TREM2-Mediated Microglial Pyroptosis: Unveiling the Neuroprotective Role of Diosmetin in Spinal Cord Ischemia-Reperfusion Injury

TREM2-Mediated Microglial Pyroptosis: Unveiling the Neuroprotective Role of Diosmetin in Spinal Cord Ischemia-Reperfusion Injury | 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 TREM2-Mediated Microglial Pyroptosis: Unveiling the Neuroprotective Role of Diosmetin in Spinal Cord Ischemia-Reperfusion Injury Sidan Liu, Yan Dong, Xinyue Zhang, Yongjian Zhou, Kexin Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4403409/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 Background Spinal cord ischemia-reperfusion injury (SCII) is a severe neurological condition marked by neuronal damage and functional impairments. The contribution of microglial pyroptosis, an inflammatory form of cell death, to SCII's development is increasingly acknowledged. Yet, the complex molecular mechanisms and potential therapeutic strategies targeting microglial pyroptosis in SCII are not fully understood. Methods Our research utilized both in vivo and in vitro models to evaluate the influence of TREM2 modulation on microglial pyroptosis and neuronal function in SCII. Principal methods included Tarlov scoring, Western blot analysis, Chromatin Immunoprecipitation (CHIP) and histological techniques, with an emphasis on proteins such as Forkhead Box O1 (FOXO1) and pyroptosis-related proteins to decipher the underlying mechanisms. Molecular docking was employed to investigate the interaction between the small molecule diosmetin and TREM2. Results We observed a marked increase in TREM2 expression following SCII, and demonstrated that TREM2 overexpression mitigated microglial pyroptosis and enhanced motor neuron functionality. Further investigation revealed that TREM2 engagement leads to the activation of Forkhead Box O1 (FOXO1) phosphorylation through the Phosphatidylinositol 3-Kinase (PI3K)/Protein Kinase B (AKT) signaling pathway. This activation sequence culminates in the downregulation of Gasdermin D (GSDMD), the primary effector of pyroptosis. Additionally, we identified diosmetin, a natural compound known for its anti-inflammatory and antioxidant effects, as a potent modulator of TREM2-mediated microglial pyroptosis. Experimental data demonstrate diosmetin's binding affinity to TREM2, conferring neuroprotection by impeding microglial pyroptosis through the TREM2/PI3K/AKT/FOXO1/GSDMD axis. Conclusion Our findings underscore the pivotal role of TREM2 in microglial pyroptosis and its therapeutic potential in SCII, positioning diosmetin as a viable pharmacological candidate for SCII prevention and therapy. Spinal cord ischemia-reperfusion injury TREM2 Diosmetin FOXO1 GSDMD Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Background Spinal cord ischemia-reperfusion injury (SCII) is a severe complication of thoracoabdominal aortic surgery that can cause paralysis [ 1 , 2 ] , which results in huge economic loss. Due to the lack of effective treatments, the precise mechanisms underlying the pathogenesis of SCII are poorly understood, making it urgent to identify the exact pathogenesis and implement effective prevention and treatment measures. After the ischemic episode, prolonged neuroinflammation hugely triggers a series of secondary injuries, finally leading to neuronal death. Emerging evidence suggests that neuroinflammation may predominantly contribute to the pathogenesis of SCII [ 3 , 4 ] . Pyroptosis, a form of programmed cell death related to inflammation, is found to be involved in SCII. Gasdermin-D (GSDMD) and caspase family executed pyroptosis and finally disrupted the cell membrane barrier and the release of inflammatory factors [ 5 ] . Microglia, the principal cell type involved in regulating inflammatory responses and undergoing pyroptosis in the central nervous system (CNS), have been found to trigger a cascade of inflammatory and oxidative stress responses when undergoing pyroptosis [ 6 , 7 ] . They release a variety of inflammatory factors and free radicals, which can directly or indirectly affect neuronal survival and function [ 8 ] . The role of microglial pyroptosis has been implicated in the pathophysiological processes of several diseases, including CNS ischemia-reperfusion injury [ 9 ] , traumatic brain injury [ 6 ] , and neurodegenerative disease. Pyroptosis in microglia inflicts damage on neurons and accelerates disease progression. Therefore, inhibiting microglial pyroptosis can attenuate subsequent neuroinflammatory responses and may become a significant therapeutic approach for CNS inflammation-related diseases, including SCII. The triggering receptor expressed on myeloid cells 2 (TREM2), predominantly expressed on the membrane of microglia in CNS, is linked with various neuropathological diseases [ 10 , 11 ] . TREM2 acts mainly through the intracellular adaptor DNAX-activation protein 12 (DAP12) and initiates downstream signaling. Previous studies of TREM2 have focused on degenerative neuropathy such as Alzheimer’s disease [ 12 , 13 ] . Recent studies have investigated the anti-inflammation role of TREM2 in diverse neurological disease models [ 14 – 16 ] . TREM2 deficiency aggravated neuroinflammation in Parkinson’s disease model [ 17 ] . Additionally, TREM2 activation attenuates neuroinflammation and neuronal apoptosis following intracerebral hemorrhage in mice [ 18 ] . TREM2 regulates the inflammatory state by modulating the phenotype of microglia and macrophages. Furthermore, TREM2 has been found to enhance pathogen clearance by inhibiting the pyroptosis of macrophages [ 19 ] . Specifically, a study found that TREM2 has the potential as a regulatory factor in mitigating the effects of cerebral infarction through the modulation of pyroptosis pathways [ 20 ] . However, its specific mechanism is unclear and has not been reported in SCII. Considering the crucial role of TREM2 in microglia, we hypothesized that it could regulate microglial pyroptosis and inhibit neuroinflammation following SCII. FOXO1, by governing the transcription of a myriad of target genes, plays a significant role in modulating the balance between cell survival and death. Recent studies have underscored the importance of FOXO1 in the context of neuroinflammation, particularly its ability to influence the pathways leading to pyroptosis. The phosphorylation status of FOXO1, often regulated by upstream signals such as the PI3K/AKT pathway, determines its nuclear translocation and subsequent gene transcription activities [ 21 ] . This regulatory mechanism is crucial in controlling the expression of GSDMD [ 22 ] . Therefore, the modulation of FOXO1 activity presents a promising therapeutic avenue in mitigating microglial pyroptosis and, by extension, reducing the neurological sequelae of SCII. Our study reveals a novel interaction between TREM2 and FOXO1, demonstrating that TREM2inhibits FOXO1 activity. This inhibition by TREM2 may play a pivotal role in reducing microglial pyroptosis, thereby mitigating neuronal damage and enhancing recovery following SCII. Studies have found that various natural plant extracts can treat or improve ischemia-reperfusion injury in many animal disease models for the antioxidant and anti-inflammatory effects. Diosmetin has been found to protect neuronal function in cerebral ischemia-reperfusion injury [ 23 , 24 ] . Recent studies have revealed that diosmetin can inhibit the activation of NLRP3 inflammasome in lipopolysaccharide (LPS)-induced acute lung injury model [ 25 ] . In an animal model of cerebral ischemia-reperfusion injury, diosmetin protects neurons by inhibiting NLRP3 inflammasome activation [ 23 ] . The precise mechanism by which diosmetin inhibits NLRP3 is unclear, as well as that in TREM2. Given the common mechanism of small-molecule compounds, which often involves binding to membrane proteins [ 26 , 27 ] , we wonder whether diosmetin interact with the transmembrane protein TREM2. We initially validated this hypothesis by molecular docking of the extracellular domain structure of TREM2 and the 3D structure of diosmetin. Subsequently, we further validated our hypothesis using molecular biology techniques. This study investigates the role of diosmetin in the pathological processes of SCII both in vivo and in vitro, providing new directions for drug therapy in the prevention and treatment of SCII. In the present study, we first identified the detrimental role of microglial pyroptosis in SCII. By investigating the influence of TREM2 expression, we explored the inhibitory effect of TREM2 on microglial pyroptosis and its neuroprotective role in SCII. Furthermore, we elucidated the neuroprotective effect of diosmetin in SCII and its close association with TREM2, suggesting its potential interaction with TREM2 as a mechanism of action. Materials and Methods Animals Sprague Dawley (SD) rats, 8–10 weeks old, weighing 250-300g, were purchased from Beijing Vital River Laboratory. Three or four rats were housed in one standard plastic cage with free access to food and water and maintained under