{"paper_id":"2c8f382f-1e10-4b84-989e-554a40b02e07","body_text":"Neuritin attenuates neuroinflammation and apoptosis in early brain injury after subarachnoid hemorrhage via endoplasmic reticulum stress-related inflammatory pathways | 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 Neuritin attenuates neuroinflammation and apoptosis in early brain injury after subarachnoid hemorrhage via endoplasmic reticulum stress-related inflammatory pathways Kunhao Ren, Linzhi Dai, Hao Zhang, Yaowen He, Bin Liu, Youjie Hu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4553300/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 Neuroinflammation is a key destructive pathophysiological process in early brain injury (EBI) following subarachnoid hemorrhage (SAH). Recent studies have discovered that endoplasmic reticulum stress-related inflammatory pathways include the IRE1α-TRAF2-NF-κB pathway, PERK-eIF2α-NF-κB pathway, and ATF6-AKT -NF-κB pathway leading to neuroinflammatory response. Neuritin is a neurotrophin that is involved in neuronal plasticity and regeneration. Studies have suggested that Neuritin has a vital role in reducing neuroinflammation, and can also decrease the expression of proteins related to endoplasmic reticulum stress following SAH. This suggests that Neuritin could be a potential therapeutic target for SAH and other neurological conditions. However, the regulatory mechanisms of Neuritin in ER stress-related inflammatory pathways after SAH are not yet fully understood. In this work, we discovered that the activation of ER stress-related inflammatory pathways leads to neuroinflammation, which further aggravates neuronal apoptosis after SAH. Our findings indicate that Neuritin overexpression play a neuroprotective role by inhibiting IRE1α-TRAF2-NF-κB pathway, PERK-eIF2α-NF-κB pathway, and ATF6-AKT-NF-κB pathway associated with endoplasmic reticulum stress. These inhibitory effects on neuroinflammation ultimately reduce nerve cell apoptosis. subarachnoid hemorrhage endoplasmic reticulum stress Neuritin neuroinflammation apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction SAH is a subtype of hemorrhagic stroke which is commonly caused by the rupture of intracranial aneurysms(Claassen & Park 2022; Etminan et al. 2019). Early brain injury (EBI) is widely recognized as the primary factor contributing to poor prognosis among SAH patients(Kusaka et al. 2004). The mechanisms underlying EBI encompass oxidative stress, neuroinflammation, blood-brain barrier destruction, microvascular dysfunction, apoptosis, and diffuse cortical depolarization(Conzen et al. 2018; Koseki et al. 2019). Neuroinflammation is a key pathophysiological process in EBI. Targeting neuroinflammation in SAH can significantly improve the prognosis(Pang et al. 2018; Suzuki 2019). Inflammatory reactions following SAH can be triggered by various factors. Oxidative stress is one of these factors, which occurs when blood enters the subarachnoid space after an aneurysm ruptures; High mobility group box 1 (HMGB1) can also mediate inflammation following SAH by activating the Toll like receptor 4 (TLR4) receptor pathway. Recent studies have shown that endoplasmic reticulum (ER) stress-related pathways can also contribute to neuroinflammation. Unlike the general inflammatory pathway, this response is mediated by ER stress-related proteins including PKR-like ER kinase (PERK), Inositol-requiring enzyme 1 (IRE1) and Activating transcription factor 6 (ATF6). Overactivation of ERS can cause glucose regulating protein 78 (GRP78) to dissociate from PERK, IRE1, and ATF6, leading to the activation of IRE1α-TRAF2, PERK-eIF2α, and ATF6-AKT pathways. These findings present a promising new avenue for targeting SAH. Recent studies have indicated that these pathways can activate the nuclear factor kappa-B (NF-κB) and promote the assembly of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome, thus linking ER stress to neuroinflammation(Xu et al. 2019). The NLRP3 inflammasome is an important component of the immune system and assumes a central role in neuroinflammation(Mangan et al. 2018). The formation of multiprotein complexes within the NLRP3 inflammasome usually begins with the recognition of pathogen-associated molecular patterns (PAMPs) and endogenous danger signals (DAMPs). Inhibition of the NLRP3 inflammasome has been shown to improve neuroinflammation, reduce neuronal pyrogenesis, and enhance the prognosis of patients suffering from cerebral hemorrhage(Zeng et al. 2017) and ischemic stroke(Luo et al. 2022). Furthermore, inhibiting of the signaling pathway that is activated by the NLRP3 inflammasome can promote neurogenesis following SAH(Dodd et al. 2021; Xu et al. 2021). We have demonstrated that Neuritin inhibited the formation of NLRP3 inflammasome following cerebral ischemia, consequently reducing neuroinflammation(Xu et al. 2023). Neuritin, an activity-induced neurotrophic factor discovered in 1993, plays a key role in promoting neurite growth. It is worth noting that Neuritin is primarily expressed in the nervous system. While most studies on Neuritin have concentrated on their role in regulating neuroprotection and regeneration; additional functions such as angiogenesis and immunomodulation have also been documented. We found that Neuritin reduced neuronal apoptosis by inhibiting ER stress-related apoptotic pathways and significantly decreased the expression of ER stress-related proteins after SAH(Zhang et al. 2021). However, the regulation of Neuritin in ER stress -related inflammatory pathways after SAH remains unclear. Our study aimed to investigate the potential of Neuritin in inhibiting ER stress-related inflammatory pathways, including the IRE1α-TRAF2 pathway, PERK-eIF2α pathway, and ATF6 pathway, with the goal of reducing neuroinflammation and neuronal apoptosis following SAH. 2. Methods 2.1 Chemical reagents BAY11-7082 and TUDCA were purchased from APExBIO; DMSO, Tween80, PEG300 were purchased from MCE; BCA protein detection kit was purchased from Thermo Scientific; ECL chemiluminescence substrate kit was purchased from Biosharp; protein phosphatase inhibitor mixture and RIPA cleavage buffer were purchased from Solarbio; ELISA kit was purchased from Jianglai Organism; and AAV-Neuritin virus was purchased from GenePharma. 2.2 Animals and SAH modeling To avoid the effect of estrogen on brain damage, we used male SD rats, weighing 250–300 g from Henan Scibes Biotechnology Co. Ltd. (Henan, China; SCXK [Yu] 2020-0005). The rodents were kept in a 12-hour day-night cycle at a temperature of approximately 22°C and a humidity level of around 60%. Throughout the experiment, they were given complimentary food and water access. Animal procedures were performed following the guidelines established by the Laboratory Animal Ethics Committee of the First Affiliated Hospital of Shihezi University (approval number A2022-189-01). Previous studies have shown that SAH was initiated through endovascular perforation. General anesthesia was induced by injecting pentobarbital sodium (40 mg/kg) intraperitoneally. Expose the right carotid artery, a 3 − 0 nylon line was carefully advanced to reach the intracranial bifurcation of the anterior and middle cerebral arteries. After probing for resistance, the insertion was continued for 2–3 mm to penetrate the vessel, resulting in SAH. The sham-operated group underwent the same process, except for vessel puncture. The rats with SAH classification < 7 were excluded from this study. (figure.1B and C) 2.3 Drug treatment AAV-Neuritin (3.67e + 13 vg /ml, 5µL) or AAV-NC (4.03e + 13 vg /ml, 5µL) was injected into the right ventricle 3 weeks prior to induction of SAH, and BAY11-7082 (5 mg/kg in 5% DMSO solution) or excipient (5% DMSO solution) was injected into the subarachnoid space within 30 min after hemorrhage using intraperitoneal injection. After subarachnoid hemorrhage, either TUDCA (dissolved in saline at a dosage of 500 mg/kg) or saline was administered via intraperitoneal injection, with a time interval of 30 minutes. (figure.1A) 2.4 Neurologic function score and bleeding degree score The modified Garcia score was used to blindly assess neurological function 24 hours after SAH. Spontaneous activity, limb movement symmetry, forepaw extension, climbing, body proprioception, and response to vibrissa touch were evaluated through six tests, with scores ranging from 1 to 3. Lower scores indicated worse neurological function. The severity of SAH was assessed 24 hours after the occurrence using the grading system for SAH severity. The basal part of the brain was divided into six sections, and each section was blindly evaluated (with scores ranging from 0to3) according to the presence of subarachnoid clots. 2.5 Brain water content (BWC) The severity of cerebral edema Twenty-four hours after SAH. SD rats were euthanized and the right hemisphere of the brain tissue was promptly dissected and separated. The wet weight was measured directly, and then the sample was dried at a temperature of 105°C for a period of 72 hours to obtain the dry weight. The ultimate mass was determined by multiplying the percentage of ([wet mass - dry mass]/wet mass) by 100%. 2.6 Evans blue (EB) staining EB staining was conducted to assess the integrity of the blood-brain barrier. After anesthesia, rats were injected intraperitoneally with 2% EB solution at a dose of 8 ml/kg and perfused with PBS via the heart after 24 h. the brains were extracted and homogenized with 50% trichloroacetic acid. The specimens were placed in a water bath at a temperature of 50°C for a duration of 48 hours, followed by centrifugation at a force of 15,000 times the acceleration due to gravity for a period of 30 minutes. Fluorescence spectrophotometry was utilized to measure the absorbance of the supernatant at 620 nm. 2.7 Immunohistochemical staining After being anesthetized, the rats went through a sequential perfusion process via the heart using PBS and 4% paraformaldehyde (PFA). The cerebral organs were extracted and transferred to 4% PFA for subsequent fixation at 4°C for 24 hours. Afterwards, the cerebral tissue was submerged in a 30% sucrose solution for 2 days, leading to the extraction of cerebral segments. After removing paraffin, the brain sections (8µm thick) were repaired using pyrolysis in citrate buffer with a pH of6.0. Subsequently, the brain sections (8µm thick) were treated with 3% hydrogen peroxide to eliminate the activity of endogenous peroxidase. Following the obstruction using 10% cow serum albumin and 0.3% Triton X-100, the cells were subjected to incubation with rabbit anti-Neuritin (1:100, Abcam, ab64186), rabbit anti-NLRP3 (1:200, Signalway Antibody, 29125-1), and rabbit anti-GRP78 (1:200 Proteintech, 11587-1-AP) at a temperature of 4°C throughout the night. On the next day, the plates were exposed to a secondary antibody at a temperature of 37°C fora duration of 2 hours. Following the plate blocking process, PI-stained nuclei were treated with an anti-fluorescent attenuating blocking agent. The Leica light microscope from Mannheim, Germany was used to capture the images. The results were analyzed using the ImageJ software. 