well and incubated at 37°C in the dark. Finally 90μL of top Buffer was added to the absorbance at a wavelength of 450 nm and measured on a microplate reader.

Longa neurobehavioral score
Longa score was added to evaluate neurological function The scoring criteria were as follows: 0 points: no neurological deficits; 1 points: left forepaw limb can not fully extend; 2 points: circle to the left; 3 points: tilt to the left when walking; 4 points: unable to walk on its own, conscious loss; 5 points: death. Score > 1 can be used as a criterion for the success of MCAO model construction.

Froze sections of brain tissue
To observe the changes of neurons following middle cerebral artery occlusion and as well as the effect of cGAS-siRNA on the MCAO model. Frozen section of 8-10μm thickness of brain tissue were prepared. Fresh tissue was fixed in 10% formaldehyde for more than 24h. Following fixation, the tissue samples were kept in a 30% sucrose solution. They were embedded and sectioned.

Tunel staining
To detect neuronal apoptosis in MCAO brain tissue, Tunnel staining was carried out in frozen brain section. First, the frozen sections were placed in a 4% paraformaldehyde fixative and fixed for 20-30 min at room temperature. Proteinase K working solution (proteinase K stock solution diluted with PBS 1:9) was added to the tissue and incubated for 30min at 37℃Finally, DAPI staining was performed for 5 min, rinsed with running water, and then the sections were sealed with glycerin.

Nissl staining
Nissl staining was used to assess changes in morphology and number of neurons in the MCAO mouse.
First, the frozen sections were washed with water for 5 minutes, and placed in a 60°C incubator for 40 min with 0.5% toluidine blue. Differentiated sections were dehydrated in ethanol, cleaned in neutral gum.

Immunofluorescence
To analyze expression changes in the cGAS-STING-pIRF3 signaling pathway in MCAO and cGAS-siRNAtreated MCAO models, brain slices was proceeded for immunofluorescence labeling. First, the frozen sections were fixed with 10% cold formaldehyde for 20 minutes and washed three times with PBS.
They were then permeabilized with 0.2% Triton X-100 for 10 minutes and washed three times with PBS. The serum from the same host as the secondary antibody was then blocked for 30 minutes and washed three times with PBS. The sections were then placed overnight in a 4℃wet box and washed three times with PBS. The sections were then incubated with a secondary antibody for 2 hours at room temperature (protected from light) and washed three times with PBS. Finally, DAPI was used to stain the nucleus for 10 min, and washed with PBS three times. Images were captured using a laser confocal microscope.
Although cGAS-STING pathway plays an important role in various diseases, it relies on the dual stimuli of local hypoglycemic and hypoxic environments but its mechanism in post-IS is still unclear. We first established an OGD cell model to simulate the ischemic environment in vivo. Along with this, BV2 cells were cultured with supernatant produced by OGD to study the changes of cGAS-STING pathway.
10 RT-PCR was used to check the mRNA expression level of cGAS, STING and p-IRF3. We found that OGD- Using western blot assay, we estimated the expression changes of cGAS, STING and p-IRF3 proteins in BV2 cells. Compared with CM and Ctrl groups, the expressions level of STING, cCAS, p-IRF3 in OGD group was significantly higher than that in other three groups, on the other hand, and the expression of IRF3 did not change significantly ( fig. 1C-D). Of note, the supernatants derived from the cell culture medium treated with glucose-free hypoxia and glucose-free hypoxia could not activate the expression of STING, cGAS and p-IRF3.
To further investigates whether mtDNA can directly activate cGAS-STING pathway, and then upregulate the expression of related genes. To authenticate the role of mtDNA in the cGAS-STING signaling pathway, we utilized dideoxycytidine (ddC), a deoxyribonucleoside analog that specifically inhibits mtDNA replication and decreases mtDNA nucleoid size [23][24][25].Treatment of OGD with ddC resulted in a significant decrease in the expression of STING, cGAS and p-IRF3, and ddC significantly inhibited the activation of the cGAS-STING pathway ( fig. 1C-D). Our results indicate that mtDNA contributes significantly to the activation of the cGAS-STING-pIRF3 signaling pathway, which may also indicate that mtDNA is an important DAMP for microglia in response to inflammatory responses.

OIDs triggers transformation of M1 and M2-like BV2 cells
To further analyze the effect of OIDs on microglia polarity, we used flow cytometry to separate different phenotypes of microglia. First, CD16/32 was stained, and then CD206 and ARG1 were costained. The results showed that the fluorescence signal of CD16/32 was significantly enhanced when stimulated by OIDs ( fig. 2A). No significant changes were observed in the Ctrl and CM groups, neither distinct changes were observed in OGD-mtDNA treated group(OGD)( fig. 2A). CD16/32 is thought to be highly expressed in M1-like microglia, this indicated that the proportion of M1 cells in BV2 cells increased after the stimulates of OIDs.