standard conditions with a 12 h light/dark cycle. All animal work was in accordance with the ethical committee of China Medical University on animal experiments. Cell culture HAPI cells (Highly Aggressively Proliferating Immortalized, a rat microglia cell line) and VSC4.1 cells (the ventral spinal cord 4.1 motor neurons) were purchased from the Chinese Academy of Sciences Shanghai Cell Bank (Shanghai, China). HAPI cells were cultured in a MEM medium (HyClone, USA) with 10% FBS (Procell Life Science & Technology, China) and 1% penicillin/streptomycin (Solarbio Science & Technology, China). VSC4.1 cells were cultured in high-glucose DMEM (HyClone, USA) with 10% FBS and 1% penicillin/streptomycin. The cells were cultured in an incubator at 5% CO2 and a temperature of 37℃. Experimental design Experiment 1 : In Vivo Investigation of Pyroptosis in SCII To investigate the role of pyroptosis in SCII, we conducted in vivo experiments using SD rats. The rats were divided into three groups: Sham, SCII+DMSO, and SCII+VX-765. We assessed motor neuron function and morphology using the Tarlov scoring system and Hematoxylin-eosin (HE) staining. Experiment 2 : In Vitro Investigation of Pyroptosis in SCII To further investigate the role of pyroptosis in SCII, we utilized HAPI cells in vitro. The cells were divided into three groups: Control, OGD/R+DMSO, and OGD/R+VX-765. Western blot analysis was performed to assess the expression of pyroptosis-related proteins at OGD (6h)/R (12h). Additionally, we examined the impact of microglial pyroptosis on neurons. Culture medium from each HAPI cell group was collected and the supernatant was used to treat VSC4.1 neuronal cells. These neuronal cells were categorized as follows: N group: Fresh complete culture medium; N+Control group: Supernatant from Control group HAPI cells; N+VX-765 group: Supernatant from Control+VX-765 group HAPI cells; N+OGD/R group: Supernatant from OGD/R group HAPI cells; N+ OGD/R+VX-765 group: Supernatant from OGD/R+VX-765 group HAPI cells. We detected changes in the expression of the anti-apoptotic protein Bcl2 in neurons using immunofluorescence. Cell toxicity in neurons was assessed using the CCK-8 assay. Experiment 3: Investigating the Role of TREM2 in Microglial Pyroptosis both in vivo and in vitro To investigate TREM2's function in SCII, we upregulated TREM2 expression in rats through Intrathecal injection of AAV particles encoding the TREM2 gene. SD rats were grouped as follows: Sham, SCII, SCII+AAV-NC (negative control), and SCII+AAV-TREM2. Motor neuron function was assessed using the Tarlov score, while HE staining was used to evaluate neuronal morphology. Neuroinflammation was detected through immunofluorescent staining for IBA-1, and Western blotting was used to assess pyroptosis-related proteins. To explore the role of TREM2 in OGD/R-induced microglial pyroptosis, HAPI cells were transduced with lentiviruses overexpressing TREM2. The cells were divided into four groups: Control, OGD/R, OGD/R+OE-NC, and OGD/R+OE-TREM2. Immunofluorescence was employed to detect the expression of TREM2 and cleaved caspase-1 in each group. Experiment 4: Investigating the Role of TREM2 in Modulating FOXO1 to Regulate Microglial Pyroptosis We explored the potential mechanism of TREM2 in regulating cell pyroptosis. FOXO1, an essential transcription factor regulating various cellular activities, was identified as a downstream effector of TREM2. In vitro experiments were designed to study the relationship between TREM2 and FOXO1. HAPI cells were grouped into Control, OGD/R, OGD/R+OE-NC, and OGD/R+OE-TREM2. Immunofluorescence and Western blotting were used to detect changes in FOXO1 expression among the groups. To further verify the relationship between TREM2 and FOXO1, HAPI cells were divided into three groups: OGD/R, OGD/R+OE-TREM2, and OGD/R+OE-TREM2+OE-FOXO1. The latter group was transfected with two lentiviruses to overexpress both TREM2 and FOXO1 before OGD/R. The expression levels of pyroptosis-related proteins were measured in these groups. Experiment 5: Investigating the Involvement of the PI3K/AKT Pathway in TREM2-Mediated Regulation of FOXO1 and Pyroptosis To determine whether TREM2 regulates FOXO1 through the PI3K/AKT pathway, HAPI cells were grouped into Control, OGD/R, OGD/R+OE-TREM2, and OGD/R+OE-TREM2+LY294002. Western blotting was employed to assess the expression levels of PI3K, AKT, phosphorylated proteins p-PI3K and p-AKT, FOXO1, and pyroptosis-related proteins in these groups. The Gene Expression Profiling Interaction Analysis (GEPIA) database was used to analyze the expression correlation between FOXO1 and GSDMD. Additionally, chromatin immunoprecipitation（CHIP）was used to investigate the transcriptional regulatory effect of FOXO1 on GSDMD. Experiment 6: Investigating the Role of Diosmetin in SCII and Its Interaction with TREM2 In this experiment, we investigated the impact of diosmetin on SCII both in vivo and in vitro. For the in vivo component, SD rats were divided into five groups: Sham, SCII, SCII+Vehicle, SCII+Dio (40mg/kg), and SCII+Dio (80mg/kg). Behavioral assessments included Tarlov scores, and H&E staining was performed to evaluate different groups. In the in vitro part, the effects of diosmetin on microglial pyroptosis were studied. HAPI cells were divided into Control, OGD/R, and OGD/R+Dio groups. Changes in pyroptosis-related proteins were assessed in these groups. To further investigate the relationship between diosmetin and TREM2, molecular docking was conducted using Autodock software to predict the interaction between the extracellular domain of TREM2 and diosmetin. To validate the docking results, HAPI cells were divided into Control, OGD/R, OGD/R+Dio, and OGD/R+Dio+si-TREM2 groups. In the OGD/R+Dio+si-TREM2 group, si-TREM2 was transfected into HAPI cells, and they were subsequently exposed to complete culture medium containing diosmetin (10μM) for one hour before OGD/R. The expression levels of pyroptosis-related proteins were observed in these groups. Rat model of spinal cord ischemia-reperfusion injury and treatment All animal experiments were approved by the Ethics Committee of China Medical University. The procedures were following the guidelines set by the Institutional Animal Care and Use Committee. Rats were anesthetized with pentobarbital (Sigma, USA) by intraperitoneal injection (35 mg/kg). Following endotracheal intubation, mechanical ventilation, and aortic arch exposition, the spinal cord ischemia-reperfusion injury model was established by blocking the aortic arch for 14 min, as previously described [28] . Rats in the sham group were subjected to the same surgery without clamping. Each postoperative rat was placed in a cage alone and kept warm. Reagent treatment of animals VX-765 is a specific pyroptosis inhibitor by targeting caspase-1. It was purchased from MCE company (CAS No.: 273404-37-8). According to the instructions, it was diluted with dimethyl sulfoxide (DMSO) to a working solution (25 mg/ml). The administration involved intraperitoneal injection at a dosage of 30 mg/kg/day for three consecutive days, with the first dose administered 30 minutes before the initiation of SCII modeling. Diosmetin (C 16 H 12 O 6 , molecular weight = 300.26, CAS NO.520-34-3, purity =99.8%,) was purchased from MCE. According to the instructions, the diosmetin powder was dissolved with 10% DMSO，40% PEG300，5% Tween-80, and 45% saline in turn to a concentration of 5mg/ml. Rats in the SCII+Diosmetin group were received intraperitoneal injections of diosmetin dilution separately at a dose of 40 mg/kg/day and 80 mg/kg/day for 3 continuous days before the model establishment. Intrathecal injection of Adeno-associated virus and si-RNAs Rats were briefly anesthetized with isoflurane and placed in a prone position with lower back elevated and flexed ventrally. A Microliter Syringes needle (Gaoge, Shanghai, China) was advanced into the subarachnoid space at L4–L6 levels. A tail-flick was the mark of the success of intrathecal injection. Once this was confirmed, the solutions were injected within 1 minute. To increase exogenous expression of TREM2 in SD rats, The AAV-TREM2 and AAV-NC (negative control) (ObiO, Shanghai, China) were delivered by intrathecal injection. The titers of AAV particles were between 1 × 10 12 and 2 × 10 12 vg/ml and 20µl of AAV-TREM2 or AAV-NC was used per rat. AAV injection was performed 1 month before SCII induction. To investigate the role of FOXO1 in SCII, FOXO1 siRNA (si-FOXO1) was intrathecally injected to knock down the expression level of FOXO1. Si-FOXO1 and a control siRNA(si-NC) were purchased from RiboBio, and the sequences were as follows: si-FOXO1, 5′-GGACAGCAAAUCAAGUUAUtt-3′, si-NC, 5′-UUCUCCGAACGUGUCACGUT-T-3′. The si-FOXO1 powder was reconstituted in enzyme-free water to a concentration of 1 nmol/20 μl. For the 3 days preceding the modeling, si-FOXO1 or si-NC was intrathecally administered to rats at a daily dose of 1 nmol per rat for 3 consecutive days. Neurological function tests Rats were acclimated to the behavioral test