2.8 Western blotting The rats were anesthetized 24 h after the SAH, perfused with PBS through the heart, and the right cerebral cortex was excised. Eight to fifteen percent SDS-PAGE was used to separate thirty micrograms of protein. Polyvinylidene fluoride membranes received the transfer of proteins. The membranes were obstructed using 5% skim milk at room temperature for 2 hours. Then, the membrane was left to incubate overnight with primary antibodies at 4°C. These antibodies include rabbit anti-NF-κB (1:2000, bioss, #bs-20355R), rabbit anti-p-NF-κB (1:2000, bioss, #bs-5661R), rabbit anti-Neuritin (1: 1000, Abcam, ab64186), rabbit anti-NLRP3 (1:1000, Signalway Antibody, 29125-1), rabbit anti-GRP78 (1:2000, Proteintech, 11587-1-AP), rabbit anti-cleaved-caspase3 (1: 500, Proteintech, 19677-1-AP), rabbit anti-TRAF2 (1:1000, Proteintech, 26846-1-AP), rabbit anti-p-PERK (1:1000, Proteintech, 29546-1-AP), rabbit anti-ATF6 (1:1000, Proteintech, #24169-1-AP), rabbit anti-p-IRE1α (1:1000, bioss, #bs-16698R), mouse anti-p-AKT (1:1000, Proteintech, #66444-1-lg), rabbit anti-p-eIF2α (1:1000, Proteintech, 28740-1-AP), and β-actin (1:8000; Proteintech). Proteintech's secondary antibodies (1:8000) were immunized for 1 h at room temperature. The ECL Plus chemiluminescence kit (Biosharp) was used to quantify proteins, and the ImageJ software was employed for protein quantification. 2.9 Enzyme-linked immunosorbent assay Blood was collected from the abdominal aorta of SD rats in each group 24 h after SAH, and serum was collected using centrifugation after 2 h of whole blood clotting. An ELISA kit was utilized to measure the concentrations of IL-1β, IL-6, and TNF-ɑ. 2.10 Statistical analysis A minimum of three trials were performed for experiments. The mean ± standard deviation was used to present the results of the statistical analysis. Statistical analysis was performed using the SPSS 26.0 statistical software. The data was evaluated for normal distribution and met the criteria for normality and chi-square requirements for one-way analysis of variance. Statistical significance was determined using a significance level of P < 0.05. The GraphPad Prism software (version 8.0) was utilized to plot the graphs. The group classification of the subjects was unknown to the researchers throughout the experiment. 3. Results 3.1 Activation of endoplasmic reticulum stress-related inflammatory pathways after SAH promotes neuroinflammation After the SAH model was established, 500 mg/kg of the ER stress inhibitor TUDCA solution was administered via the peritoneum into SD rats to study the impact of ER stress on neuroinflammation in SAH-affected rats. In the control group, SD rats received an equal injection of saline. After a day, the cerebral cortical tissues were removed from the area around the puncture sites. Western blotting was used to determine the levels of NF-κB, p-NF-κB, NLRP3, GRP78, p-PERK, TRAF2, ATF6, p-IRE1α, p-eIF2α, and p-AKT. The findings indicate that in the SAH group, the levels of NLRP3, p-NF-κB/NF-κB ratio, GRP78, p-PERK, TRAF2, p-IRE1α, ATF6, and p-eIF2α were higher compared to the sham group. the p-AKT levels were lower in the SAH group. Furthermore, the p-NF-κB/NF-κB ratio, NLRP3, cleaved-caspase3, GRP78, p-PERK, TRAF2, p-IRE1α, p-eIF2α, and ATF6 protein levels were significantly reduced in the SAH + TUDCA group compared to the SAH group. Notably, the p-AKT levels were significantly elevated in the SAH + TUDCA group. (figure.2A-K) 3.2 Neuroinflammatory response after SAH aggravated neuronal apoptosis To examine the impact of neuroinflammatory reaction on neuronal apoptosis in rats with SAH, 5 mg/kg NF-κB inhibitor BAY11-7082 solution was injected intraperitoneally after the establishment of the SAH model in SD rats. Then, 5% DMSO solution was injected into the control group of SD rats, western blotting was performed to assess the levels of NF-κB, p-NF-κB, NLRP3, and cleaved-caspase3 in the cerebral cortex. The levels of p-NF-κB/NF-κB ratio, NLRP3, and cleaved-caspase3 was significantly higher in the SAH group than in the sham group. The p-NF-κB/NF-κB ratio, NLRP3, and cleaved-caspase3 levels were lower in the SAH + BAY11-7082 group than in the SAH group. (figure.3A-F) It is suggested that the NF-κB inhibitor BAY11-7082 suppressed neuroinflammation and reduced neuronal apoptosis in SAH rats. 3.3 Neuritin overexpression attenuates neuroinflammatory responses and thus reduces neuronal apoptosis by inhibiting the endoplasmic reticulum stress-related inflammatory pathway 3.3.1 Neuritin overexpression attenuates neuroinflammatory response and reduces neuronal apoptosis In order to investigate the effect of Neuritin on neuronal apoptosis in rats with SAH through neuroinflammation, the AAV-Neuritin virus was injected into the brains of SD rats using a stereotactic device 21 days before the establishment of the SAH model, and AAV-NC was injected into the sham group. After establishment of the SAH model, 500 mg/kg of the ER stress inhibitor TUDCA solution was intraperitoneally injected into the SD rats, while the Sham group got an equivalent amount of saline. Immunofluorescence was used to observe the expression and distribution of Neuritin. Western blotting was utilized to detect changes in the levels of NF-κB, p-NF-κB, NLRP3 and cleaved-caspase3 in the cerebral cortex near the puncture point. The results of the western blotting and immunofluorescence indicated that compared to the SAH group and the SAH + AAV-NC group, the Neuritin expression in the SAH + AAV-Neuritin group was considerably higher. while the difference of Neuritin expression in the SAH group and SAH + AAV-NC group was not statistically significant (figure.4A-D). Western blotting results showed that the p-NF-κB/NF-κB ratio levels, NLRP3, and cleaved-caspase3 in the SAH group was significantly higher than in the sham group. The p-NF-κB/NF-κB ratio, NLRP3, and cleaved-caspase3 protein levels were significantly lower in the SAH + AAV-Neuritin and SAH + AAV-Neuritin + TUDCA groups than in the SAH group. There was no statistically significant difference in the protein expression between the two groups. TUNEL labelling showed a substantial increase in the number of positive cells in the SAH group than in the sham group, and a decrease in the number of positive cells in the SAH + AAV-Neuritin and SAH + AAV-Neuritin + TUDCA groups than in the SAH + AAV-NC group. However, there was no discernible difference between the two groups' positive cell counts. (figure.5A-M) 3.3.2 Neuritin overexpression attenuates neuroinflammatory responses and thus reduces neuronal apoptosis by inhibiting the endoplasmic reticulum stress-related inflammatory pathway To investigate the effect of Neuritin on the ER stress-related inflammatory pathway, an SD rat SAH model was established using stereotactic injection of AAV-Neuritin viral transfection for 21 days, while the sham group was injected with AAV-NC. After establishment of the SAH model in SD rats, 500 mg/kg of TUDCA solution was intraperitoneally injected into the SD rats, and an equal amount of saline was injected into the control group. After 24 h, 4% paraformaldehyde solution was used to perfuse the brain for immunofluorescence observation of NLRP3 and GRP78. The cerebral cortical tissues from the vicinity of puncture points were used for the observation of NF-κB, p-NF-κB, NLRP3, cleaved-caspase3, GRP78, p-PERK, TRAF2, ATF6, p-IRE1α, p-eIF2α, and p-AKT using western blotting. The SAH group exhibited significantly greater levels of p-NF-κB/NF-κB ratio, NLRP3, cleaved-caspase3, GRP78, p-PERK, TRAF2, p-IRE1α, p-eIF2α, and ATF6 compared to the sham group. p-Akt expression was lower in the SAH group than in the sham group. The p-NF-κB/NF-κB ratio, NLRP3, cleaved-caspase3, GRP78, p-PERK, TRAF2, p-IRE1α, p-eIF2α, and ATF6 levels were markedly lower in the SAH + AAV-Neuritin and SAH + AAV-Neuritin + TUDCA groups than in the SAH group. p-AKT levels were significantly higher in the SAH + AAV-Neuritin and SAH + AAV-Neuritin + TUDCA groups than in the SAH group. The SAH group had considerably more TUNEL-stained positive cells than the sham group, while the SAH + AAV-Neuritin and SAH + AAV-Neuritin + TUDCA groups had fewer positive cells than the SAH group. (figure.6A-K) These findings were based on the results of TUNEL staining. However, there was no significant difference in the number of positive cells between the two groups. 4. Discussion In this study, we found that ER stress-related inflammatory pathways including IRE1α-TRAF2 pathway, PERK- eIF2α pathway, and ATF6 pathway activate NF-κB phosphorylation after SAH. Hence, inhibition of ER stress-related inflammatory pathways could reduce neuroinflammation. We further verified that inhibition of neuroinflammation reduced neuronal apoptosis after SAH, and found that Neuritin improved ER stress-related neuroinflammation by reducing apoptosis and inhibiting ER stress-related inflammatory pathways, including IRE1α-TRAF2 pathway, PERK- eIF2α pathway, and ATF6 pathway, which led to the reduction of neurological deficits and cerebral edema. Hyperactivation of ER stress after SAH promotes neuroinflammatory response(Marciniak et al. 2021; Ren et al. 2021). The massive influx of blood into the subarachnoid space after aneurysm rupture also causes damage and dysfunction of the endoplasmic reticulum during cellular persistence. ER stress maintains homeostasis in vivo through the UPR response. However, whether this response restores endoplasmic reticulum homeostasis depends on the stimulus intensity and duration. If the stimulus is too strong or lasts too long, it can disrupt the protective mechanism of UPR, resulting in the build-up and accumulation of unfolded proteins, thus promoting an inflammatory response. ER stress is over-activated in EBI after SAH, and its signature proteins, GRP78, ATF6, IRE1α, and PERK, are all increased at 72 h after hemorrhage, with peaks occurring at 24 h. ERS-related inflammatory pathways included IRE1α-TRAF2-NF-κB pathway, PERK-eIF2α-NF-κB pathway, and ATF6-AKT-NF-κB pathway. These three pathways exacerbate neuroinflammation after SAH by activating NF-κB to promote NLRP3 inflammasome generation(Li et al. 2020). Our experiments also showed that ER stress and neuroinflammation occurred in rats’ cortex with endovascular perforation-induced SAH, and the application of the ER stress inhibitor TUDCA reduced the expression of ER stress signature proteins GRP78, IRE1, and PERK as well as their downstream TRAF2 and eIF2α proteins, and the phosphorylation levels of NF-κB, which is an indicator of neuroinflammation, and the expression of NLRP3 inflammatory vesicle expression were also decreased. This outcome is in line with what earlier research has shown. The trend for ATF6 was unstable. In SAH and cerebral ischemia rat models, ATF6 expression was elevated in the model group(Fei et al. 2021; Xu et al. 2018), which agreed with the outcomes of our experiment. However, some researchers have observed a decrease in ATF6 expression in the SAH group in immunofluorescence experiments using an SAH mouse model(Wu et al. 2023). In addition, some researchers found no statistically significant difference in ATF6 expression when the expression of other endoplasmic reticulum stress-related proteins was downregulated on day 14 in the CPSP model(T. Liu et al. 2021). Based on this, under endoplasmic reticulum stress, ATF6 (ATF6p90) is transferred from the endoplasmic reticulum and broken down by site 1 and site 2 proteases at the Golgi. and at this time, although there is an elevation of ATF6 protein expression, it cannot catch up with the speed of its shearing, which is manifested as a decrease of ATF6 protein expression. If the endoplasmic reticulum stress is in the over-activated state, the Golgi apparatus will also be damaged to a certain extent. In this situation, the speed of ATF6 being sheared is not as fast as the speed of ATF6 production, which is manifested as elevated ATF6 protein expression. Therefore, we believe that ATF6 may be a more accurate indicator of the over-activation of endoplasmic reticulum stress than other endoplasmic reticulum