11
Arg1-positive cells decreased significantly after stimulation with OIDs, and remained at a low level in the OGD (fig. 2B). The results indicated that Arg1 gene expression was decreased after OIDs stimulation, CD206 expression was not significantly affected by OIDs, it is well documented that CD206 is expressed more in M2 microglia likewise Arg1 expression is more pronounced in M2 microglia than in M1. Overall, the signal of Arg1-CD206 co-stained cells decreased after OIDs treatment, moreover, the ratio of M1/M2 was markly increased. On the other hand, in OGD+ddC, RFS of M1/M2 was significantly decreased (fig. 2C). These results indicated that OID triggered and promoted phenotype transformation from M2 to M1 phenotype, ddC treatment reversed the transformation from M2 to M1 and confirming that mtDNA is involved in the process. After that, flow cytometry was performed in the si-cGAS group and the Ctrl group. As described above, CD16/32 staining and CD206-Arg1 co-staining were performed. The results showed that CD16/32 signal was not altered significantly after si-cGAS treatment( fig. 4B). This may suggest that the cGAS-STING pathway is not directly related to the expression of CD16/32. In the CD206-Arg1 double-staining system, we found that the signals of Arg1 and CD206 were significantly enhanced after si-cGAS( fig. 4C), suggesting that after inhibiting the cGAS-STING-IRF3 signaling pathway, the frenquency of M2 cells was increased but that of M1 cells was decreased. Additionally, the ratio of M1/M2 was less than 1 after cGAS-siRNA treatment, indicating that the incidence of M2 BV2 cells was increased( fig. 4E).
The expression of IL-10 and TGF-β then followed which showed that it was significantly increased (p < 0.01, P < 0.05, Student's t test), however, the expression of TNFα was significantly decreased (p <0.05, Student's t test), and iNOS expression was not significantly decreased( fig. 4D). This suggests that following inhibit cGAS expression, the expression of inflammation-related genes decreased, while that IL-10 and TGF-β in activated microglia increased and indeed shifted for the repair type. This suggest that cGAS is potential target in treatment of IS. In other words, inhibition of cGAS expression may attenuate microglia-mediated inflammation and prevent neuronal damage.

Establishment of mouse ischemic stroke model
In vitro we have confirmed our hypothesis that the cGAS-STING signaling pathway in activated BV2 microglia affects the expression of downstream inflammatory factors. It promotes the MI phenotype polarization, which may exacerbate inflammation. Moreover, we demonstrated that inhibiting cGAS expression can effectively reduce the expression level of inflammation-related genes. We then constructed a mouse MCAO model to further demonstrate the role of cGAS-STING pathway in vivo. At 3, 5 and 7 days after MCAO, brain section were prepared and processed for immunefluorescence.
Images were captured and shown ( fig. 5). In the control group, there was only modulate cGAS, STING and p-IRF3 immunefluorescence in activated microglia double labeled with iba1. In MCAO group at 3, 5 and 7 days, cGAS, STING and p-IRF3 immunefluorescence in iba1 labeled microglia was noticeably augmented( fig. 5).
Longa neurological score(Table1) showed that scores of neurological MCAO model at 1 day and 3 day were 2.78 ±0.67 and 1.89 ± 0.6, respectively. This shows that the MCAO model we constructed is reliable for further assessment.

si-cGAS inhibited MIDs-induced apoptosis
OIDs  6A). In Nissl stained section in the ctrl-MCAO, many neurons appears atrophic,also the number of neurons was greatly reduced. In cGAS-siRNA lentivirus-transfected mice brain tissue ( fig. 6B). the neurons appears normal in external morphology. In summary, the ctrl-MCAO group showed decreased in neural population however, after inhibiting the expression of cGAS, the number of neurons was increased significantly (fig. 6C).

si-cGAS reduced MIDs-activated gene expression
To further explore the molecular mechanism of MIDs-induced apoptosis, we performed immunofluorescence experiments on three groups of brain tissue sections mentioned above. cGAS, STING and p-IRF3 gene immunofluorescence was located separated in area containing many iba1 positive cells. cGAS, STING, and p-IRF3 signals were absent in the control group, but intense cGAS, STING, and p-IRF3 immunofluorescence signals were detected in area rich in iba1 positive cells in the 14 ctrl-MCAO group ( fig. 7A). Such an expression pattern immunofluorescence induced by MIDs was significantly inhibited by cGAS-siRNA, .WB and ELISA assays confirmed the expression of cGAS, STING, and p-IRF3 as detected by immunofluorescence fig. 7B,C .