environment at least 1h before testing. Tarlov scale was performed to measure the neurological function of the animals 24h following SCII induction. Modified Tarlov criteria were used as follows: 0 = no voluntary hind-limb function; 1 = only perceptible joint movement; 2 = active movement but unable to stand; 3 = able to stand but unable to walk; or 4 = completely normal hind-limb motor function. Hematoxylin and eosin (H&E) staining HE staining was conducted on spinal cord sections. Briefly, lumbar enlargement segments of spinal cord were embedded in paraffin and sectioned at 4 um thickness. Deparaffinization and rehydration: Sections were deparaffinized in xylene (2 x 10 min) and rehydrated through a graded ethanol series (100%, 95%, 80%, and 70%; 5 min each), followed by distilled water rinse. Antigen retrieval: Sections were submerged in 10 mM sodium citrate buffer (pH 6.0) and heated in a pressure cooker for 3 min at full pressure. After cooling at room temperature for 20 min, sections were washed with PBS (2 x 5 min).Permeabilization: Sections were treated with 0.1% Triton X-100 in PBS for 10 min, followed by PBS washes (2 x 5 min).All other staining steps were performed following standard protocols. TUNEL staining TUNEL staining was conducted using the Cy3 TUNEL cell apoptosis assay kit (Bioscience, China). Following deparaffinization and antigen retrieval, sections were incubated with the TUNEL reaction mixture for 60 min at 37°C in a humidified chamber, protected from light. Counterstaining: Sections were counterstained with DAPI (4’,6-diamidino-2-phenylindole) for 10 min, followed by a brief rinse in PBS. Fluorescence microscopy was used to visualize TUNEL-positive cells (red) and nuclei (blue). Oxygen-glucose deprivation/reperfusion model and cell cytotoxicity assay A cellular model of oxygen-glucose deprivation/reperfusion was performed to mimic SCII in vitro. After normal culture for at least 24h, HAPI cells with glucose-free medium were transferred into an incubation chamber flushed with a gas mixture of 95% N2 and 5% for 15 minutes. The chamber was then sealed and placed into a humidified incubator at 37°C 5% CO2 for 6h. Culturing was continued for 0-24h before further measurement. The cytotoxicity of neurons was measured by Cell Counting Kit-8 (CCK-8; Beyotime Biotechnology , Shanghai, China) according to the instructions provided with the reagents. VSC4.1 Neuronal Cell Culture with Conditioned Microglia Media To co-culture VSC4.1 neuronal cells with conditioned microglia media, HAPI cell culture media were collected following various interventions and centrifuged to obtain the supernatant. VSC4.1 neuronal cells prepared a day in advance, were washed with sterile PBS and added with supernatant to co-culture for another 12h. Cell Transfection To meet experimental requirements, HAPI cells were stably transfected with lentiviral vectors (LV) for TREM2 overexpression. The TREM2 lentiviral particles were purchased from Synthbio, and the cells were stably transfected as previously described [29] . The stable cell line overexpressing TREM2 was selected by exposure to puromycin for 3 days. Additionally, HAPI cells were transiently transfected with TREM2 siRNA using Lipo3000 in a 12-well plate. The culture media were changed 24 hours post-transfection. Drug application in vitro VX-765, purchased from MCE company (CAS No.: 273404-37-8, 10 mM * 1 mL in DMSO) was diluted in a complete culture medium to a final concentration of 1 μM and applied before the onset of OGD/R. LY294002 (Sigma), a competitive PI3K-AKT pathway inhibitor, were dissolved in DMSO and diluted in saline. HAPI cells were pretreated with LY294002 (20μM) 1 h before OGD/R. Western blotting Protein samples were obtained from L1-L3 spinal cord segment and HAPI cells. All the protein samples were lysed in RIPA buffer, and quantified by using a BCA protein assay kit following sonication. Equal amounts of protein (30 µg/lane) were separated on SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat dry milk (Nestle) and incubated at 4℃ overnight with the following antibodies: goat anti-rabbit TREM2 (1:1000, Abmart, China), goat anti-rabbit FOXO1 (1:2000, Proteintech, China), goat anti-mouse IL-1β (1:1000, Abmart, China), goat anti-rabbit cleaved-caspase 1(1:1000, Abmart, China), GSDMD, NLRP3 Docking and molecular dynamics The 3D structures of TREM2 ectodomains was retrieved from the PDB database (http://www.rcsb.org/pdb ), and the small-molecule ligand Diosmetin was obtained from the ZINC database (https://zinc.docking.org/). Both two 3D structures were used to perform molecular docking following hydrogenation and dehydration with Autocock Tools software. The docking results were analyzed and visualized with PyMol software. Statistical analyses Statistical analyses were performed using GraphPad Prism 7.0. We tested the normality of the data using the Kolmogorov-Smirnov test. For data that followed a normal distribution, data was expressed as mean ± SEM. Depending on the grouping of experiments and the characteristics of different data, the independent samples t-test was used for comparisons between two groups, and one-way ANOVA or two-way ANOVA for repeated measurements were used for comparisons among multiple groups. For non-normally distributed data, the Mann-Whitney U test was used. A p value of less than 0.05 was considered to indicate statistically significant differences. Results Pyroptosis occurred in SD rats following SCII By searching the GEO database, we identified one experimental animal model data (GSE138966) that was relevant to our study. Using Metascape, we performed enrichment analysis on the top 500 differentially expressed genes, which revealed that the inflammatory response was the most significant pathological process in rats after spinal cord ischemia-reperfusion injury. We further examined the changes in inflammatory factors after SCII in rats. Western blot analysis revealed an increased expression of IL-1β, which increased at 12h and peaked at 24h post-SCII ( p <0.05, p <0.01). Detection of other pyroptosis-related proteins at 24h post-SCII showed elevated expression levels of NLRP3, TNF-α and cleaved caspase-1 ( p <0.01). The above results demonstrate significant inflammation and pyroptosis occurring after SCII. Pyroptosis inhibitor VX-765 mitigated the neural damage caused by SCII Investigating the impact of pyroptosis on motor neurons, we first observed behavioral changes in different groups of rats by utilizing Tarlov scores for lower limb motor function evaluation. The Tarlov score results showed a significant decrease in the SCII+DMSO group compared to the Sham group ( p <0.01), indicating impaired motor function caused by SCII. However, the rats pretreated with VX-765 exhibited an increased score ( p <0.01), indicating VX-765 reversed SCII-induced motor neuronal dysfunction. HE staining of the rat spinal cord ventral horn revealed intact neurons in the different groups. The number of intact neurons reflected the neuronal damage. The rats in SCII group had fewer intact neurons ( p <0.01), indicating neuronal damage. However, the rats with VX-765 treatment had more intact neurons ( p <0.01). These findings suggest that VX-765 improved SCII-induced motor dysfunction, highlighting the role of pyroptosis in the SCII pathology. OGD/R induced microglial pyroptosis Firstly, through immunofluorescence staining, we evaluated the change of IL-1β expression levels after OGD/R. The results showed that OGD/R induced a significant increase of IL-1β ( p <0.01), as shown in Figure 3. A-B. However, VX-765 treatment was able to inhibit the elevation of IL-1β triggered by OGD/R ( p <0.01). Moreover, we also observed the morphological changes caused by OGD/R: the cells became swollen and round, and the cell boundaries were blurred. These changes were consistent with the characteristics of pyroptosis. We examined changes in pyroptosis-related proteins in each group. The results showed that OGD/R led to a significant increase of NLRP3, IL-1β and cleaved caspase-1 in microglia ( p <0.01). However, VX-765 treatment was able to reverse these effects ( p <0.01). The above results indicated that OGD/R induced pyroptosis. OGD/R-induced microglial pyroptosis enhanced neuronal damage. We previously demonstrated that OGD/R could induce pyroptosis in microglia. To further verify its effects on neuronal cells, we treated the neuronal cells with the supernatant of microglial cells from different groups, and detected changes in the expression of the anti-apoptotic protein Bcl2 in the neuronal cells. Bcl2 expression was reduced following OGD/R (N+OGD/R vs N+Control, p <0.01). However, treatment with VX-765 appears to restore Bcl2 intensity towards normal levels (N+OGD/R+VX-75 vs N+OGD/R, p <0.01), indicating a protective effect against OGD/R-induced apoptosis. The CCK-8 assay was utilized to clarify the impact of OGD/R on the viability of VSC 4.1 neuronal cells (Fig. 4C). The results indicated that cell viability was impaired by OGD/R treatment and improved by VX-765 