stress signature proteins. Numerous studies have shown that neuroinflammation is a significant factor in the pathophysiological process of EBI after SAH(Dienel et al. 2021; Hu et al. 2021; Xu et al. 2019). We explored the complex relationship between neuroinflammation and apoptosis after SAH, and demonstrated that neuroinflammatory responses occur in rats’ cortex with SAH induced by endovascular perforation. The ER stress-related inflammatory pathway mediated the involvement of NF-κB in the neuroinflammatory process. Application of the NF-κB inhibitor BAY11-7082 suppressed NF-κB phosphorylation and the expression of NLRP3 and cleaved-caspase3. The number of TUNEL-stained positive cells in the cerebral cortex was also significantly decreased, which is consistent with the findings of an earlier investigation. We verified that the neuroinflammatory response following SAH exacerbates neuronal apoptosis. Our previous studies on the mechanism of Neuritin on the apoptotic pathway of ER stress found that ER stress is not only closely related to apoptosis(Zhang et al. 2017), but also has a strong link with inflammation(Xu et al. 2023). Neuritin is a member of the neurotrophic factor family, which can be triggered by neuronal activity and neurotrophic factors to promote neural synapse growth and branching. It is crucial for the regeneration and plasticity of neurons. There is a direct interaction between Neuritin and AMPA receptors(Schwenk et al. 2012; Subramanian et al. 2019). Combined application of AMPA and PepA up-regulates BDNF via the LynERK1/2-CREB signaling pathway expression that could protect neurons in hippocampal CA1 region from cerebral ischemia-induced death. AMPA-TrkB mediates the activation of the PI3K/Akt signaling pathway, which is related to the MAPK pathway, and protects CA1 neurons from ischemic injury by inhibiting JNK3 activity and Capase-3 activity(Zhang et al. 2009). In addition, activation of the neuronal BDNF/PI3K/AKT pathway protects neurons from neuroinflammation(B. Liu et al. 2021; Yan et al. 2022). AMPA activation upregulates expression of the BDNF/PI3K/AKT pathway. Based on the direct relationship between Neuritin and AMPA, and the role of the AMPA-related pathway, we speculate that Neuritin may have anti-inflammatory effects. This result is consistent with our previous experimental results, which indicated that endocortical ERS was overactivated, and neuroinflammation and neuronal apoptosis were exacerbated in the cerebral cortex of the SAH rat model induced by endovascular perforation. However, overexpression of Neuritin by brain stereotactic technique using adenoviral transfection significantly suppressed the expression of ER stress-related proteins and inflammation- and apoptosis-related proteins in the cerebral cortex of the SAH rats. ELISA results indicated a decrease in the levels of TNF-α, IL-6, and IL-1β, and that TUNEL staining revealed a significant reduction of positive cells in the cerebral cortex. Thus, we demonstrated that Neuritin significantly reduced neuroinflammation by inhibiting the ER stress-associated inflammatory pathway after SAH, thereby reducing neuronal apoptosis, alleviating neurological dysfunction and cerebral edema, and repairing the BBB function. This study had some limitations. A conditional knockout mouse model may have been more appropriate to exclude many of the uncontrolled variables in the mic. We only evaluated the anti-inflammatory and anti-apoptotic properties of Neuritin, without talking about how it contributes to oxidative stress or how other signalling networks might affect this defence mechanism. Future research ought to concentrate on how these pathways interact with one another. 5. Conclusions In summary(figure.7), the endoplasmic reticulum stress-related inflammatory pathway activates neuroinflammation-mediated neuronal apoptosis after SAH, and Neuritin alleviates neuroinflammatory toxicity by inhibiting the endoplasmic reticulum stress-related inflammatory pathway to achieve cerebroprotective effects. Thus, Neuritin may be a potential therapeutic target for the treatment of SAH. Declarations Funding: This study was supported by grants from National Natural Science Foundation of China (No. 81960222), the Hospital-level Project of the First Affiliated Hospital of School of Medicine, Shihezi University (No. BS202103), the Shihezi University Science Research Foundation (No. ZZZC202181), and the Shihezi University University-level Project Fund (No. ZZZC202064A). Conflict of Interest: The authors declare that there is no competing interest. Ethical approval: The study was approved by the Laboratory Animal Ethics Committee of the First Affiliated Hospital of Shihezi University (approval number A2022-189-01). Author contribution:Dong Zhao,Ketao Ma and Weidong Tian contributed to the study design and revised the manuscript.Kunhao Ren, Linzhi Dai,Hao Zhang, and Yaowen He conducted the experiment and collected and analyzed the data. Bin Liu and Youjie Hu provided assistance in experiment performing.Kunhao Ren and Linzhi Dai wrote the manuscript. All authors reviewed and approved the final manuscript. Data Availability: Data is provided within the manuscript or supplementary information files. References Claassen, J., & Park, S. (2022). Spontaneous subarachnoid haemorrhage. The Lancet , 400 (10355), 846-862. https://doi.org/10.1016/s0140-6736(22)00938-2 Conzen, C., Becker, K., Albanna, W., Weiss, M., Bach, A., Lushina, N., Steimers, A., Pinkernell, S., Clusmann, H., Lindauer, U., & Schubert, G. A. (2018). The Acute Phase of Experimental Subarachnoid Hemorrhage: Intracranial Pressure Dynamics and Their Effect on Cerebral Blood Flow and Autoregulation. Translational Stroke Research , 10 (5), 566-582. https://doi.org/10.1007/s12975-018-0674-3 Dienel, A., Veettil, R. A., Matsumura, K., Savarraj, J. P. J., Choi, H. A., Kumar T, P., Aronowski, J., Dash, P., Blackburn, S. L., & McBride, D. W. (2021). α7-Acetylcholine Receptor Signaling Reduces Neuroinflammation After Subarachnoid Hemorrhage in Mice. Neurotherapeutics , 18 (3), 1891-1904. https://doi.org/10.1007/s13311-021-01052-3 Dodd, W. S., Noda, I., Martinez, M., Hosaka, K., & Hoh, B. L. (2021). NLRP3 inhibition attenuates early brain injury and delayed cerebral vasospasm after subarachnoid hemorrhage. Journal of Neuroinflammation , 18 (1). https://doi.org/10.1186/s12974-021-02207-x Etminan, N., Chang, H.-S., Hackenberg, K., de Rooij, N. K., Vergouwen, M. D. I., Rinkel, G. J. E., & Algra, A. (2019). Worldwide Incidence of Aneurysmal Subarachnoid Hemorrhage According to Region, Time Period, Blood Pressure, and Smoking Prevalence in the Population. JAMA Neurology , 76 (5). https://doi.org/10.1001/jamaneurol.2019.0006 Fei, H., Xiang, P., Luo, W., Tan, X., Gu, C., Liu, M., Chen, M., Wang, Q., & Yang, J. (2021). CTRP1 Attenuates Cerebral Ischemia/Reperfusion Injury via the PERK Signaling Pathway. Frontiers in Cell and Developmental Biology , 9 . https://doi.org/10.3389/fcell.2021.700854 Hu, X., Yan, J., Huang, L., Araujo, C., Peng, J., Gao, L., Liu, S., Tang, J., Zuo, G., & Zhang, J. H. (2021). INT-777 attenuates NLRP3-ASC inflammasome-mediated neuroinflammation via TGR5/cAMP/PKA signaling pathway after subarachnoid hemorrhage in rats. Brain, Behavior, and Immunity , 91 , 587-600. https://doi.org/10.1016/j.bbi.2020.09.016 Koseki, H., Miyata, H., Shimo, S., Ohno, N., Mifune, K., Shimano, K., Yamamoto, K., Nozaki, K., Kasuya, H., Narumiya, S., & Aoki, T. (2019). Two Diverse Hemodynamic Forces, a Mechanical Stretch and a High Wall Shear Stress, Determine Intracranial Aneurysm Formation. Translational Stroke Research , 11 (1), 80-92. https://doi.org/10.1007/s12975-019-0690-y Kusaka, G., Ishikawa, M., Nanda, A., Granger, D. N., & Zhang, J. H. (2004). Signaling Pathways for Early Brain Injury after Subarachnoid Hemorrhage. Journal of Cerebral Blood Flow & Metabolism , 24 (8), 916-925. https://doi.org/10.1097/01.Wcb.0000125886.48838.7e Li, W., Cao, T., Luo, C., Cai, J., Zhou, X., Xiao, X., & Liu, S. (2020). Crosstalk between ER stress, NLRP3 inflammasome, and inflammation. Applied Microbiology and Biotechnology , 104 (14), 6129-6140. https://doi.org/10.1007/s00253-020-10614-y Liu, B., Zhang, Y., Yang, Z., Liu, M., Zhang, C., Zhao, Y., & Song, C. (2021). ω-3 DPA Protected Neurons from Neuroinflammation by Balancing Microglia M1/M2 Polarizations through Inhibiting NF-κB/MAPK p38 Signaling and Activating Neuron-BDNF-PI3K/AKT Pathways. Marine Drugs , 19 (11). https://doi.org/10.3390/md19110587 Liu, T., Li, T., Chen, X., Li, Z., Feng, M., Yao, W., Wan, L., Zhang, C., & Zhang, Y. (2021). EETs/sEHi alleviates nociception by blocking the crosslink between endoplasmic reticulum stress and neuroinflammation in a central poststroke pain model. Journal of Neuroinflammation , 18 (1). https://doi.org/10.1186/s12974-021-02255-3 Luo, L., Liu, M., Fan, Y., Zhang, J., Liu, L., Li, Y., Zhang, Q., Xie, H., Jiang, C., Wu, J., Xiao, X., & Wu, Y. (2022). Intermittent theta-burst stimulation improves motor function by inhibiting neuronal pyroptosis and regulating microglial polarization via TLR4/NFκB/NLRP3 signaling pathway in cerebral ischemic mice. Journal of Neuroinflammation , 19 (1). https://doi.org/10.1186/s12974-022-02501-2 Mangan, M. S. J., Olhava, E. J., Roush, W. R., Seidel, H. M., Glick, G. D., & Latz, E. (2018). Targeting the NLRP3 inflammasome in inflammatory diseases. Nat Rev Drug Discov , 17 (8), 588-606. https://doi.org/10.1038/nrd.2018.97 Marciniak, S. J., Chambers, J. E., & Ron, D. (2021). Pharmacological targeting of endoplasmic reticulum stress in disease. Nature Reviews Drug Discovery , 21 (2), 115-140. https://doi.org/10.1038/s41573-021-00320-3 Pang, J., Peng, J., Matei, N., Yang, P., Kuai, L., Wu, Y., Chen, L., Vitek, M. P., Li, F., Sun, X., Zhang, J. H., & Jiang, Y. (2018). Apolipoprotein E Exerts a Whole-Brain Protective Property by Promoting M1? Microglia Quiescence After Experimental Subarachnoid Hemorrhage in Mice. Translational Stroke Research , 9 (6), 654-668. https://doi.org/10.1007/s12975-018-0665-4 Ren, J., Bi, Y., Sowers, J. R., Hetz, C., & Zhang, Y. (2021). Endoplasmic reticulum stress and unfolded protein response in cardiovascular diseases. Nature Reviews Cardiology , 18 (7), 499-521. https://doi.org/10.1038/s41569-021-00511-w Schwenk, J., Harmel, N., Brechet, A., Zolles, G., Berkefeld, H., Müller, Catrin S., Bildl, W., Baehrens, D., Hüber, B., Kulik, A., Klöcker, N., Schulte, U., & Fakler, B. (2012). High-Resolution Proteomics Unravel Architecture and Molecular Diversity of Native AMPA Receptor Complexes. Neuron , 74 (4), 621-633. https://doi.org/10.1016/j.neuron.2012.03.034 Subramanian, J., Michel, K., Benoit, M., & Nedivi, E. (2019). CPG15/Neuritin Mimics Experience in Selecting Excitatory Synapses for Stabilization by Facilitating PSD95 Recruitment. Cell Reports , 28 (6), 1584-1595.e1585. https://doi.org/10.1016/j.celrep.2019.07.012 Suzuki, H. (2019). Inflammation: a Good Research Target to Improve Outcomes of Poor-Grade Subarachnoid Hemorrhage. Translational Stroke Research , 10 (6), 597-600. https://doi.org/10.1007/s12975-019-00713-y Wu, K., Chen, L., Qiu, Z., Zhao, B., Hou, J., Lei, S., Jiang, M., & Xia, Z. (2023). Protective Effect and Mechanism of Xbp1s Regulating HBP/O-GlcNAcylation through GFAT1 on Brain Injury after SAH. Biomedicines , 11 (5). https://doi.org/10.3390/biomedicines11051259 Xu, H., Dong, J., Li, Y., Zhang, L., Yin, J., Zhu, C., Wang, X., Ren, K., Zhang, H., & Zhao, D. (2023). Neuritin has a neuroprotective role in the rat model of acute ischemia stroke by inhibiting neuronal apoptosis and NLRP3 inflammasome. J Stroke Cerebrovasc Dis , 32 (12), 