si-cGAS suppressed the expression of pro-inflammatory factors in MCAO models
Recent

Discussion
Inflammatory mechanism plays a crucial role in the pathological response of ischemic stroke, and such aseptic inflammation is essential for the repair of initial cerebral tissue lesion.  [16], which activates STING by synthesizing cyclic GMP-AMP (cGAMP) and this would further lead to downstream activation of transcription factors such as NF-kappa B and IRF3 that induce the production of type I interferons (IFNs) [16]. Relevant to this is the report that cGAS is strongly activated in myocardial ischemic injury, triggering downstream events mediated by STING cascade, and promoting macrophages to transform into M1 phenotype.
Remarkably, inactivation of this pathway promotes the transformation of macrophages to M2-like subtypes, which promotes wound healing, angiogenesis, reduces pathological cardiac remodeling and increases survival [11]. We have shown in this study the critical role of cGAS-STING in controlling microglial polarity transition and demonstrated the potential of cGAS as a therapeutic target for ischemic stroke.
Arginine metabolism has gradually become a core factor in inflammatory response. The major arginine-catabolizing enzymes involved in inflammatory immune responses are the isoforms of NOS (NOS1-3) and arginase (Arg1 and 2); however, only NOS2 (iNOS) and Arg1 are induced by multiple types of cytokines in macrophages. iNOS is controlled through inducible transcription in response to pro-inflammatory cytokines such as interferon γ (IFNγ), TNFα, and IL-1β [38,39]. Similarly, Arg1 expression is also induced by many cytokines, including IL-10 [40], granulocyte-macrophage colonystimulating factor (GM-CSF) [41], TGF-β [42], prostaglandin E2 (PGE2) [43], cyclic adenosine monophosphate (cAMP) [44] and toll-like receptor (TLR) agonists [45]. Hence, the distinct expression of Arg1 and iNOS largely determines the polar transformation of macrophages/microglia. The present results have shown that OGD cells released a large numbers of OIDs in the culture medium, and that they can activate the cGAS-STING-IRF3 signaling pathway. Similarly, in MCAO, the damage brain tissues release abundant MIDs which could also trigger the activation of cGAS-STING pathway.
Activated cGAS-STING-IRF3 resulted in increased expression of iNOS and TNFα, with a concomitant increase in cell proportions labeled with CD16/32. We believe that OIDs/MIDs had activated the cGAS-STING-IRF3 signaling pathway, which further stimulated the expression of downstream transcription factors such as TNFα and induced elevation of iNOS. It has been reported that activated microglia mediate neuronal damage through NO [46], which may affect the process of inflammation, including cGMP, NF-κB-dependent signal transduction pathway, or the expression of other inflammatory factors.
In contrast, the expression of Arg1 and TGF-β was inhibited, and the cell signal signed by Arg1-CD206 is attenuated. These findings suggest that the polarization state of microglia is closely associated with the metabolism of arginine. The present demonstration of changes in inflammatory cytokines and microglia phenotypes, is therefore consistent with the phenotypic transformation of macrophages in myocardial infarction [11,47].
The cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP [16], cGAS can activate transcription factor IRF3 in a STING-dependent manner. Studies have shown that injury of tissue can release DAMPs from mitochondria. mtDNA DAMPs is a distinct sequence which contains unmethylated CpG motifs [48]. This particular mtDNA can activate neutrophils through Toll-like receptor 9 (TLR-9), triggering inflammation and leading to organ damage [48]. It is well documented that mislocalization of both genomic and mitochondrial DNA can trigger cGAS-STING signaling [15,17,18,49,50]. In this study, we extracted mtDNA from the OGD cells and used it to stimulate the BV2 microglia. However, the exogenous mtDNA from the OGD cells barely triggers the activation of cGAS-STING pathway, indicating that the process may be more complex than expected. But interestingly, the activation of cGAS-STING pathway was significantly inhibited when we treated OGD with ddC. In other words, the outcome indicates that the single cyclic mtDNA can not be considered as an effective DAMPs which can be recognized by cGAS; however, the reduction of mtDNA copy number effectively inhibited the activation of cGAS-STING pathway.
Genetic ablation of cGAS or STING can alleviate or treat life-shortening inflammatory phenotypes, which has been corroborated in the mouse model [49,[51][52][53]. It is generally accepted that MI can drive inflammation and aggravate mortality, and the cGAS-STING pathway mediates the activation of IFN I signaling in cardiac macrophages and thus leading to massive death of cardiomyocytes [10,11].
Blocking-up of cGAS, IRF3, and IFNAR1 significantly improved early survival of animals in the MI model. Therefore, these findings suggest that components of cGAS-STING pathway represent promising targets for developing pharmacological intervention for MI and other pathway-related pathologies. We inhibited the expression of cGAS in microglia by siRNA technology and found that STING and p-IRF3 gene expression was inhibited. However, CD206-Arg1 labeled cell signaling was significantly enhanced. This indicates that the proportion of reparation microglia is significantly increased after inhibition of the cGAS-STING pathway. It is noteworthy that the expression of TNFα and iNOS and other inflammation-related factors was decreased in varying degrees, while the expression levels of while IL-10 and TGF-β were significantly increased. Upregulation of Arg1 is a means by which macrophages limit the availability of arginine and regulate nitric oxide production [54]. Moreover, arginine can regulate the post-transcriptional translation of iNOS [55]. In summary, we believe that inhibition if cGAS can increase the expression of M2 microglia phenotype and production of cytokines such as IL-10 and TGF-β, and further increase the level of Arg1 [40,42].
Arg1 can reduce iNOS expression through substrate competition mechanism and translational regulation, effectively reduce the expression of inflammatory factors and turn microglia to reparative phenotype.
Ischemic stroke remains a major account for brain damage, which is caused by insufficient blood supply or insufficient blood flow in specific parts of the brain, especially in the middle cerebral artery region [56]. MCAO followed by reperfusion is widely accepted to mimic stroke in basic medical research [57]. In cerebral ischemia microglia respond to inflammation followed by release of a wide range of cytotoxic agents, for example pro-inflammatory cytokines, arachidonic acid derivatives, glutamate, and free radicals which then leads to neuronal damage [58]. We have demonstrated unequivocally that activation of the cGAS-STING signaling pathway leads to the expression of downstream pro-inflammatory factors, which in turn leads to the transformation of microglia polarity in vitro. In the MCAO model, we found that the cGAS-STING-pIRF3 signaling pathway was strongly activated. More importantly, we found that the expression of M1-type cytokines such as iNOS and TNFα was significantly increased in microglia, which is consistent with our results in vitro.
The present results with labeling show that MIDs cause a large number of neuronal apoptosis, which can be suppressed by cGAS-siRNA. Furthermore, we found that cGAS-siRNA could reduce the level of iNOS and TNFα significantly in mouse microglia, as well as increase the level of Arg1.