treatment. These results suggest that neuronal cells suffer damage cultured in the supernatant from microglia post-OGD/R. However, the use of the pyroptosis inhibitor VX-765 can mitigate the neuronal damage caused by OGD/R. TREM2 improved the neurological deficit triggered by SCII To investigate whether TREM2 is involved in the process of SCII, we measured the expression levels of TREM2 at different time points following SCII. The results showed that TREM2 expression increased at 12h, peaked at 24h following SCII induction ( p <0.05, p <0.01 respectively). We assessed TREM2's potential to protect motor neurons during SCII in rats by Tarlov scores and HE staining. Based on previous experimental results, 24h post-SCII was selected as the observation time point. The upregulation of TREM2 via AAV in rat spinal cord was confirmed by Western blotting. Rats with TREM2 overexpression showed a higher Tarlov score ( p <0.01), indicating improved motor function. Similarly, HE staining revealed that overexpression of TREM2 improved motor neuron damage induced by SCII. When compared with Sham group, SCII group had a lower number of intact neurons ( p <0.01). Rats with TREM2 overexpression were able to reverse the loss of motor neurons induced by SCII ( p <0.01). These results indicated a protective effect of TREM2 overexpression on motor neurons. TREM2 inhibited microglial activation induced by SCII Microglia play a crucial role in regulating inflammation and pyroptosis in the CNS. Our study examined their activation after SCII and the effect of TREM2 on this process. Findings revealed that SCII induced an increase in IBA-1 staining intensity, indicating enhanced microglial activation ( p <0.01). However, the TREM2-overexpressing rats showed reduced IBA-1 staining intensity ( p <0.01), as displayed in Fig 6. These findings imply that TREM2 overexpression can suppress microglia activation induced by SCII, underscoring the involvement of microglial activation in spinal cord ischemia-reperfusion injury. TREM2 inhibit ed the pyroptosis caused by SCII in vivo To discern whether TREM2's neuroprotective effects against SCII was associated with pyroptosis, we examined the impact of TREM2 on pyroptosis-related proteins. Western blot analysis revealed enhanced expression of GSDMD, IL-1β, and cleaved caspase-1 following SCII ( p <0.01). However, in the TREM2 overexpressed SCII+AAV-TREM2 group, these expression levels were reduced when compared with AAV-NC group. Furthermore, we investigated the expression of apoptosis-related proteins Bcl2 and Bax. SCII caused increased pro-apoptotic Bax expression ( p <0.01) and decreased anti-apoptotic Bcl2 expression ( p <0.05). Interestingly, the TREM2 overexpressed SCII+AAV-TREM2 group exhibited decreased expression of Bax and elevated level of Bcl2 ( p <0.01). These experimental findings suggest that SCII induced pyroptosis and apoptosis in spinal cord tissue, while TREM2 overexpression reversed these changes. TREM2 overexpression inhibited cleaved caspase-1 expression caused by OGD/R in vitro Cleaved caspase-1 serves as a critical biomarker for detecting pyroptosis. We measured the levels of cleaved caspase-1 in HAPI cells across various groups to assess the occurrence and severity of pyroptosis. To overexpress TREM2 in HAPI cells, we utilized lentiviruses, and confirmed the transfection efficiency via Western blot analysis, as shown in Figure 9A-B. Exposure to OGD/R resulted in increased expression levels of both TREM2 and cleaved caspase-1 ( p <0.05, p <0.01 respectively). The TREM2 overexpression (OE-TREM2) group displayed a substantial increase in TREM2 levels ( p <0.01), confirming successful lentiviral transfection, along with a reduced level of cleaved caspase-1 compared to the control group (OE-NC) ( p <0.01). The expression levels of TREM2 and cleaved caspase-1 in both the OGD/R group and the OGD/R+OE-NC group showed no significant differences. TREM2 inhibited the activity of FOXO1 caused by OGD/R Given that TREM2 has been shown to inhibit GSDMD expression, as previously verified in Figure 8A-B, and considering earlier studies suggesting FOXO1's role in regulating GSDMD transcription [22] , our research focused on whether TREM2 influences GSDMD expression through FOXO1 regulation.. To delve deeper into the relationship between TREM2 and FOXO1, we examined the impact of TREM2 overexpression on FOXO1 expression in vitro. The results from immunofluorescence (Fig. 9A-B) revealed a significant increase in FOXO1 expression in the OGD/R group compared to the control group ( p <0.01). Notably, HAPI cells overexpressing TREM2 through lentiviral transfection were able to reverse the OGD/R-induced upregulation of FOXO1 ( p <0.01). This pattern was similarly observed in the Western blot analysis. As depicted in Fig. 9C-D, FOXO1 expression significantly rose in the OGD/R group in contrast to the Control group ( p <0.01). Conversely, FOXO1 expression in the OE-TREM2+OGD/R group was notably lower compared to the OGD/R group ( p <0.01). These findings demonstrate that TREM2 overexpression effectively counteract the OGD/R-induced increase in FOXO1 expression. The immunofluorescence results, as shown in Figure 9A-B, indicated a significant elevation in FOXO1 expression in the OGD/R group compared to the control group ( p <0.01). Remarkably, HAPI cells overexpressing TREM2 via lentiviral transfection successfully mitigated the OGD/R-induced upregulation of FOXO1 ( p <0.01). This trend was consistently observed in Western blot analyses; Figure 9C-D illustrates that FOXO1 expression was substantially higher in the OGD/R group than in the control group ( p <0.01). However, in the group overexpressing TREM2 and subjected to OGD/R (OE-TREM2+OGD/R), FOXO1 levels were significantly reduced compared to the OGD/R group alone ( p <0.01). These results suggest that TREM2 overexpression effectively counteracts the OGD/R-induced increase in FOXO1 expression. The rescue experiment provided further confirmation of TREM2's suppression of FOXO1 To clarify the relationship between TREM2 and FOXO1 in the context of anti-pyroptosis, we co-overexpressed both genes in a single group of HAPI cells. Subsequently, we assessed the changes in inflammation and pyroptosis-associated markers across the groups. Initially, we confirmed the successful overexpression of TREM2 (Fig. 8A-B) and FOXO1 (Fig. 10A-B). As shown in Figure 10C-G, the Western blot analysis revealed that, compared to the OGD/R group, the expression levels of NLRP3, GSDMD, IL-1β, and cleaved caspase-1 were significantly reduced in the OGD/R+OE-TREM2 group ( p <0.01, p <0.01, p <0.05, p <0.05, respectively). Furthermore, relative to the OGD/R+OE-TREM2 group, increased expression levels of NLRP3, GSDMD, IL-1β, and cleaved caspase-1 were observed in the OGD/R+OE-TREM2+OE-FOXO1 group ( p <0.01, p <0.01, p <0.05, p <0.05, respectively). These findings strongly support the role of TREM2 in modulating FOXO1 activity, highlighting its influence on the regulation of inflammation and pyroptosis-related markers. TREM2 inhibited pyroptosis via PI3K/AKT/FOXO1/GSDMD pathway To delve deeper into how TREM2 suppresses microglial pyroptosis and its interaction with FOXO1, we investigated the primary downstream signaling pathway of TREM2, specifically the PI3K/AKT pathway. Additionally, we utilized the PI3K inhibitor LY294002 in a rescue experiment to substantiate the underlying mechanisms further. We examined the impact of TREM2 overexpression in HAPI cells on the PI3K/AKT signaling pathway and its downstream effector, FOXO1, following OGD/R. The results showed that phosphorylation levels of PI3K and AKT were significantly increased in the OGD/R+OE-TREM2 group compared to the OGD/R group ( p <0.01), while FOXO1 expression was markedly decreased ( p <0.01). Pre-treatment with LY294002 in the TREM2-overexpressing HAPI cells led to reduced phosphorylation levels of PI3K and AKT relative to the OGD/R+OE-TREM2 group ( p <0.01). These findings suggest that TREM2 inhibits microglial pyroptosis via the PI3K/AKT/FOXO1 signaling pathway. Moreover, the downregulation of FOXO1 corresponded with decreased expressions of NLRP3, IL-1β, and particularly GSDMD ( p <0.01), as depicted in Figure 11G-J. We also conducted a correlation analysis between FOXO1 and GSDMD by GEPIA database, which yielded significant P and R values (Fig. 11K). Furthermore, a CHIP assay confirmed the binding between FOXO1 and the promoter region of GSDMD (Fig. 11L). These comprehensive approaches underscore the critical role of TREM2 in modulating key inflammatory pathways and its therapeutic potential in reducing microglial pyroptosis. Diosmetin pretreatment improved the neurological deficit triggered by SCII Firstly, we explored the impact of diosmetin on SCII outcomes in vivo by administering varying concentrations of the compound. The Tarlov scores for the SCII group and the SCII+Dio (40 mg/kg) group were lower compared to the sham group ( p <0.01). Rats treated with diosmetin in both concentrations showed significant improvements in behavioral