107391. https://doi.org/10.1016/j.jstrokecerebrovasdis.2023.107391 Xu, P., Tao, C., Zhu, Y., Wang, G., Kong, L., Li, W., Li, R., Li, J., Zhang, C., Wang, L., Liu, X., Sun, W., & Hu, W. (2021). TAK1 mediates neuronal pyroptosis in early brain injury after subarachnoid hemorrhage. Journal of Neuroinflammation , 18 (1). https://doi.org/10.1186/s12974-021-02226-8 Xu, W., Gao, L., Li, T., Zheng, J., Shao, A., & Zhang, J. (2018). Apelin-13 Alleviates Early Brain Injury after Subarachnoid Hemorrhage via Suppression of Endoplasmic Reticulum Stress-mediated Apoptosis and Blood–Brain Barrier Disruption: Possible Involvement of ATF6/CHOP Pathway. Neuroscience , 388 , 284-296. https://doi.org/10.1016/j.neuroscience.2018.07.023 Xu, W., Li, T., Gao, L., Zheng, J., Yan, J., Zhang, J., & Shao, A. (2019). Apelin-13/APJ system attenuates early brain injury via suppression of endoplasmic reticulum stress-associated TXNIP/NLRP3 inflammasome activation and oxidative stress in a AMPK-dependent manner after subarachnoid hemorrhage in rats. Journal of Neuroinflammation , 16 (1). https://doi.org/10.1186/s12974-019-1620-3 Yan, J., Zhang, Y., Wang, L., Li, Z., Tang, S., Wang, Y., Gu, N., Sun, X., & Li, L. (2022). TREM2 activation alleviates neural damage via Akt/CREB/BDNF signalling after traumatic brain injury in mice. Journal of Neuroinflammation , 19 (1). https://doi.org/10.1186/s12974-022-02651-3 Zeng, J., Chen, Y., Ding, R., Feng, L., Fu, Z., Yang, S., Deng, X., Xie, Z., & Zheng, S. (2017). Isoliquiritigenin alleviates early brain injury after experimental intracerebral hemorrhage via suppressing ROS- and/or NF-κB-mediated NLRP3 inflammasome activation by promoting Nrf2 antioxidant pathway. Journal of Neuroinflammation , 14 (1). https://doi.org/10.1186/s12974-017-0895-5 Zhang, H., He, X., Wang, Y., Sun, X., Zhu, L., Lei, C., Yin, J., Li, X., Hou, F., He, W., & Zhao, D. (2017). Neuritin attenuates early brain injury in rats after experimental subarachnoid hemorrhage. International Journal of Neuroscience , 127 (12), 1087-1095. https://doi.org/10.1080/00207454.2017.1337013 Zhang, L., Wang, Y., Pan, R.-l., Li, Y., Hu, Y.-q., Xv, H., Zhu, C., Wang, X., Yin, J.-w., Ma, K.-t., & Zhao, D. (2021). Neuritin attenuates oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal injury by promoting autophagic flux. Experimental Cell Research , 407 (2). https://doi.org/10.1016/j.yexcr.2021.112832 Zhang, Q. G., Han, D., Hu, S. Q., Li, C., Yu, C. Z., Wang, R., & Zhang, G. Y. (2009). Positive modulation of AMPA receptors prevents downregulation of GluR2 expression and activates the Lyn‐ERK1/2‐CREB signaling in rat brain ischemia. Hippocampus , 20 (1), 65-77. https://doi.org/10.1002/hipo.20593 Additional Declarations No competing interests reported. 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4553300\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":316778595,\"identity\":\"349557da-81fa-4407-b0e8-fd3d93ee50c9\",\"order_by\":0,\"name\":\"Kunhao Ren\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Department of Neurosurgery, the First Affiliated Hospital of Shihezi University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Kunhao\",\"middleName\":\"\",\"lastName\":\"Ren\",\"suffix\":\"\"},{\"id\":316778598,\"identity\":\"954a5fef-8b5b-44e8-8424-22f9cd934d28\",\"order_by\":1,\"name\":\"Linzhi Dai\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Department of Neurosurgery, the First Affiliated Hospital of Shihezi University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Linzhi\",\"middleName\":\"\",\"lastName\":\"Dai\",\"suffix\":\"\"},{\"id\":316778600,\"identity\":\"dd5de90e-d256-4a35-b607-b8129335bf1c\",\"order_by\":2,\"name\":\"Hao Zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Department of Neurosurgery, the First Affiliated Hospital of Shihezi University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hao\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":316778602,\"identity\":\"74789c13-70f6-4043-814e-f538ee8fb196\",\"order_by\":3,\"name\":\"Yaowen He\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Department of Neurosurgery, the First Affiliated Hospital of Shihezi University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yaowen\",\"middleName\":\"\",\"lastName\":\"He\",\"suffix\":\"\"},{\"id\":316778605,\"identity\":\"80dffe03-1848-4359-8872-d7bf484d0d6a\",\"order_by\":4,\"name\":\"Bin Liu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Department of Neurosurgery, the First Affiliated Hospital of Shihezi University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Bin\",\"middleName\":\"\",\"lastName\":\"Liu\",\"suffix\":\"\"},{\"id\":316778606,\"identity\":\"177f10bb-6796-456a-ac2c-c94bf46da4be\",\"order_by\":5,\"name\":\"Youjie Hu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Department of Neurosurgery, the First Affiliated Hospital of Shihezi University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Youjie\",\"middleName\":\"\",\"lastName\":\"Hu\",\"suffix\":\"\"},{\"id\":316778607,\"identity\":\"2a1bc5a5-3294-4a62-a3fa-862de5dfba0d\",\"order_by\":6,\"name\":\"Ketao Ma\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shihezi University School of Medicine\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ketao\",\"middleName\":\"\",\"lastName\":\"Ma\",\"suffix\":\"\"},{\"id\":316778610,\"identity\":\"ebcbd79d-06b1-42f6-8a8e-647ef1506cdd\",\"order_by\":7,\"name\":\"Weidong Tian\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shihezi University School of Medicine\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Weidong\",\"middleName\":\"\",\"lastName\":\"Tian\",\"suffix\":\"\"},{\"id\":316778612,\"identity\":\"1f7c1208-e580-4286-88bb-cb0c499c1e6a\",\"order_by\":8,\"name\":\"Dong Zhao\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYDACCQYGZiDF2AAkDiRU2PDw8zcQr4XxwIMzaTKSMw4Qr4X54MO2wzYGDQn4dcjPbn/8uaDGRrZfuv3CgQS28zwGDAcYP3zMwa3F4M6BBOMZx9KMZ845U3Aggec2jzlzA7PkzG14tEgkHEjmYTucuOFGTsKBBInbPJYNB9iYefFokZ+R2HCY5x9Mi8E5HgOg8/BqYbiRzNjM2wbSkn7gQELCAcJaDG6kMTPz9gH9MiMHGC9AR0rOONiM1y/yM9Iff+b5BgwxifTHH3/+s7Pn528++OEjPochADB4IQCcEogC7A+IVTkKRsEoGAUjDAAAJEtcJNLnAeQAAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Department of Neurosurgery, the First Affiliated Hospital of Shihezi University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Dong\",\"middleName\":\"\",\"lastName\":\"Zhao\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-06-09 09:53:20\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4553300/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4553300/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":59174438,\"identity\":\"04edbcbd-0110-40ac-b684-dad810b42072\",\"added_by\":\"auto\",\"created_at\":\"2024-06-27 09:13:09\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1392615,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e(A) Timeline of different treatments and animal groups. (B-C) Identification of SAH model.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4553300/v1/4f0a0472dbeb014b6df21bd5.png\"},{\"id\":59174443,\"identity\":\"52118521-2e4e-4a8c-a62a-11f0739ef066\",\"added_by\":\"auto\",\"created_at\":\"2024-06-27 09:13:09\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1782839,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEndoplasmic reticulum stress-related inflammatory pathways promote neuroinflammation after SAH. (A-H) Representative images and quantitative analysis of p-PERK, p-IRE1α, ATF6, GRP78, p-AKT, TRAF2, p-eIF2α.(I-K) Representative images and quantitative analysis of NF-κB, p-NF-κB, NLRP3. \\u003csup\\u003e# \\u003c/sup\\u003ecompared to the Sham group; (\\u003csup\\u003e###\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.001, \\u003csup\\u003e##\\u003c/sup\\u003e\\u003csup\\u003e\\u003cem\\u003e \\u003c/em\\u003e\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.01,\\u003csup\\u003e #\\u003c/sup\\u003e\\u003cem\\u003e P\\u003c/em\\u003e\\u0026lt;0.05); * compared to the SAH + Vehcile group (***\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt;0.001, **\\u003cem\\u003e P\\u003c/em\\u003e \\u0026lt;0.01, *\\u003cem\\u003e P\\u003c/em\\u003e\\u0026lt;0.05).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4553300/v1/e9d34f53e30154bf1ebde95e.png\"},{\"id\":59175119,\"identity\":\"db9b4b01-4d8a-481e-ad26-9aae2e63ebc4\",\"added_by\":\"auto\",\"created_at\":\"2024-06-27 09:21:09\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1372051,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eNeuroinflammatory response exacerbates neuronal apoptosis after SAH. (A) Representative images of TUNEL. scale bar = 20μm. (B) Representative images of NLRP3, NF-κB, p-NF-κB, Cleaved-caspase3. (C) Quantitative analysis of neuronal TUNEL. (D-F) Quantitative analysis of NLRP3, NF-κB, p-NF-κB, Cleaved-caspase3. \\u003csup\\u003e# \\u003c/sup\\u003ecompared to the Sham group; (\\u003csup\\u003e###\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.001, \\u003csup\\u003e##\\u003c/sup\\u003e\\u003csup\\u003e\\u003cem\\u003e \\u003c/em\\u003e\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.01,\\u003csup\\u003e #\\u003c/sup\\u003e\\u003cem\\u003e P\\u003c/em\\u003e\\u0026lt;0.05); * compared to the SAH + Vehcile group (***\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt;0.001, **\\u003cem\\u003e P\\u003c/em\\u003e \\u0026lt;0.01, *\\u003cem\\u003e P\\u003c/em\\u003e\\u0026lt;0.05).\\u0026nbsp;\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4553300/v1/9fba16b68e8363d2b978297d.png\"},{\"id\":59174439,\"identity\":\"6585bd31-3299-473f-b598-8cc3a303c2e8\",\"added_by\":\"auto\",\"created_at\":\"2024-06-27 09:13:09\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":268479,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAAV-Neuritin overexpression validation. (A-B) Typical images of Neuritin. scale bar = 20μm. (C-D) Quantitative analysis of Neuritin. \\u003csup\\u003e#\\u003c/sup\\u003e compared to the SAH group; (\\u003csup\\u003e###\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.001, \\u003csup\\u003e##\\u003c/sup\\u003e\\u003csup\\u003e\\u003cem\\u003e \\u003c/em\\u003e\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.01).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4553300/v1/a448edc0a726336bedc1bd05.png\"},{\"id\":59174440,\"identity\":\"7caf8512-4fb6-4e00-911f-d470e3fa8f04\",\"added_by\":\"auto\",\"created_at\":\"2024-06-27 09:13:09\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1808929,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eNeuritin overexpression attenuates the neuroinflammatory response and thus reduces neuronal apoptosis. (A-D) Modified Carcia score after SAH, SAH score, BWC and Evans Blue staining were used as indicators to analyze neurological dysfunction. (E-F) Typical images of TUNEL and quantitative analysis. Scale bar = 20μm (G-I) Quantitative analysis of IL-1β, IL-6, and TNF-ɑ (ELISA). (J-M) Representative images and quantitative analysis of NF-κB, p-NF-κB, NLRP3, Cleaved-caspase3. \\u003csup\\u003e#\\u003c/sup\\u003e compared to the Sham group (\\u003csup\\u003e###\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.001, \\u003csup\\u003e##\\u003c/sup\\u003e\\u003csup\\u003e\\u003cem\\u003e \\u003c/em\\u003e\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.01,\\u003csup\\u003e #\\u003c/sup\\u003e\\u003cem\\u003e P\\u003c/em\\u003e\\u0026lt;0.05). * compared to the SAH + AAV-NC group (***\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt;0.001, **\\u003cem\\u003e P\\u003c/em\\u003e \\u0026lt;0.01, *\\u003cem\\u003e P\\u003c/em\\u003e \\u0026lt;0.05).