Conclusions
The present results have demonstrated the beneficial or protective role of cGAS-STING-IRF3 pathway 18 in polarization of microglia (Fig. 9). Inhibition of the cGAS-STING pathway can effectively reduce the release of proinflammatory factors during stroke, prompting microglia to switch to a reparative phenotype.
Very strikingly, we inhibiting cGAS can effectively reduce the apoptosis of neurons and neurological decline caused by OIDs/MIDs. This suggests that cGAS may be a potential drug target for preventing brain injury. Thus inhibition of the cGAS-STING pathway with its inhibitor and analogues can be applied for treatment of brain diseases such as ischemic stroke.

Availability of data and materials
Data is available upon reasonable request.

Ethics approval and consent to participate
All experimental procedures were approved by the Ethics Committee of Kunming Medical University, in accordance with the "Regulations on the Management of Experimental Animals" promulgated by the National Science and Technology Commission of the People's Republic of China.

Consent for publication
Not applicable.     Immunefluorescence signals of cGAS, STING, p-IRF3 in iba1 labeled microglia in MCAO mice.

Figure 6
Estimation of MIDs-induced apoptosis. A: DAPI and TUNEL staining of brain in 3 groups, sham represents normal mouse brain tissue, ctrl-MCAO represents mouse brain injected with ctrl-siRNA, and cGAS-MCAO represents mouse brain injected with cGAS-siRNA. B: Nissl staining of brain section in three groups.C: Bar graph showing the number of surviving neurons of three groups in sham, ctrl-MCAO and cGAS-MCAO whose number density was 121.0±20.27,27.00±10.54 and 60.67±18.50, respectively.

Figure 9
The activation and inhibition of cGAS-STING signaling pathway is closely related to the levels of neuroinflammatory factors. When cGAS-STING is activated, the levels of iNOS and TNFα increase, while the levels of IL-10 and TGFβ decrease. Conversely, after the cGAS-STING pathway is inhibited, the levels of IL-10 and TGFβ increase and the levels of iNOS and TNFα decrease. Changes in these proinflammatory factors and anti-inflammatory factors regulate the polarity of microglia, and changes the proportion of M1 / M2 microglia in the brain.