scores relative to those in the SCII group ( p <0.01). Notably, the SCII+Dio (80 mg/kg) group showed a more pronounced improvement compared to the SCII+Dio (40 mg/kg) group ( p <0.01). There was no statistical difference between the SCII group and the SCII+NC group. In line with these behavioral findings, H&E staining analysis indicated a significant reduction in the number of intact neurons in the SCII group compared to the sham group ( p <0.01). The administration of diosmetin effectively counteracted this decrease ( p <0.01), with the high-concentration SCII+Dio (80 mg/kg) group exhibiting the greater degree of amelioration( p <0.01). The results suggest that diosmetin has a protective effect on neurons against spinal cord ischemia-reperfusion injury. Diosmetin inhibited pyroptosis-related protein changes induced by SCII . To explore whether the neuroprotective effect of diosmetin is related to pyroptosis, the expression levels of pyroptosis-related proteins were detected by Western blot analysis. Based on the results of the previous part, 80mg/kg was used as the dosage of diosmetin for subsequent experiments. As shown in Fig. 13A-E, the results revealed that the levels of FOXO1, NLRP3, IL-1β, and cleaved caspase-1 were significantly increased in SCII group compared to sham group ( p <0.01). However, in the SCII+Dio group pretreated with diosmetin, the expression levels of FOXO1, NLRP3, IL-1β, and cleaved caspase-1 were significantly decreased compared to SCII group ( p <0.05, p <0.01, p <0.01 p <0.01, respectively). Additionally, in vitro experiments also found that diosmetin could inhibit GSDMD elevation caused by OGD/R (Fig. 13F-G). Diosmetin played the anti-pyroptosis role via TREM2 /PI3K/AKT/FOXO1 pathway To explore whether the anti-pyroptosis effect of diosmetin was related to its binding to TREM2, we first performed molecular docking analysis between them. We obtained the 3D structure of the extracellular domain of TREM2 (PDB: 6YYE) from the Protein Data Bank (PDB) and the 3D structure of the small molecule compound Diosmetin (ZINC5733652) from the ZINC database (zinc.docking.org/). Subsequently, we performed dehydration and hydrogenation treatments on the protein and the small molecule compound separately using AutoDock software. We then conducted docking simulations and selected the binding site with the highest negative binding energy for analysis. The results indicated that the extracellular domain structure of TREM2 and Diosmetin formed hydrogen bonds at the GLU-220 and ILE-246 residues, with a binding energy of -5.46 kJ/mol. To further investigate whether diosmetin's anti-pyroptosis effect is related to TREM2, we utilized siRNA technology to silence TREM2 expression in HAPI cells. Expression levels of GSDMD, IL-1β, and cleaved caspase-1 in different experimental groups were detected. As shown in Fig. 14C-G, compared to the OGD/R group, the OGD/R+Dio group exhibited a reduction in the expression levels of GSDMD, FOXO1, IL-1β, and cleaved caspase-1 ( p <0.05, p <0.01, p <0.05, p <0.05), consistent with our previous results. To elucidate the specific mechanism of diosmetin, we used a PI3K inhibitor to detect the changes in phosphorylation and expression levels of PI3K/AKT/FOXO1 proteins in different groups. The results showed that compared with OGD/R group, the phosphorylation levels of PI3K and AKT were increased ( p <0.01), while the expression level of FOXO1 was decreased ( p <0.01) in OGD/R+Dio group. Compared with OGD/R+Dio group, the phosphorylation levels of PI3K and AKT were decreased ( p <0.01, p <0.05), while the expression level of FOXO1 was increased ( p <0.05) in OGD/R+Dio+LY294002 group. These results suggested that diosmetin regulated pyroptosis through the PI3K/AKT/FOXO1 pathway. Discussion In the current study of SCII, we underscore the significant role of microglial pyroptosis in contributing to neuronal damage and functional impairments. Our findings suggest that modulating microglial behavior can reduce pyroptosis, thereby improving neuronal function. Additionally, we have identified a natural compound that acts as a modulator, offering neuroprotection by reducing microglial pyroptosis. These discoveries open new pharmacological research directions for the treatment and prevention of SCII. Microglia pyroptosis has been identified as a critical factor in neuroinflammation, leading to significant neuronal damage and impairment of neuronal survival参考文献. Numerous studies have demonstrated that pyroptosis in microglia plays a pivotal role in central nervous system ischemia-reperfusion injury and oxygen-glucose deprivation-reoxygenation models [ 30 – 32 ] . It has been shown that inhibiting microglial pyroptosis can ameliorate neuronal cell damage [ 33 – 36 ] . In this study, we focused on the role of microglial pyroptosis in SCII and found that SCII induces microglial activation characterized by the upregulation of NLRP3, caspase-1, and IL-1β, which are crucial in exacerbating neuronal damage. The significant finding of our research is that inhibition of microglial pyroptosis through VX-765, a caspase-1 inhibitor, markedly reduces SCII-induced motor neuron injury and neurological dysfunction. VX-765 served effectively to establish a benchmark for the inhibition of microglial pyroptosis, confirming the pathway's critical role in neuronal injury and the potential for therapeutic intervention. We chose VX-765 for its proven effectiveness and tolerability in animal studies, and its unique ability to reduce inflammation without the side effects typical of similar compounds [ 37 , 38 ] . These outcomes imply that microglial pyroptosis plays a role in the pathogenesis of SCII and may represent a promising therapeutic target. TREM2 has been implicated in the process of pyroptosis [ 19 ] . Enhanced TREM2 signaling has been shown to reduce microglial activation, decrease neuroinflammation, and subsequently reduce neuronal damage and neurodegeneration [ 18 , 29 , 39 , 40 ] . Several reports have highlighted the role of TREM2 in mitigating neuroinflammation by inhibiting the NRLP3 inflammasome in the CNS [ 19 , 41 – 43 ] .. We observed an increase in TREM2 expression following SCII, which was time-dependent and consistent with increases noted in other disease models [ 15 , 18 , 44 ] . The change of TREM2 expression implies that it is involved in the pathophysiological process of SCII. Additionally, we found that TREM2 overexpression could inhibit microglia activation and improve motor neuron function damage caused by SCII. Further research has revealed that TREM2 can suppress the expression of proteins associated with pyroptosis. In vitro experiments utilizing immunofluorescence assays have demonstrated that microglia overexpressing TREM2 exhibit reduced levels of cleaved-caspase-1 following OGD/R, further confirming the inhibitory effect of TREM2 on microglial pyroptosis. Prior studies have indicated that TREM2 enhances corneal resistance to damage by inhibiting caspase-1-dependent pyroptosis [ 45 ] and promotes pathogen clearance by preventing macrophage pyroptosis [ 19 ] . Moreover, macrophages with TREM2 deficiency are more vulnerable to pyroptotic death resulting in increased inflammation [ 42 ] . Taken together, these findings, including ours, suggest that TREM2 can regulate its host cells to inhibit pyroptosis, providing a multi-faceted approach to controlling inflammatory processes in neurodegenerative conditions. FOXO1 is involved in the anti-pyroptosis effect of TREM2. FOXO1 plays a critical role in regulating genes associated with metabolic disorders, autophagy, and oxidative stress. It has been observed to alleviate myocardial and renal ischemia-reperfusion injuries by dampening inflammatory responses and protecting mitochondrial function [ 46 , 47 ] . However, in cases of hepatic ischemia-reperfusion injury, FOXO1 triggers inflammation and cell death through the XBP1-Foxo1 axis [ 48 ] . Additionally, one study found that neutrophils expressing high levels of FOXO1 infiltrate the brain in both acute and chronic phase after traumatic brain injury (TBI), exacerbating acute inflammatory brain injury and promoting depression caused by late TBI [ 49 ] . Hence, the role of FOXO1 in ischemia-reperfusion injuries can vary depending on the tissue, organ, signaling pathways, and regulatory factors involved. FOXO1’s activity and function are modulated by various post-translational modifications, with phosphorylation being a predominant modification that affects its subcellular localization and transcriptional activity. Our study unveiled that TREM2 suppressed FOXO1 activity, which was verified by co-overexpressing both TREM2 and FOXO1 genes to rescue. We subsequently verified that TREM2 phosphorylates FOXO1 via the PI3K/AKT pathway by utilizing the PI3K inhibitor LY294002. This led to FOXO's nuclear translocation and the inhibition of its transcriptional functions. Additionally, our research confirmed that FOXO1 is capable of regulating the transcription of GSDMD, a pivotal executor of pyroptosis, as has been