\\u0026nbsp;\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4553300/v1/2fe0a00ddd8ae468d806bf9f.png\"},{\"id\":59174444,\"identity\":\"d29000fe-bb1e-4679-9d44-c9ce5b09dd7f\",\"added_by\":\"auto\",\"created_at\":\"2024-06-27 09:13:10\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":2479545,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eNeuritin overexpression attenuates the neuroinflammatory response and thus reduces neuronal apoptosis by inhibiting the endoplasmic reticulum stress-related inflammatory pathway. (A-C) Typical images and quantitative analysis of NLRP3 and GRP78. Scale bar = 50μm.(D-K) Representative images and quantitative analysis of p-PERK, p-IRE1α, ATF6, GRP78, p-AKT, TRAF2, p-eIF2α. # compared to the Sham group (\\u003csup\\u003e###\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.001, \\u003csup\\u003e##\\u003c/sup\\u003e\\u003csup\\u003e\\u003cem\\u003e \\u003c/em\\u003e\\u003c/sup\\u003e\\u003cem\\u003eP\\u003c/em\\u003e\\u0026lt;0.01). * compared to the SAH + AAV-NC group (***\\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt;0.001, **\\u003cem\\u003e P\\u003c/em\\u003e \\u0026lt;0.01, *\\u003cem\\u003e P\\u003c/em\\u003e \\u0026lt;0.05).\\u0026nbsp;\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4553300/v1/8b2ce4a0b408509998fb174a.png\"},{\"id\":59174441,\"identity\":\"edb01008-152e-449d-b2c9-814ecb49ffea\",\"added_by\":\"auto\",\"created_at\":\"2024-06-27 09:13:09\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":827184,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eNeuritin overexpression attenuates the neuroinflammatory response and thus reduces neuronal apoptosis by inhibiting the endoplasmic reticulum stress-related inflammatory pathway.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4553300/v1/1aef52279b1f226cdd544f0d.png\"},{\"id\":59812378,\"identity\":\"d200d019-a42e-48ab-a535-79cb07bbe049\",\"added_by\":\"auto\",\"created_at\":\"2024-07-07 22:01:36\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":10450833,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4553300/v1/0a645380-4555-4683-87f8-c0a04f528bf9.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Neuritin attenuates neuroinflammation and apoptosis in early brain injury after subarachnoid hemorrhage via endoplasmic reticulum stress-related inflammatory pathways\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eSAH is a subtype of hemorrhagic stroke which is commonly caused by the rupture of intracranial aneurysms(Claassen \\u0026amp; Park 2022; Etminan et al. 2019). Early brain injury (EBI) is widely recognized as the primary factor contributing to poor prognosis among SAH patients(Kusaka et al. 2004). The mechanisms underlying EBI encompass oxidative stress, neuroinflammation, blood-brain barrier destruction, microvascular dysfunction, apoptosis, and diffuse cortical depolarization(Conzen et al. 2018; Koseki et al. 2019). Neuroinflammation is a key pathophysiological process in EBI. Targeting neuroinflammation in SAH can significantly improve the prognosis(Pang et al. 2018; Suzuki 2019).\\u003c/p\\u003e \\u003cp\\u003eInflammatory reactions following SAH can be triggered by various factors. Oxidative stress is one of these factors, which occurs when blood enters the subarachnoid space after an aneurysm ruptures; High mobility group box 1 (HMGB1) can also mediate inflammation following SAH by activating the Toll like receptor 4 (TLR4) receptor pathway. Recent studies have shown that endoplasmic reticulum (ER) stress-related pathways can also contribute to neuroinflammation. Unlike the general inflammatory pathway, this response is mediated by ER stress-related proteins including PKR-like ER kinase (PERK), Inositol-requiring enzyme 1 (IRE1) and Activating transcription factor 6 (ATF6). Overactivation of ERS can cause glucose regulating protein 78 (GRP78) to dissociate from PERK, IRE1, and ATF6, leading to the activation of IRE1α-TRAF2, PERK-eIF2α, and ATF6-AKT pathways. These findings present a promising new avenue for targeting SAH. Recent studies have indicated that these pathways can activate the nuclear factor kappa-B (NF-κB) and promote the assembly of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome, thus linking ER stress to neuroinflammation(Xu et al. 2019). The NLRP3 inflammasome is an important component of the immune system and assumes a central role in neuroinflammation(Mangan et al. 2018). The formation of multiprotein complexes within the NLRP3 inflammasome usually begins with the recognition of pathogen-associated molecular patterns (PAMPs) and endogenous danger signals (DAMPs). Inhibition of the NLRP3 inflammasome has been shown to improve neuroinflammation, reduce neuronal pyrogenesis, and enhance the prognosis of patients suffering from cerebral hemorrhage(Zeng et al. 2017) and ischemic stroke(Luo et al. 2022). Furthermore, inhibiting of the signaling pathway that is activated by the NLRP3 inflammasome can promote neurogenesis following SAH(Dodd et al. 2021; Xu et al. 2021).\\u003c/p\\u003e \\u003cp\\u003eWe have demonstrated that Neuritin inhibited the formation of NLRP3 inflammasome following cerebral ischemia, consequently reducing neuroinflammation(Xu et al. 2023). Neuritin, an activity-induced neurotrophic factor discovered in 1993, plays a key role in promoting neurite growth. It is worth noting that Neuritin is primarily expressed in the nervous system. While most studies on Neuritin have concentrated on their role in regulating neuroprotection and regeneration; additional functions such as angiogenesis and immunomodulation have also been documented. We found that Neuritin reduced neuronal apoptosis by inhibiting ER stress-related apoptotic pathways and significantly decreased the expression of ER stress-related proteins after SAH(Zhang et al. 2021). However, the regulation of Neuritin in ER stress -related inflammatory pathways after SAH remains unclear.\\u003c/p\\u003e \\u003cp\\u003eOur study aimed to investigate the potential of Neuritin in inhibiting ER stress-related inflammatory pathways, including the IRE1α-TRAF2 pathway, PERK-eIF2α pathway, and ATF6 pathway, with the goal of reducing neuroinflammation and neuronal apoptosis following SAH.\\u003c/p\\u003e\"},{\"header\":\"2. Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1 Chemical reagents\\u003c/h2\\u003e \\u003cp\\u003eBAY11-7082 and TUDCA were purchased from APExBIO; DMSO, Tween80, PEG300 were purchased from MCE; BCA protein detection kit was purchased from Thermo Scientific; ECL chemiluminescence substrate kit was purchased from Biosharp; protein phosphatase inhibitor mixture and RIPA cleavage buffer were purchased from Solarbio; ELISA kit was purchased from Jianglai Organism; and AAV-Neuritin virus was purchased from GenePharma.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2 Animals and SAH modeling\\u003c/h2\\u003e \\u003cp\\u003eTo avoid the effect of estrogen on brain damage, we used male SD rats, weighing 250\\u0026ndash;300 g from Henan Scibes Biotechnology Co. Ltd. (Henan, China; SCXK [Yu] 2020-0005). The rodents were kept in a 12-hour day-night cycle at a temperature of approximately 22\\u0026deg;C and a humidity level of around 60%. Throughout the experiment, they were given complimentary food and water access. Animal procedures were performed following the guidelines established by the Laboratory Animal Ethics Committee of the First Affiliated Hospital of Shihezi University (approval number A2022-189-01). Previous studies have shown that SAH was initiated through endovascular perforation. General anesthesia was induced by injecting pentobarbital sodium (40 mg/kg) intraperitoneally.\\u003c/p\\u003e \\u003cp\\u003eExpose the right carotid artery, a 3\\u0026thinsp;\\u0026minus;\\u0026thinsp;0 nylon line was carefully advanced to reach the intracranial bifurcation of the anterior and middle cerebral arteries. After probing for resistance, the insertion was continued for 2\\u0026ndash;3 mm to penetrate the vessel, resulting in SAH. The sham-operated group underwent the same process, except for vessel puncture. The rats with SAH classification\\u0026thinsp;\\u0026lt;\\u0026thinsp;7 were excluded from this study. (figure.1B and C)\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3 Drug treatment\\u003c/h2\\u003e \\u003cp\\u003eAAV-Neuritin (3.67e\\u0026thinsp;+\\u0026thinsp;13 vg /ml, 5\\u0026micro;L) or AAV-NC (4.03e\\u0026thinsp;+\\u0026thinsp;13 vg /ml, 5\\u0026micro;L) was injected into the right ventricle 3 weeks prior to induction of SAH, and BAY11-7082 (5 mg/kg in 5% DMSO solution) or excipient (5% DMSO solution) was injected into the subarachnoid space within 30 min after hemorrhage using intraperitoneal injection. After subarachnoid hemorrhage, either TUDCA (dissolved in saline at a dosage of 500 mg/kg) or saline was administered via intraperitoneal injection, with a time interval of 30 minutes. (figure.1A)\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4 Neurologic function score and bleeding degree score\\u003c/h2\\u003e \\u003cp\\u003eThe modified Garcia score was used to blindly assess neurological function 24 hours after SAH. Spontaneous activity, limb movement symmetry, forepaw extension, climbing, body proprioception, and response to vibrissa touch were evaluated through six tests, with scores ranging from 1 to 3. Lower scores indicated worse neurological function. The severity of SAH was assessed 24 hours after the occurrence using the grading system for SAH severity. The basal part of the brain was divided into six sections, and each section was blindly evaluated (with scores ranging from 0to3) according to the presence of subarachnoid clots.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.5 Brain water content (BWC)\\u003c/h2\\u003e \\u003cp\\u003eThe severity of cerebral edema Twenty-four hours after SAH. SD rats were euthanized and the right hemisphere of the brain tissue was promptly dissected and separated. The wet weight was measured directly, and then the sample was dried at a temperature of 105\\u0026deg;C for a period of 72 hours to obtain the dry weight. The ultimate mass was determined by multiplying the percentage of ([wet mass - dry mass]/wet mass) by 100%.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.6 Evans blue (EB) staining\\u003c/h2\\u003e \\u003cp\\u003eEB staining was conducted to assess the integrity of the blood-brain barrier. After anesthesia, rats were injected intraperitoneally with 2% EB solution at a dose of 8 ml/kg and perfused with PBS via the heart after 24 h. the brains were extracted and homogenized with 50% trichloroacetic acid. The specimens were placed in a water bath at a temperature of 50\\u0026deg;C for a duration of 48 hours, followed by centrifugation at a force of 15,000 times the acceleration due to gravity for a period of 30 minutes. Fluorescence spectrophotometry was utilized to measure the absorbance of the supernatant at 620 nm.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.7 Immunohistochemical staining\\u003c/h2\\u003e \\u003cp\\u003eAfter being anesthetized, the rats went through a sequential perfusion process via the heart using PBS and 4% paraformaldehyde (PFA). The cerebral organs were extracted and transferred to 4% PFA for subsequent fixation at 4\\u0026deg;C for 24 hours. Afterwards, the cerebral tissue was submerged in a 30% sucrose solution for 2 days, leading to the extraction of cerebral segments. After removing paraffin, the brain sections (8\\u0026micro;m thick) were repaired using pyrolysis in citrate buffer with a pH of6.0. Subsequently, the brain sections (8\\u0026micro;m thick) were treated with 3% hydrogen peroxide to eliminate the activity of endogenous peroxidase. Following the obstruction using 10% cow serum albumin and 0.3% Triton X-100, the cells were subjected to incubation with rabbit anti-Neuritin (1:100, Abcam, ab64186), rabbit anti-NLRP3 (1:200, Signalway Antibody, 29125-1), and rabbit anti-GRP78 (1:200 Proteintech, 11587-1-AP) at a temperature of 4\\u0026deg;C throughout the night. On the next day, the plates were exposed to a secondary antibody at a temperature of 37\\u0026deg;C fora duration of 2 hours. Following the plate blocking process, PI-stained nuclei were treated with an anti-fluorescent attenuating blocking agent. The Leica light microscope from Mannheim, Germany was used to capture the images. The results were analyzed using the ImageJ software.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.8 Western blotting\\u003c/h2\\u003e \\u003cp\\u003eThe rats were anesthetized 24 h after the SAH, perfused with PBS through the heart, and the right cerebral cortex was excised. Eight to fifteen percent SDS-PAGE was used to separate thirty micrograms of protein. Polyvinylidene fluoride membranes received the transfer of proteins. The membranes were obstructed using 5% skim milk at room temperature for 2 hours. Then, the membrane was left to incubate overnight with primary antibodies at 4\\u0026deg;C. These antibodies include rabbit anti-NF-κB (1:2000, bioss, #bs-20355R), rabbit anti-p-NF-κB (1:2000, bioss, #bs-5661R), rabbit anti-Neuritin (1: 1000, Abcam, ab64186), rabbit anti-NLRP3 (1:1000, Signalway Antibody, 29125-1), rabbit anti-GRP78 (1:2000, Proteintech, 11587-1-AP), rabbit anti-cleaved-caspase3 (1: 500, Proteintech, 19677-1-AP), rabbit anti-TRAF2 (1:1000, Proteintech, 26846-1-AP), rabbit anti-p-PERK (1:1000, Proteintech, 29546-1-AP), rabbit anti-ATF6 (1:1000, Proteintech, #24169-1-AP), rabbit anti-p-IRE1α (1:1000, bioss, #bs-16698R), mouse anti-p-AKT (1:1000, Proteintech, #66444-1-lg), rabbit anti-p-eIF2α (1:1000, Proteintech, 28740-1-AP), and β-actin (1:8000; Proteintech). Proteintech's secondary antibodies (1:8000) were immunized for 1 h at room temperature. The ECL Plus chemiluminescence kit (Biosharp) was used to quantify proteins, and the ImageJ software was employed for protein quantification.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.9 Enzyme-linked immunosorbent assay\\u003c/h2\\u003e \\u003cp\\u003eBlood was collected from the abdominal aorta of SD rats in each group 24 h after SAH, and serum was collected using centrifugation after 2 h of whole blood clotting. An ELISA kit was utilized to measure the concentrations of IL-1β, IL-6, and TNF-ɑ.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.10 Statistical analysis\\u003c/h2\\u003e \\u003cp\\u003eA minimum of three trials were performed for experiments. The mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard deviation was used to present the results of the statistical analysis. Statistical analysis was performed using the SPSS 26.0 statistical software. The data was evaluated for normal distribution and met the criteria for normality and chi-square requirements for one-way analysis of variance. Statistical significance was determined using a significance level of \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05. The GraphPad Prism software (version 8.0) was utilized to plot the graphs. The group classification of the subjects was unknown to the researchers throughout the experiment.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results\",\"content\":\"\\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.1 Activation of endoplasmic reticulum stress-related inflammatory pathways after SAH promotes neuroinflammation\\u003c/h2\\u003e \\u003cp\\u003eAfter the SAH model was established, 500 mg/kg of the ER stress inhibitor TUDCA solution was administered via the peritoneum into SD rats to study the impact of ER stress on neuroinflammation in SAH-affected rats. In the control group, SD rats received an equal injection of saline. After a day, the cerebral cortical tissues were removed from the area around the puncture sites. Western blotting was used to determine the levels of NF-κB, p-NF-κB, NLRP3, GRP78, p-PERK, TRAF2, ATF6, p-IRE1α, p-eIF2α, and p-AKT. The findings indicate that in the SAH group, the levels of NLRP3, p-NF-κB/NF-κB ratio, GRP78, p-PERK, TRAF2, p-IRE1α, ATF6, and p-eIF2α were higher compared to the sham group. the p-AKT levels were lower in the SAH group. Furthermore, the p-NF-κB/NF-κB ratio, NLRP3, cleaved-caspase3, GRP78, p-PERK, TRAF2, p-IRE1α, p-eIF2α, and ATF6 protein levels were significantly reduced in the SAH\\u0026thinsp;+\\u0026thinsp;TUDCA group compared to the SAH group. Notably, the p-AKT levels were significantly elevated in the SAH\\u0026thinsp;+\\u0026thinsp;TUDCA group. (figure.2A-K)\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.2 Neuroinflammatory response after SAH aggravated neuronal apoptosis\\u003c/h2\\u003e \\u003cp\\u003eTo examine the impact of neuroinflammatory reaction on neuronal apoptosis in rats with SAH, 5 mg/kg NF-κB inhibitor BAY11-7082 solution was injected intraperitoneally after the establishment of the SAH model in SD rats. Then, 5% DMSO solution was injected into the control group of SD rats, western blotting was performed to assess the levels of NF-κB, p-NF-κB, NLRP3, and cleaved-caspase3 in the cerebral cortex. The levels of p-NF-κB/NF-κB ratio, NLRP3, and cleaved-caspase3 was significantly higher in the SAH group than in the sham group. The p-NF-κB/NF-κB ratio, NLRP3, and cleaved-caspase3 levels were lower in the SAH\\u0026thinsp;+\\u0026thinsp;BAY11-7082 group than in the SAH group. (figure.3A-F) It is suggested that the NF-κB inhibitor BAY11-7082 suppressed neuroinflammation and reduced neuronal apoptosis in SAH rats.\\u003c/p\\u003e \\u003cp\\u003e3.3 Neuritin overexpression attenuates neuroinflammatory responses and thus reduces neuronal apoptosis by inhibiting the endoplasmic reticulum stress-related inflammatory pathway\\u003c/p\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.3.1 Neuritin overexpression attenuates neuroinflammatory response and reduces neuronal apoptosis\\u003c/h2\\u003e \\u003cp\\u003eIn order to investigate the effect of Neuritin on neuronal apoptosis in rats with SAH through neuroinflammation, the AAV-Neuritin virus was injected into the brains of SD rats using a stereotactic device 21 days before the establishment of the SAH model, and AAV-NC was injected into the sham group. After establishment of the SAH model, 500 mg/kg of the ER stress inhibitor TUDCA solution was intraperitoneally injected into the SD rats, while the Sham group got an equivalent amount of saline. Immunofluorescence was used to observe the expression and distribution of Neuritin. Western blotting was utilized to detect changes in the levels of NF-κB, p-NF-κB, NLRP3 and cleaved-caspase3 in the cerebral cortex near the puncture point. The results of the western blotting and immunofluorescence indicated that compared to the SAH group and the SAH\\u0026thinsp;+\\u0026thinsp;AAV-NC group, the Neuritin expression in the SAH\\u0026thinsp;+\\u0026thinsp;AAV-Neuritin group was considerably higher. while the difference of Neuritin expression in the SAH group and SAH\\u0026thinsp;+\\u0026thinsp;AAV-NC group was not statistically significant (figure.4A-D). Western blotting results showed that the p-NF-κB/NF-κB ratio levels, NLRP3, and cleaved-caspase3 in the SAH group was significantly higher than in the sham group. The p-NF-κB/NF-κB ratio, NLRP3, and cleaved-caspase3 protein levels were significantly lower in the SAH\\u0026thinsp;+\\u0026thinsp;AAV-Neuritin and SAH\\u0026thinsp;+\\u0026thinsp;AAV-Neuritin\\u0026thinsp;+\\u0026thinsp;TUDCA groups than in the SAH group. There was no statistically significant difference in the protein expression between the two groups. TUNEL labelling showed a substantial increase in the number of positive cells in the SAH group than in the sham group, and a decrease in the number of positive cells in the SAH\\u0026thinsp;+\\u0026thinsp;AAV-Neuritin and SAH\\u0026thinsp;+\\u0026thinsp;AAV-Neuritin\\u0026thinsp;+\\u0026thinsp;TUDCA groups than in the SAH\\u0026thinsp;+\\u0026thinsp;AAV-NC group. However, there was no discernible difference between the two groups' positive cell counts. (figure.5A-M)\\u003c/p\\u003e \\u003cp\\u003e3.3.2 Neuritin overexpression attenuates neuroinflammatory responses and thus reduces neuronal apoptosis by inhibiting the endoplasmic reticulum stress-related inflammatory pathway\\u003c/p\\u003e \\u003cp\\u003eTo investigate the effect of Neuritin on the ER stress-related inflammatory pathway, an SD rat SAH model was established using stereotactic injection of AAV-Neuritin viral transfection for 21 days, while the sham group was injected with AAV-NC. After establishment of the SAH model in SD rats, 500 mg/kg of TUDCA solution was intraperitoneally injected into the SD rats, and an equal amount of saline was injected into the control group. After 24 h, 4% paraformaldehyde solution was used to perfuse the brain for immunofluorescence observation of NLRP3 and GRP78. The cerebral cortical tissues from the vicinity of puncture points were used for the observation of NF-κB, p-NF-κB, NLRP3, cleaved-caspase3, GRP78, p-PERK, TRAF2, ATF6, p-IRE1α, p-eIF2α, and p-AKT using western blotting. The SAH group exhibited significantly greater levels of p-NF-κB/NF-κB ratio, NLRP3, cleaved-caspase3, GRP78, p-PERK, TRAF2, p-IRE1α, p-eIF2α, and ATF6 compared to the sham group. p-Akt expression was lower in the SAH group than in the sham group. The p-NF-κB/NF-κB ratio, NLRP3, cleaved-caspase3, GRP78, p-PERK, TRAF2, p-IRE1α, p-eIF2α, and ATF6 levels were markedly lower in the SAH\\u0026thinsp;+\\u0026thinsp;AAV-Neuritin and SAH\\u0026thinsp;+\\u0026thinsp;AAV-Neuritin\\u0026thinsp;+\\u0026thinsp;TUDCA groups than in the SAH group. p-AKT levels were significantly higher in the SAH\\u0026thinsp;+\\u0026thinsp;AAV-Neuritin and SAH\\u0026thinsp;+\\u0026thinsp;AAV-Neuritin\\u0026thinsp;+\\u0026thinsp;TUDCA groups than in the SAH group. The SAH group had considerably more TUNEL-stained positive cells than the sham group, while the SAH\\u0026thinsp;+\\u0026thinsp;AAV-Neuritin and SAH\\u0026thinsp;+\\u0026thinsp;AAV-Neuritin\\u0026thinsp;+\\u0026thinsp;TUDCA groups had fewer positive cells than the SAH group. (figure.6A-K) These findings were based on the results of TUNEL staining. However, there was no significant difference in the number of positive cells between the two groups.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e\"},{\"header\":\"4. Discussion\",\"content\":\"\\u003cp\\u003eIn this study, we found that ER stress-related inflammatory pathways including IRE1α-TRAF2 pathway, PERK- eIF2α pathway, and ATF6 pathway activate NF-κB phosphorylation after SAH. Hence, inhibition of ER stress-related inflammatory pathways could reduce neuroinflammation. We further verified that inhibition of neuroinflammation reduced neuronal apoptosis after SAH, and found that Neuritin improved ER stress-related neuroinflammation by reducing apoptosis and inhibiting ER stress-related inflammatory pathways, including IRE1α-TRAF2 pathway, PERK- eIF2α pathway, and ATF6 pathway, which led to the reduction of neurological deficits and cerebral edema.