validated in other disease models [ 22 , 46 ] . These observations strongly indicate that FOXO1’s role in the transcriptional regulation of GSDMD is both consistent and widespread across different disease states. Diosmetin exhibits anti-pyroptotic properties and is associated with TREM2 Our study revealed that pre-treatment with diosmetin significantly ameliorated SCII-induced motor neuron deficits, aligning with the findings of previous research that showcased diosmetin's ability to protect neurons in cases of cerebral ischemia-reperfusion injury [ 23 ] . Furthermore, our in vitro experiments demonstrated that diosmetin mitigated OGD/R-induced pyroptosis of microglia. These outcomes introduce new prospects for the prevention and treatment of SCII in clinical practice. To elucidate the working mechanism of diosmetin, we explored its interaction with TREM2. It is known that TREM2 is a membrane protein exclusively expressed on the surface of microglia in the CNS, where it plays a role in inhibiting pyroptosis. Our investigation indicated that diosmetin may bind to TREM2, thereby exerting its anti-pyroptotic and neuroprotective effects through this pathway. By using Autodock software for molecular docking, we identified a potential binding relationship between diosmetin and the extracellular domain structure of TREM2, with a binding energy of -5.46 kJ/mol. Recent studies have suggested that a binding energy of less than − 5 kJ/mol signifies a strong interaction between small molecule compounds and proteins [ 50 , 51 ] . Additionally, when we knocked down TREM2 in HAPI cells in vitro, the neuroprotective impact of diosmetin significantly diminished, further substantiating the hypothesis that diosmetin exerts its effects through binding to TREM2. While these findings offer new insights into the roles of diosmetin and TREM2 in SCII, our study does come with certain limitations. One limitation pertains to the utilization of cell lines instead of primary cells, which may not entirely capture the characteristics of microglial cells. Nevertheless, the use of cell lines is well-documented in prior studies and can still provide valuable insights into microglial biology and function. Additionally, we require more experimental evidence to definitively confirm the precise binding relationship between diosmetin and TREM2. Furthermore, the pharmacological properties, side effects, and safety profile of diosmetin warrant further investigation. More clinical studies are also essential in the future to validate our findings and explore the potential application of diosmetin in the clinical prevention and treatment of SCII. Conclusion Our results suggest that the TREM2-mediated PI3K/AKT/FOXO1/GSDMD pathway holds promise as a therapeutic target for SCII. Furthermore, diosmetin emerges as a promising candidate for the treatment of spinal cord I/R injury, with its anti-pyroptosis actions offering potential therapeutic benefits. Subsequent studies are necessary to validate these findings and fully unlock the therapeutic potential of diosmetin in SCII treatment. Abbreviations AAV: Adeno-associated virus AKT: /protein kinase B CNS: central nervous system DMSO: Dimethyl sulfoxide FOXO1: forkhead box protein O1 GSDMD: gasdermin D LV: Lentivirus OGD/R: Oxygen-glucose deprivation/reoxygenation PI3K: phosphatidylinositol 3-kinase SCII: Spinal cord ischemia-reperfusion injury TREM2: Triggering receptor expressed on myeloid cells 2 Declarations Ethics declarations All animal experiments were conducted in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals and received approval from the Institutional Animal Care and Use Committee of China Medical University Consent for publication All authors are consent for publication. Funding This work was supported by National Natural Science Foundation of China [grant numbers 82371287]. Authors' contributions HM and SDL designed the experiment. TF and SDL drafted the main manuscript, and HM revised and proofread the manuscript. SDL and XYZ performed the experiments, YD prepared all the figures. KXW and YJZ analyzed the data and organized the original experimental materials. YD and TF provided technical guidance for the experiments. All authors reviewed the manuscript. Acknowledgements Not applicable. Declaration of Competing Interest The authors declare that there is no conflict of interest on the article. Availability of data and materials The data that support the findings of this study are available from the corresponding author upon reasonable request. References HIRAOKA T, KOMIYA T, TSUNEYOSHI H, et al. Risk factors for spinal cord ischaemia after thoracic endovascular aortic repair [J]. Interact Cardiovasc Thorac Surg, 2018, 27(1): 54-9. ZHU P, LI J X, FUJINO M, et al. Development and treatments of inflammatory cells and cytokines in spinal cord ischemia-reperfusion injury [J]. Mediators Inflamm, 2013, 2013: 701970. MI J, YANG Y, YAO H, et al. Inhibition of heat shock protein family A member 8 attenuates spinal cord ischemia-reperfusion injury via astrocyte NF-kappaB/NLRP3 inflammasome pathway : HSPA8 inhibition protects spinal ischemia-reperfusion injury [J]. J Neuroinflammation, 2021, 18(1): 170. FU J, SUN H, WEI H, et al. Astaxanthin alleviates spinal cord ischemia-reperfusion injury via activation of PI3K/Akt/GSK-3beta pathway in rats [J]. J Orthop Surg Res, 2020, 15(1): 275. HSU S K, LI C Y, LIN I L, et al. Inflammation-related pyroptosis, a novel programmed cell death pathway, and its crosstalk with immune therapy in cancer treatment [J]. Theranostics, 2021, 11(18): 8813-35. GU L, SUN M, LI R, et al. Microglial pyroptosis: Therapeutic target in secondary brain injury following intracerebral hemorrhage [J]. Front Cell Neurosci, 2022, 16: 971469. GU L, SUN M, LI R, et al. Didymin Suppresses Microglia Pyroptosis and Neuroinflammation Through the Asc/Caspase-1/GSDMD Pathway Following Experimental Intracerebral Hemorrhage [J]. Front Immunol, 2022, 13: 810582. LI Y, SONG W, TONG Y, et al. Isoliquiritin ameliorates depression by suppressing NLRP3-mediated pyroptosis via miRNA-27a/SYK/NF-kappaB axis [J]. J Neuroinflammation, 2021, 18(1): 1. LIU X, ZHANG M, LIU H, et al. Bone marrow mesenchymal stem cell-derived exosomes attenuate cerebral ischemia-reperfusion injury-induced neuroinflammation and pyroptosis by modulating microglia M1/M2 phenotypes [J]. Exp Neurol, 2021, 341: 113700. KOBER D L, BRETT T J. TREM2-Ligand Interactions in Health and Disease [J]. J Mol Biol, 2017, 429(11): 1607-29. JAY T R, VON SAUCKEN V E, LANDRETH G E. TREM2 in Neurodegenerative Diseases [J]. Mol Neurodegener, 2017, 12(1): 56. QIN Q, TENG Z, LIU C, et al. TREM2, microglia, and Alzheimer's disease [J]. Mech Ageing Dev, 2021, 195: 111438. DEL-AGUILA J L, BENITEZ B A, LI Z, et al. TREM2 brain transcript-specific studies in AD and TREM2 mutation carriers [J]. Mol Neurodegener, 2019, 14(1): 18. CIGNARELLA F, FILIPELLO F, BOLLMAN B, et al. TREM2 activation on microglia promotes myelin debris clearance and remyelination in a model of multiple sclerosis [J]. Acta Neuropathol, 2020, 140(4): 513-34. ZHANG Y, FENG S, NIE K, et al. TREM2 modulates microglia phenotypes in the neuroinflammation of Parkinson's disease [J]. Biochem Biophys Res Commun, 2018, 499(4): 797-802. TREM2 inhibits inflammatory responses in mouse microglia by suppressing the PI3K/NF-κB signaling [J]. Cell Biology International. GUO Y, WEI X, YAN H, et al. TREM2 deficiency aggravates alpha-synuclein-induced neurodegeneration and neuroinflammation in Parkinson's disease models [J]. FASEB J, 2019, 33(11): 12164-74. CHEN S, PENG J, SHERCHAN P, et al. TREM2 activation attenuates neuroinflammation and neuronal apoptosis via PI3K/Akt pathway after intracerebral hemorrhage in mice [J]. J Neuroinflammation, 2020, 17(1): 168. WANG Y, CAO C, ZHU Y, et al. TREM2/beta-catenin attenuates NLRP3 inflammasome-mediated macrophage pyroptosis to promote bacterial clearance of pyogenic bacteria [J]. Cell Death Dis, 2022, 13(9): 771. LIANG S, XU L, XIN X, et al. Study on pyroptosis-related genes Casp8, Gsdmd and Trem2 in mice with cerebral infarction [J]. PeerJ, 2024, 12: e16818. HUANG L K, ZENG X S, JIANG Z W, et al. Echinacoside alleviates glucocorticoid induce osteonecrosis of femoral head in rats through PI3K/AKT/FOXO1 pathway [J]. Chem Biol Interact, 2024, 391: 110893. XU S, WANG J, ZHONG J, et al. CD73 alleviates GSDMD-mediated microglia pyroptosis in spinal cord injury through PI3K/AKT/Foxo1 signaling [J]. Clin Transl Med, 2021, 11(1): e269. SHI M, WANG J, BI F, et al. Diosmetin alleviates cerebral ischemia-reperfusion injury through Keap1-mediated Nrf2/ARE signaling pathway activation and NLRP3 inflammasome inhibition [J]. Environ Toxicol, 2022, 37(6): 1529-42. MEI Z, DU L, LIU X, et al. Diosmetin alleviated cerebral ischemia/reperfusion injury in vivo and in vitro by inhibiting oxidative stress via the SIRT1/Nrf2 signaling pathway [J]. Food Funct, 2022, 13(1): 198-212. XIA J, LI J, DENG M, et al. Diosmetin alleviates acute lung injury caused by lipopolysaccharide by targeting barrier function [J]. Inflammopharmacology, 2023: 1-11. YIN H, FLYNN A D. Drugging Membrane Protein Interactions [J]. Annu Rev Biomed Eng, 2016, 18: 51-76. GONG J, CHEN Y, PU F, et al. Understanding Membrane Protein Drug Targets in Computational Perspective [J]. Curr Drug Targets, 2019, 20(5): 551-64. LI X Q, YU Q, FANG B, et al. Knockdown of the AIM2 molecule attenuates ischemia-reperfusion-induced spinal neuronal pyroptosis by inhibiting AIM2 inflammasome activation and subsequent release of cleaved caspase-1 and IL-1beta [J]. Neuropharmacology, 2019, 160: 107661. LIU S, CAO X, WU Z, et al. TREM2 improves neurological dysfunction and attenuates neuroinflammation, TLR signaling and neuronal apoptosis in the acute phase of intracerebral hemorrhage [J]. Frontiers in Aging Neuroscience, 2022, 14. YU P, ZHANG X, LIU N, et al. Pyroptosis: mechanisms and diseases [J]. Signal Transduct Target Ther, 2021, 6(1): 128. RAN Y, SU W, GAO F, et al. Curcumin Ameliorates White Matter Injury after Ischemic Stroke by Inhibiting Microglia/Macrophage Pyroptosis through NF-kappaB Suppression and NLRP3 Inflammasome Inhibition [J]. Oxid Med Cell Longev, 2021, 2021: 1552127. DING R, LI H, LIU Y, et al. Activating cGAS-STING axis contributes to neuroinflammation in CVST mouse model and induces inflammasome activation and microglia pyroptosis [J]. J Neuroinflammation, 2022, 19(1): 137. WAN P, SU W, ZHANG Y, et al. LncRNA H19 initiates microglial pyroptosis and neuronal death in retinal ischemia/reperfusion injury [J]. Cell Death Differ, 2020, 27(1): 176-91. HU Z, YUAN Y, ZHANG X, et al. Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Attenuate Oxygen-Glucose Deprivation/Reperfusion-Induced Microglial Pyroptosis by Promoting FOXO3a-Dependent Mitophagy [J]. Oxid Med Cell Longev, 2021, 2021: 6219715. CHANG Y, ZHU J, WANG D, et al. NLRP3 inflammasome-mediated microglial pyroptosis is critically involved in the development of post-cardiac arrest brain injury [J]. J Neuroinflammation, 2020, 17(1): 219. ZHANG X, ZHANG Y, WANG B, et al. Pyroptosis-mediator GSDMD promotes Parkinson's disease pathology via microglial activation and dopaminergic neuronal death [J]. Brain Behav Immun, 2024, 119: 129-45. WEN S, DENG F, LI L, et al. VX-765 ameliorates renal injury and fibrosis in diabetes by regulating caspase-1-mediated pyroptosis and inflammation [J]. J Diabetes Investig, 2022, 13(1): 22-33. LI N, WANG Y, WANG X, et al. Pathway network of pyroptosis and its potential inhibitors in acute kidney injury [J]. Pharmacol Res, 2022, 175: 106033. WANG Y, LIN Y, WANG L, et al. TREM2 ameliorates neuroinflammatory response and cognitive impairment via PI3K/AKT/FoxO3a signaling pathway in Alzheimer's disease mice [J]. Aging (Albany NY), 2020, 12(20): 20862-79. WU R, LI X, XU P, et al. TREM2 protects against cerebral ischemia/reperfusion injury [J]. Mol Brain, 2017, 10(1): 20. LI Y, LONG W, GAO M, et al. TREM2 Regulates High Glucose-Induced Microglial Inflammation via the NLRP3 Signaling Pathway [J]. Brain Sci, 2021, 11(7). YANG S, YANG Y, WANG F, et al. TREM2 Dictates Antibacterial Defense and Viability of Bone Marrow-derived Macrophages during Bacterial Infection [J]. Am J Respir Cell Mol Biol, 2021, 65(2): 176-88. JIANG W, LIU F, LI H, et al. TREM2 ameliorates anesthesia and surgery-induced cognitive impairment by regulating mitophagy and NLRP3 inflammasome in aged C57/BL6 mice [J]. Neurotoxicology, 2022, 90: 216-27. CAO C, DING J, CAO D, et al. TREM2 modulates neuroinflammation with elevated IRAK3 expression and plays a neuroprotective role after experimental SAH in rats [J]. Neurobiol Dis, 2022, 171: 105809. QU W, WANG Y, WU Y, et al. Triggering Receptors Expressed on Myeloid Cells 2 Promotes Corneal Resistance Against Pseudomonas aeruginosa by Inhibiting Caspase-1-Dependent Pyroptosis [J]. Front Immunol, 2018, 9: 1121. ZHANG B, SUN C, LIU Y, et al. Exosomal miR-27b-3p Derived from Hypoxic Cardiac Microvascular Endothelial Cells Alleviates Rat Myocardial Ischemia/Reperfusion Injury through Inhibiting Oxidative Stress-Induced Pyroptosis via Foxo1/GSDMD Signaling [J]. Oxid Med Cell Longev, 2022, 2022: 8215842. WANG D, WANG Y, ZOU X, et al. FOXO1 inhibition prevents renal ischemia-reperfusion injury via cAMP-response element binding protein/PPAR-gamma coactivator-1alpha-mediated mitochondrial biogenesis [J]. Br J Pharmacol, 2020, 177(2): 432-48. ZHOU M, LIU Y-W-Y, HE Y-H, et al. FOXO1 reshapes neutrophils to aggravate acute brain damage and promote late depression after traumatic brain injury [J]. Military Medical Research, 2024, 11(1). QU X, YANG T, WANG X, et al. Macrophage RIPK3 triggers inflammation and cell death via the XBP1–Foxo1 axis in liver ischaemia–reperfusion injury [J]. JHEP Reports, 2023, 5(11). ZAHRA N, ZESHAN B, ISHAQ M. Carbapenem resistance gene crisis in A. baumannii: a computational analysis [J]. BMC Microbiol, 2022, 22(1): 290. ISMAIL S, ABBASI S W, YOUSAF M, et al. Design of a Multi-Epitopes Vaccine against Hantaviruses: An Immunoinformatics and Molecular Modelling Approach [J]. Vaccines (Basel), 2022, 10(3). Additional Declarations No competing interests reported. Supplementary Files Uncroppedpicture.zip 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-4403409","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":310287386,"identity":"6d73c11d-ca15-470b-ae72-c1c7ed2aa1d2","order_by":0,"name":"Sidan Liu","email":"","orcid":"","institution":"the First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Sidan","middleName":"","lastName":"Liu","suffix":""},{"id":310287389,"identity":"9bd347c4-f31c-4bcf-b808-90b569b320b1","order_by":1,"name":"Yan Dong","email":"","orcid":"","institution":"the First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Dong","suffix":""},{"id":310287390,"identity":"4a2aaa1f-0a63-4862-b4c9-693fdeee9865","order_by":2,"name":"Xinyue Zhang","email":"","orcid":"","institution":"the First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinyue","middleName":"","lastName":"Zhang","suffix":""},{"id":310287392,"identity":"68040e12-8e60-4a2b-854b-8382d055aa3c","order_by":3,"name":"Yongjian Zhou","email":"","orcid":"","institution":"the First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yongjian","middleName":"","lastName":"Zhou","suffix":""},{"id":310287394,"identity":"c73c6d5b-16c6-413b-8468-a1c41db6f385","order_by":4,"name":"Kexin Wang","email":"","orcid":"","institution":"the First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Kexin","middleName":"","lastName":"Wang","suffix":""},{"id":310287395,"identity":"76a52fbc-dfba-470e-b02f-184c23328c06","order_by":5,"name":"Hong Ma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYFACHhDxj5mfvfnAgQ8VxGs5wC7Zcyzx4IwzJGjhN5jhY3yYt4UIDbrtZw8+Lvh1R9pAgufDAd4GBnl+sQP4tZidyUs2ntn3zNhcunfDAckdDIYzZycQ0HIgx0yat4c52XLO2Q0HDM8wJBjcJqTl/BuwlvoNN3IeHEhsI0bLDaAtPD8OMxvcyGE4cJA4LW+MjXkb0piBgWxwsOGMBBF+OZ9j+Jjnjw0oKh9//lNhI88vTUALGDC2wZkSRCgHgz/EKhwFo2AUjIIRCQC9UU0jFmdAPQAAAABJRU5ErkJggg==","orcid":"","institution":"the First Hospital of China Medical University","correspondingAuthor":true,"prefix":"","firstName":"Hong","middleName":"","lastName":"Ma","suffix":""},{"id":310287396,"identity":"4be13dee-6306-41db-8208-4f586e2b68ac","order_by":6,"name":"Te Fang","email":"","orcid":"","institution":"the First Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Te","middleName":"","lastName":"Fang","suffix":""}],"badges":[],"createdAt":"2024-05-11 04:08:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4403409/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4403409/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57891425,"identity":"301250e6-fdf1-442f-bac9-28bd16e81786","added_by":"auto","created_at":"2024-06-07 06:35:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":780372,"visible":true,"origin":"","legend":"\u003cp\u003eSignificant pyroptosis occurred following SCII in SD rats. A. Bioinformatics analysis data. B-C. Changes in the expression of IL-1β at different time points after SCII. D-G. Changes in the expression of pyroptosis-related proteins at 24 hour after SCII.\u003c/p\u003e","description":"","filename":"Figure1..png","url":"https://assets-eu.researchsquare.com/files/rs-4403409/v1/0793c0a229a81443872b2f51.png"},{"id":57891427,"identity":"a73b2626-c9ae-4682-a75e-c9549bd69445","added_by":"auto","created_at":"2024-06-07 06:35:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1564098,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of pyroptosis on motor neurons. A. Tarlov score results of lower limb motor function in different groups. B-C. HE staining results of spinal cord tissue in different groups of SD rats. Vs sham group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; vs SCII+DMSO group, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, n=5, scale bar=100μm.