\\u003c/p\\u003e \\u003cp\\u003eHyperactivation of ER stress after SAH promotes neuroinflammatory response(Marciniak et al. 2021; Ren et al. 2021). The massive influx of blood into the subarachnoid space after aneurysm rupture also causes damage and dysfunction of the endoplasmic reticulum during cellular persistence. ER stress maintains homeostasis in vivo through the UPR response. However, whether this response restores endoplasmic reticulum homeostasis depends on the stimulus intensity and duration. If the stimulus is too strong or lasts too long, it can disrupt the protective mechanism of UPR, resulting in the build-up and accumulation of unfolded proteins, thus promoting an inflammatory response. ER stress is over-activated in EBI after SAH, and its signature proteins, GRP78, ATF6, IRE1α, and PERK, are all increased at 72 h after hemorrhage, with peaks occurring at 24 h. ERS-related inflammatory pathways included IRE1α-TRAF2-NF-κB pathway, PERK-eIF2α-NF-κB pathway, and ATF6-AKT-NF-κB pathway. These three pathways exacerbate neuroinflammation after SAH by activating NF-κB to promote NLRP3 inflammasome generation(Li et al. 2020). Our experiments also showed that ER stress and neuroinflammation occurred in rats\\u0026rsquo; cortex with endovascular perforation-induced SAH, and the application of the ER stress inhibitor TUDCA reduced the expression of ER stress signature proteins GRP78, IRE1, and PERK as well as their downstream TRAF2 and eIF2α proteins, and the phosphorylation levels of NF-κB, which is an indicator of neuroinflammation, and the expression of NLRP3 inflammatory vesicle expression were also decreased. This outcome is in line with what earlier research has shown.\\u003c/p\\u003e \\u003cp\\u003eThe trend for ATF6 was unstable. In SAH and cerebral ischemia rat models, ATF6 expression was elevated in the model group(Fei et al. 2021; Xu et al. 2018), which agreed with the outcomes of our experiment. However, some researchers have observed a decrease in ATF6 expression in the SAH group in immunofluorescence experiments using an SAH mouse model(Wu et al. 2023). In addition, some researchers found no statistically significant difference in ATF6 expression when the expression of other endoplasmic reticulum stress-related proteins was downregulated on day 14 in the CPSP model(T. Liu et al. 2021). Based on this, under endoplasmic reticulum stress, ATF6 (ATF6p90) is transferred from the endoplasmic reticulum and broken down by site 1 and site 2 proteases at the Golgi. and at this time, although there is an elevation of ATF6 protein expression, it cannot catch up with the speed of its shearing, which is manifested as a decrease of ATF6 protein expression. If the endoplasmic reticulum stress is in the over-activated state, the Golgi apparatus will also be damaged to a certain extent. In this situation, the speed of ATF6 being sheared is not as fast as the speed of ATF6 production, which is manifested as elevated ATF6 protein expression. Therefore, we believe that ATF6 may be a more accurate indicator of the over-activation of endoplasmic reticulum stress than other endoplasmic reticulum stress signature proteins.\\u003c/p\\u003e \\u003cp\\u003eNumerous studies have shown that neuroinflammation is a significant factor in the pathophysiological process of EBI after SAH(Dienel et al. 2021; Hu et al. 2021; Xu et al. 2019). We explored the complex relationship between neuroinflammation and apoptosis after SAH, and demonstrated that neuroinflammatory responses occur in rats\\u0026rsquo; cortex with SAH induced by endovascular perforation. The ER stress-related inflammatory pathway mediated the involvement of NF-κB in the neuroinflammatory process. Application of the NF-κB inhibitor BAY11-7082 suppressed NF-κB phosphorylation and the expression of NLRP3 and cleaved-caspase3. The number of TUNEL-stained positive cells in the cerebral cortex was also significantly decreased, which is consistent with the findings of an earlier investigation. We verified that the neuroinflammatory response following SAH exacerbates neuronal apoptosis.\\u003c/p\\u003e \\u003cp\\u003eOur previous studies on the mechanism of Neuritin on the apoptotic pathway of ER stress found that ER stress is not only closely related to apoptosis(Zhang et al. 2017), but also has a strong link with inflammation(Xu et al. 2023). Neuritin is a member of the neurotrophic factor family, which can be triggered by neuronal activity and neurotrophic factors to promote neural synapse growth and branching. It is crucial for the regeneration and plasticity of neurons. There is a direct interaction between Neuritin and AMPA receptors(Schwenk et al. 2012; Subramanian et al. 2019). Combined application of AMPA and PepA up-regulates BDNF via the LynERK1/2-CREB signaling pathway expression that could protect neurons in hippocampal CA1 region from cerebral ischemia-induced death. AMPA-TrkB mediates the activation of the PI3K/Akt signaling pathway, which is related to the MAPK pathway, and protects CA1 neurons from ischemic injury by inhibiting JNK3 activity and Capase-3 activity(Zhang et al. 2009). In addition, activation of the neuronal BDNF/PI3K/AKT pathway protects neurons from neuroinflammation(B. Liu et al. 2021; Yan et al. 2022). AMPA activation upregulates expression of the BDNF/PI3K/AKT pathway. Based on the direct relationship between Neuritin and AMPA, and the role of the AMPA-related pathway, we speculate that Neuritin may have anti-inflammatory effects. This result is consistent with our previous experimental results, which indicated that endocortical ERS was overactivated, and neuroinflammation and neuronal apoptosis were exacerbated in the cerebral cortex of the SAH rat model induced by endovascular perforation. However, overexpression of Neuritin by brain stereotactic technique using adenoviral transfection significantly suppressed the expression of ER stress-related proteins and inflammation- and apoptosis-related proteins in the cerebral cortex of the SAH rats. ELISA results indicated a decrease in the levels of TNF-α, IL-6, and IL-1β, and that TUNEL staining revealed a significant reduction of positive cells in the cerebral cortex. Thus, we demonstrated that Neuritin significantly reduced neuroinflammation by inhibiting the ER stress-associated inflammatory pathway after SAH, thereby reducing neuronal apoptosis, alleviating neurological dysfunction and cerebral edema, and repairing the BBB function.\\u003c/p\\u003e \\u003cp\\u003eThis study had some limitations. A conditional knockout mouse model may have been more appropriate to exclude many of the uncontrolled variables in the mic. We only evaluated the anti-inflammatory and anti-apoptotic properties of Neuritin, without talking about how it contributes to oxidative stress or how other signalling networks might affect this defence mechanism. Future research ought to concentrate on how these pathways interact with one another.\\u003c/p\\u003e\"},{\"header\":\"5. Conclusions\",\"content\":\"\\u003cp\\u003eIn summary(figure.7), the endoplasmic reticulum stress-related inflammatory pathway activates neuroinflammation-mediated neuronal apoptosis after SAH, and Neuritin alleviates neuroinflammatory toxicity by inhibiting the endoplasmic reticulum stress-related inflammatory pathway to achieve cerebroprotective effects. Thus, Neuritin may be a potential therapeutic target for the treatment of SAH.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003eFunding: This study was supported by grants from National Natural Science Foundation of China (No. 81960222), the Hospital-level Project of the First Affiliated Hospital of School of Medicine, Shihezi University (No. BS202103), the Shihezi University Science Research Foundation (No. ZZZC202181), and the Shihezi University University-level Project Fund (No. ZZZC202064A).\\u003c/p\\u003e\\n\\u003cp\\u003eConflict of Interest: The authors declare that there is no competing interest.\\u003c/p\\u003e\\n\\u003cp\\u003eEthical approval: The study was approved by the Laboratory Animal Ethics Committee of the First Affiliated Hospital of Shihezi University (approval number A2022-189-01).\\u003c/p\\u003e\\n\\u003cp\\u003eAuthor contribution:Dong Zhao,Ketao Ma and Weidong Tian contributed to the study design and revised the manuscript.Kunhao Ren, Linzhi Dai,Hao Zhang, \\u0026nbsp; and Yaowen He conducted the experiment and collected and analyzed the data. Bin Liu and Youjie Hu provided assistance in experiment performing.Kunhao Ren and \\u0026nbsp;Linzhi Dai wrote the manuscript. All authors reviewed and approved the final manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003eData Availability: Data is provided within the manuscript or supplementary information files.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eClaassen, J., \\u0026amp; Park, S. (2022). Spontaneous subarachnoid haemorrhage. \\u003cem\\u003eThe Lancet\\u003c/em\\u003e,\\u003cem\\u003e 400\\u003c/em\\u003e(10355), 846-862. https://doi.org/10.1016/s0140-6736(22)00938-2 \\u003c/li\\u003e\\n\\u003cli\\u003eConzen, C., Becker, K., Albanna, W., Weiss, M., Bach, A., Lushina, N., Steimers, A., Pinkernell, S., Clusmann, H., Lindauer, U., \\u0026amp; Schubert, G. A. (2018). The Acute Phase of Experimental Subarachnoid Hemorrhage: Intracranial Pressure Dynamics and Their Effect on Cerebral Blood Flow and Autoregulation. \\u003cem\\u003eTranslational Stroke Research\\u003c/em\\u003e,\\u003cem\\u003e 10\\u003c/em\\u003e(5), 566-582. https://doi.org/10.1007/s12975-018-0674-3 \\u003c/li\\u003e\\n\\u003cli\\u003eDienel, A., Veettil, R. A., Matsumura, K., Savarraj, J. P. J., Choi, H. A., Kumar T, P., Aronowski, J., Dash, P., Blackburn, S. L., \\u0026amp; McBride, D. W. (2021). \\u0026alpha;7-Acetylcholine Receptor Signaling Reduces Neuroinflammation After Subarachnoid Hemorrhage in Mice. \\u003cem\\u003eNeurotherapeutics\\u003c/em\\u003e,\\u003cem\\u003e 18\\u003c/em\\u003e(3), 1891-1904. https://doi.org/10.1007/s13311-021-01052-3 \\u003c/li\\u003e\\n\\u003cli\\u003eDodd, W. S., Noda, I., Martinez, M., Hosaka, K., \\u0026amp; Hoh, B. L. (2021). NLRP3 inhibition attenuates early brain injury and delayed cerebral vasospasm after subarachnoid hemorrhage. \\u003cem\\u003eJournal of Neuroinflammation\\u003c/em\\u003e,\\u003cem\\u003e 18\\u003c/em\\u003e(1). https://doi.org/10.1186/s12974-021-02207-x \\u003c/li\\u003e\\n\\u003cli\\u003eEtminan, N., Chang, H.-S., Hackenberg, K., de Rooij, N. K., Vergouwen, M. D. I., Rinkel, G. J. E., \\u0026amp; Algra, A. (2019). Worldwide Incidence of Aneurysmal Subarachnoid Hemorrhage According to Region, Time Period, Blood Pressure, and Smoking Prevalence in the Population. \\u003cem\\u003eJAMA Neurology\\u003c/em\\u003e,\\u003cem\\u003e 76\\u003c/em\\u003e(5). https://doi.org/10.1001/jamaneurol.2019.0006 \\u003c/li\\u003e\\n\\u003cli\\u003eFei, H., Xiang, P., Luo, W., Tan, X., Gu, C., Liu, M., Chen, M., Wang, Q., \\u0026amp; Yang, J. (2021). CTRP1 Attenuates Cerebral Ischemia/Reperfusion Injury via the PERK Signaling Pathway. \\u003cem\\u003eFrontiers in Cell and Developmental Biology\\u003c/em\\u003e,\\u003cem\\u003e 9\\u003c/em\\u003e. https://doi.org/10.3389/fcell.2021.700854 \\u003c/li\\u003e\\n\\u003cli\\u003eHu, X., Yan, J., Huang, L., Araujo, C., Peng, J., Gao, L., Liu, S., Tang, J., Zuo, G., \\u0026amp; Zhang, J. H. (2021). INT-777 attenuates NLRP3-ASC inflammasome-mediated neuroinflammation via TGR5/cAMP/PKA signaling pathway after subarachnoid hemorrhage in rats. \\u003cem\\u003eBrain, Behavior, and Immunity\\u003c/em\\u003e,\\u003cem\\u003e 91\\u003c/em\\u003e, 587-600. https://doi.org/10.1016/j.bbi.2020.09.016 \\u003c/li\\u003e\\n\\u003cli\\u003eKoseki, H., Miyata, H., Shimo, S., Ohno, N., Mifune, K., Shimano, K., Yamamoto, K., Nozaki, K., Kasuya, H., Narumiya, S., \\u0026amp; Aoki, T. (2019). Two Diverse Hemodynamic Forces, a Mechanical Stretch and a High Wall Shear Stress, Determine Intracranial Aneurysm Formation. \\u003cem\\u003eTranslational Stroke Research\\u003c/em\\u003e,\\u003cem\\u003e 11\\u003c/em\\u003e(1), 80-92. https://doi.org/10.1007/s12975-019-0690-y \\u003c/li\\u003e\\n\\u003cli\\u003eKusaka, G., Ishikawa, M., Nanda, A., Granger, D. N., \\u0026amp; Zhang, J. H. (2004). Signaling Pathways for Early Brain Injury after Subarachnoid Hemorrhage. \\u003cem\\u003eJournal of Cerebral Blood Flow \\u0026amp; Metabolism\\u003c/em\\u003e,\\u003cem\\u003e 24\\u003c/em\\u003e(8), 916-925. https://doi.org/10.1097/01.Wcb.0000125886.48838.7e \\u003c/li\\u003e\\n\\u003cli\\u003eLi, W., Cao, T., Luo, C., Cai, J., Zhou, X., Xiao, X., \\u0026amp; Liu, S. (2020). Crosstalk between ER stress, NLRP3 inflammasome, and inflammation. \\u003cem\\u003eApplied Microbiology and Biotechnology\\u003c/em\\u003e,\\u003cem\\u003e 104\\u003c/em\\u003e(14), 6129-6140. https://doi.org/10.1007/s00253-020-10614-y \\u003c/li\\u003e\\n\\u003cli\\u003eLiu, B., Zhang, Y., Yang, Z., Liu, M., Zhang, C., Zhao, Y., \\u0026amp; Song, C. (2021). \\u0026omega;-3 DPA Protected Neurons from Neuroinflammation by Balancing Microglia M1/M2 Polarizations through Inhibiting NF-\\u0026kappa;B/MAPK p38 Signaling and Activating Neuron-BDNF-PI3K/AKT Pathways. \\u003cem\\u003eMarine Drugs\\u003c/em\\u003e,\\u003cem\\u003e 19\\u003c/em\\u003e(11). https://doi.org/10.3390/md19110587 \\u003c/li\\u003e\\n\\u003cli\\u003eLiu, T., Li, T., Chen, X., Li, Z., Feng, M., Yao, W., Wan, L., Zhang, C., \\u0026amp; Zhang, Y. (2021). EETs/sEHi alleviates nociception by blocking the crosslink between endoplasmic reticulum stress and neuroinflammation in a central poststroke pain model. \\u003cem\\u003eJournal of Neuroinflammation\\u003c/em\\u003e,\\u003cem\\u003e 18\\u003c/em\\u003e(1). https://doi.org/10.1186/s12974-021-02255-3 \\u003c/li\\u003e\\n\\u003cli\\u003eLuo, L., Liu, M., Fan, Y., Zhang, J., Liu, L., Li, Y., Zhang, Q., Xie, H., Jiang, C., Wu, J., Xiao, X., \\u0026amp; Wu, Y. (2022). Intermittent theta-burst stimulation improves motor function by inhibiting neuronal pyroptosis and regulating microglial polarization via TLR4/NF\\u0026kappa;B/NLRP3 signaling pathway in cerebral ischemic mice. \\u003cem\\u003eJournal of Neuroinflammation\\u003c/em\\u003e,\\u003cem\\u003e 19\\u003c/em\\u003e(1). https://doi.org/10.1186/s12974-022-02501-2 \\u003c/li\\u003e\\n\\u003cli\\u003eMangan, M. S. J., Olhava, E. J., Roush, W. R., Seidel, H. M., Glick, G. D., \\u0026amp; Latz, E. (2018). Targeting the NLRP3 inflammasome in inflammatory diseases. \\u003cem\\u003eNat Rev Drug Discov\\u003c/em\\u003e,\\u003cem\\u003e 17\\u003c/em\\u003e(8), 588-606. https://doi.org/10.1038/nrd.2018.97 \\u003c/li\\u003e\\n\\u003cli\\u003eMarciniak, S. J., Chambers, J. E., \\u0026amp; Ron, D. (2021). Pharmacological targeting of endoplasmic reticulum stress in disease. \\u003cem\\u003eNature Reviews Drug Discovery\\u003c/em\\u003e,\\u003cem\\u003e 21\\u003c/em\\u003e(2), 115-140. https://doi.org/10.1038/s41573-021-00320-3 \\u003c/li\\u003e\\n\\u003cli\\u003ePang, J., Peng, J., Matei, N., Yang, P., Kuai, L., Wu, Y., Chen, L., Vitek, M. P., Li, F., Sun, X., Zhang, J. H., \\u0026amp; Jiang, Y. (2018). Apolipoprotein E Exerts a Whole-Brain Protective Property by Promoting M1? Microglia Quiescence After Experimental Subarachnoid Hemorrhage in Mice. \\u003cem\\u003eTranslational Stroke Research\\u003c/em\\u003e,\\u003cem\\u003e 9\\u003c/em\\u003e(6), 654-668. https://doi.org/10.1007/s12975-018-0665-4 \\u003c/li\\u003e\\n\\u003cli\\u003eRen, J., Bi, Y., Sowers, J. R., Hetz, C., \\u0026amp; Zhang, Y. (2021). Endoplasmic reticulum stress and unfolded protein response in cardiovascular diseases. \\u003cem\\u003eNature Reviews Cardiology\\u003c/em\\u003e,\\u003cem\\u003e 18\\u003c/em\\u003e(7), 499-521. https://doi.org/10.1038/s41569-021-00511-w \\u003c/li\\u003e\\n\\u003cli\\u003eSchwenk, J., Harmel, N., Brechet, A., Zolles, G., Berkefeld, H., M\\u0026uuml;ller, Catrin S., Bildl, W., Baehrens, D., H\\u0026uuml;ber, B., Kulik, A., Kl\\u0026ouml;cker, N., Schulte, U., \\u0026amp; Fakler, B. (2012). High-Resolution Proteomics Unravel Architecture and Molecular Diversity of Native AMPA Receptor Complexes. \\u003cem\\u003eNeuron\\u003c/em\\u003e,\\u003cem\\u003e 74\\u003c/em\\u003e(4), 621-633. https://doi.org/10.1016/j.neuron.2012.03.034 \\u003c/li\\u003e\\n\\u003cli\\u003eSubramanian, J., Michel, K., Benoit, M., \\u0026amp; Nedivi, E. (2019). CPG15/Neuritin Mimics Experience in Selecting Excitatory Synapses for Stabilization by Facilitating PSD95 Recruitment. \\u003cem\\u003eCell Reports\\u003c/em\\u003e,\\u003cem\\u003e 28\\u003c/em\\u003e(6), 1584-1595.e1585. https://doi.org/10.1016/j.celrep.2019.07.012 \\u003c/li\\u003e\\n\\u003cli\\u003eSuzuki, H. (2019). Inflammation: a Good Research Target to Improve Outcomes of Poor-Grade Subarachnoid Hemorrhage. \\u003cem\\u003eTranslational Stroke Research\\u003c/em\\u003e,\\u003cem\\u003e 10\\u003c/em\\u003e(6), 597-600. https://doi.org/10.1007/s12975-019-00713-y \\u003c/li\\u003e\\n\\u003cli\\u003eWu, K., Chen, L., Qiu, Z., Zhao, B., Hou, J., Lei, S., Jiang, M., \\u0026amp; Xia, Z. (2023). Protective Effect and Mechanism of Xbp1s Regulating HBP/O-GlcNAcylation through GFAT1 on Brain Injury after SAH. \\u003cem\\u003eBiomedicines\\u003c/em\\u003e,\\u003cem\\u003e 11\\u003c/em\\u003e(5). https://doi.org/10.3390/biomedicines11051259 \\u003c/li\\u003e\\n\\u003cli\\u003eXu, H., Dong, J., Li, Y., Zhang, L., Yin, J., Zhu, C., Wang, X., Ren, K., Zhang, H., \\u0026amp; Zhao, D. (2023). Neuritin has a neuroprotective role in the rat model of acute ischemia stroke by inhibiting neuronal apoptosis and NLRP3 inflammasome. \\u003cem\\u003eJ Stroke Cerebrovasc Dis\\u003c/em\\u003e,\\u003cem\\u003e 32\\u003c/em\\u003e(12), 107391. https://doi.org/10.1016/j.jstrokecerebrovasdis.2023.107391 \\u003c/li\\u003e\\n\\u003cli\\u003eXu, P., Tao, C., Zhu, Y., Wang, G., Kong, L., Li, W., Li, R., Li, J., Zhang, C., Wang, L., Liu, X., Sun, W., \\u0026amp; Hu, W. (2021). TAK1 mediates neuronal pyroptosis in early brain injury after subarachnoid hemorrhage. \\u003cem\\u003eJournal of Neuroinflammation\\u003c/em\\u003e,\\u003cem\\u003e 18\\u003c/em\\u003e(1). https://doi.org/10.1186/s12974-021-02226-8 \\u003c/li\\u003e\\n\\u003cli\\u003eXu, W., Gao, L., Li, T., Zheng, J., Shao, A., \\u0026amp; Zhang, J. (2018). Apelin-13 Alleviates Early Brain Injury after Subarachnoid Hemorrhage via Suppression of Endoplasmic Reticulum Stress-mediated Apoptosis and Blood\\u0026ndash;Brain Barrier Disruption: Possible Involvement of ATF6/CHOP Pathway. \\u003cem\\u003eNeuroscience\\u003c/em\\u003e,\\u003cem\\u003e 388\\u003c/em\\u003e, 284-296. https://doi.org/10.1016/j.neuroscience.2018.07.023 \\u003c/li\\u003e\\n\\u003cli\\u003eXu, W., Li, T., Gao, L., Zheng, J., Yan, J., Zhang, J., \\u0026amp; Shao, A. (2019). Apelin-13/APJ system attenuates early brain injury via suppression of endoplasmic reticulum stress-associated TXNIP/NLRP3 inflammasome activation and oxidative stress in a AMPK-dependent manner after subarachnoid hemorrhage in rats. \\u003cem\\u003eJournal of Neuroinflammation\\u003c/em\\u003e,\\u003cem\\u003e 16\\u003c/em\\u003e(1). https://doi.org/10.1186/s12974-019-1620-3 \\u003c/li\\u003e\\n\\u003cli\\u003eYan, J., Zhang, Y., Wang, L., Li, Z., Tang, S., Wang, Y., Gu, N., Sun, X., \\u0026amp; Li, L. (2022). TREM2 activation alleviates neural damage via Akt/CREB/BDNF signalling after traumatic brain injury in mice. \\u003cem\\u003eJournal of Neuroinflammation\\u003c/em\\u003e,\\u003cem\\u003e 19\\u003c/em\\u003e(1). https://doi.org/10.1186/s12974-022-02651-3 \\u003c/li\\u003e\\n\\u003cli\\u003eZeng, J., Chen, Y., Ding, R., Feng, L., Fu, Z., Yang, S., Deng, X., Xie, Z., \\u0026amp; Zheng, S. (2017). Isoliquiritigenin alleviates early brain injury after experimental intracerebral hemorrhage via suppressing ROS- and/or NF-\\u0026kappa;B-mediated NLRP3 inflammasome activation by promoting Nrf2 antioxidant pathway. \\u003cem\\u003eJournal of Neuroinflammation\\u003c/em\\u003e,\\u003cem\\u003e 14\\u003c/em\\u003e(1). https://doi.org/10.1186/s12974-017-0895-5 \\u003c/li\\u003e\\n\\u003cli\\u003eZhang, H., He, X., Wang, Y., Sun, X., Zhu, L., Lei, C., Yin, J., Li, X., Hou, F., He, W., \\u0026amp; Zhao, D. (2017). Neuritin attenuates early brain injury in rats after experimental subarachnoid hemorrhage. \\u003cem\\u003eInternational Journal of Neuroscience\\u003c/em\\u003e,\\u003cem\\u003e 127\\u003c/em\\u003e(12), 1087-1095. https://doi.org/10.1080/00207454.2017.1337013 \\u003c/li\\u003e\\n\\u003cli\\u003eZhang, L., Wang, Y., Pan, R.-l., Li, Y., Hu, Y.-q., Xv, H., Zhu, C., Wang, X., Yin, J.-w., Ma, K.-t., \\u0026amp; Zhao, D. (2021). Neuritin attenuates oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal injury by promoting autophagic flux. \\u003cem\\u003eExperimental Cell Research\\u003c/em\\u003e,\\u003cem\\u003e 407\\u003c/em\\u003e(2). https://doi.org/10.1016/j.yexcr.2021.112832 \\u003c/li\\u003e\\n\\u003cli\\u003eZhang, Q. G., Han, D., Hu, S. Q., Li, C., Yu, C. Z., Wang, R., \\u0026amp; Zhang, G. Y. (2009). Positive modulation of AMPA receptors prevents downregulation of GluR2 expression and activates the Lyn‐ERK1/2‐CREB signaling in rat brain ischemia. \\u003cem\\u003eHippocampus\\u003c/em\\u003e,\\u003cem\\u003e 20\\u003c/em\\u003e(1), 65-77. https://doi.org/10.1002/hipo.20593 \\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\":\"info@researchsquare.com\",\"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, endoplasmic reticulum stress, Neuritin, neuroinflammation, apoptosis\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4553300/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4553300/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eNeuroinflammation is a key destructive pathophysiological process in early brain injury (EBI) following subarachnoid hemorrhage (SAH). Recent studies have discovered that endoplasmic reticulum stress-related inflammatory pathways include the IRE1α-TRAF2-NF-κB pathway, PERK-eIF2α-NF-κB pathway, and ATF6-AKT -NF-κB pathway leading to neuroinflammatory response. Neuritin is a neurotrophin that is involved in neuronal plasticity and regeneration. Studies have suggested that Neuritin has a vital role in reducing neuroinflammation, and can also decrease the expression of proteins related to endoplasmic reticulum stress following SAH. This suggests that Neuritin could be a potential therapeutic target for SAH and other neurological conditions. However, the regulatory mechanisms of Neuritin in ER stress-related inflammatory pathways after SAH are not yet fully understood. In this work, we discovered that the activation of ER stress-related inflammatory pathways leads to neuroinflammation, which further aggravates neuronal apoptosis after SAH. Our findings indicate that Neuritin overexpression play a neuroprotective role by inhibiting IRE1α-TRAF2-NF-κB pathway, PERK-eIF2α-NF-κB pathway, and ATF6-AKT-NF-κB pathway associated with endoplasmic reticulum stress. These inhibitory effects on neuroinflammation ultimately reduce nerve cell apoptosis.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Neuritin attenuates neuroinflammation and apoptosis in early brain injury after subarachnoid hemorrhage via endoplasmic reticulum stress-related inflammatory pathways\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-06-27 09:13:05\",\"doi\":\"10.21203/rs.3.rs-4553300/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"ff2d6f01-e9a8-47d4-b1e7-853b4e7f2e4a\",\"owner\":[],\"postedDate\":\"June 27th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-07-07T21:53:24+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-06-27 09:13:05\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4553300\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4553300\",\"identity\":\"rs-4553300\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}