\u003c/p\u003e","description":"","filename":"Figure2..png","url":"https://assets-eu.researchsquare.com/files/rs-4403409/v1/2d129163f834d4f7cb916c2e.png"},{"id":57890903,"identity":"5785ed24-cb2d-4d31-9390-f6b010d81684","added_by":"auto","created_at":"2024-06-07 06:27:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1535903,"visible":true,"origin":"","legend":"\u003cp\u003eOGD/R caused an increase in the expression of pyroptosis-related proteins in microglia, and pyroptosis inhibitor VX-765 reversed this effect. A-B. Immunofluorescence results of the expression of IL-1β in different groups. Vs control group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; vs OGD/R group, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, n=3, scale bar=100μm. C-F. Western-blot results and representive bar graph of NLRP3, IL-1β, and cleaved caspase-1 in different groups. Vs control group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; vs OGD/R+DMSO group, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, n=3.\u003c/p\u003e","description":"","filename":"Figure3..png","url":"https://assets-eu.researchsquare.com/files/rs-4403409/v1/5dbe1330e4af43b50320b32d.png"},{"id":57890905,"identity":"c78cce4c-254d-4f82-b20b-97b150aaf5d6","added_by":"auto","created_at":"2024-06-07 06:27:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1374377,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of conditioned microglial media on neurons. A-B. Immunofluorescence staining was used to detect the expression of the anti-apoptotic protein Bcl2 in neurons from different groups, vs N+Control group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; vs N+OGD/R group, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, n=3. C. The CCK-8 assay detected cytotoxicity in neurons from different groups. Vs N+Control group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; vs N+OGD/R group, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, n=3, scale bar=50μm.\u003c/p\u003e","description":"","filename":"Figure4..png","url":"https://assets-eu.researchsquare.com/files/rs-4403409/v1/525ca34fbfd2698aa05664e9.png"},{"id":57891881,"identity":"751715c7-f1f4-4021-9966-2a2dd3aebca7","added_by":"auto","created_at":"2024-06-07 06:43:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2551068,"visible":true,"origin":"","legend":"\u003cp\u003eTREM2 expression levels at different time points following SCII. Vs sham group, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, n=3. \u0026nbsp;The impact of TREM2 overexpression on motor neurons in rats. A-B. Western blotting confirmed successful overexpression of TREM2 in rat spinal cord. Vs AAV-NC, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, n=5. C. Tarlov scoring was used to evaluate the motor function of the lower limbs in rats. D-E. HE staining to detect changes in the number of anterior horn motor neurons in the spinal cord of SD rats. Vs sham group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01; vs AAV-NC group, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, n=5, scale bar=100μm.\u003c/p\u003e","description":"","filename":"Figure5..png","url":"https://assets-eu.researchsquare.com/files/rs-4403409/v1/f1e8a103adab61ee755cb231.png"},{"id":57891428,"identity":"570ab8d0-16c8-415a-860b-36f867005fb8","added_by":"auto","created_at":"2024-06-07 06:35:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3070309,"visible":true,"origin":"","legend":"\u003cp\u003eResults of IBA-1 immunofluorescence staining. A. Images of IBA-1 fluorescence staining for different groups (two views per group, 10× and 20× respectively). B. Bar graph quantifying the average fluorescence intensity of IBA-1. Vs sham group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt;0.01; vs SCII group, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, n=3, scale bar=100μm.\u003c/p\u003e","description":"","filename":"Figure6..png","url":"https://assets-eu.researchsquare.com/files/rs-4403409/v1/ad2eb08073399e293e604c33.png"},{"id":57890909,"identity":"f4e1f019-31a4-49d0-88a6-8c915ab3d7f7","added_by":"auto","created_at":"2024-06-07 06:27:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":706801,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blotting was performed to detect the levels of pyroptosis-related and apoptosis-related proteins in different groups. The quantification of protein expression was represented by bar graphs. Vs sham group, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 and \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01; vs AAV-NC group, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01. n=3.\u003c/p\u003e","description":"","filename":"Figure7..png","url":"https://assets-eu.researchsquare.com/files/rs-4403409/v1/ea69be13d6a918f248f11a3c.png"},{"id":57891883,"identity":"9a5ade7c-ec4f-46cb-baa3-bef31d113c72","added_by":"auto","created_at":"2024-06-07 06:43:52","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1442510,"visible":true,"origin":"","legend":"\u003cp\u003eTREM2 overexpression inhibited cleaved caspase-1 expression caused by OGD/R in HAPI cells. A-B. TREM2 overexpression was verified by Western blotting, n=3. C-E. The expression levels of TREM2 and cleaved caspase-1 were detected by cellular immunofluorescence in different groups and their corresponding quantization bars. Vs control group, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; vs OE-NC group, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, n=3, scale bar=100μm.\u003c/p\u003e","description":"","filename":"Figure8..png","url":"https://assets-eu.researchsquare.com/files/rs-4403409/v1/6d0d3cbe93f772234975bc3a.png"},{"id":57890908,"identity":"1e7a87ac-4d1a-4782-86df-32926094c412","added_by":"auto","created_at":"2024-06-07 06:27:50","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1343187,"visible":true,"origin":"","legend":"\u003cp\u003eUpregulating TREM2 suppressed OGD/R-induced FOXO1 expression. A-B. Cell immunofluorescence assay was used to detect FOXO1 expression in different groups. Vs control group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; vs OE-NC group, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. n=3, scale bar=100μm. C-D. Western blot assay was used to detect FOXO1 expression after TREM2 overexpression. Vs control group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; vs OGD/R group, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. n=3.\u003c/p\u003e","description":"","filename":"Figure9..png","url":"https://assets-eu.researchsquare.com/files/rs-4403409/v1/cc4dd4d8e282a1fdd3f44f58.png"},{"id":57890915,"identity":"0974279a-95c8-45f9-b588-29e07f864bcc","added_by":"auto","created_at":"2024-06-07 06:27:52","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":769862,"visible":true,"origin":"","legend":"\u003cp\u003eCo-overexpression of TREM2 and FOXO1 further verified that TREM2 inhibited FOXO1. A-B. The effect of FOXO1 overexpression was verified, vs control group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, n=3. D-G. Western blot analysis detected NLRP3, GSDMD, IL-1β, and cleaved caspase-1 expression levels in different groups. Vs OGD/R group, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; vs OGD/R+OE-TREM2 group \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, n=3.\u003c/p\u003e","description":"","filename":"Figure10..png","url":"https://assets-eu.researchsquare.com/files/rs-4403409/v1/6bc8a02ad0287b54743e709d.png"},{"id":57891430,"identity":"11338f40-3df4-4106-8c88-2c05fece42ea","added_by":"auto","created_at":"2024-06-07 06:35:50","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":1014144,"visible":true,"origin":"","legend":"\u003cp\u003eTREM2 played the anti-pyroptosis role through PI3K/AKT/FOXO1/GSDMD pathway. A-F. Western-blot results of PI3K/AKT/FOXO1 pathway and pyroptosis-related proteins. Vs sham group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; vs OGD/R group, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; vs OGD/R+OE-TREM2 group, \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, n=3. G-J. Western-blot results of NLRP3, GSDMD and IL-1β. Vs si-NC group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, n=3. K. The correlation analysis of FOXO1 and GSDMD (GEPIA). L. ChIP analysis confirmed FOXO1 is directly bound to the GSDMD promoter.\u003c/p\u003e","description":"","filename":"Figure11..png","url":"https://assets-eu.researchsquare.com/files/rs-4403409/v1/f31de0ade5b1f42d1f734577.png"},{"id":57890916,"identity":"307e3a37-a06a-4404-80bd-4fdae9f5ee55","added_by":"auto","created_at":"2024-06-07 06:27:52","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":2640548,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of diosmetin on spinal cord anterior horn neurons following ischemia-reperfusion injury in SD rats. A. Tarlov scores in different experimental groups, n=5. Vs sham group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05; Vs SCII+NC group, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; Vs SCII+Dio (40mg/kg) group, \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01. B-C. HE staining results of spinal cord sections and corresponding quantification of intact neuron numbers in different groups are presented as bar graphs. Vs sham group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; vs SCII group, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; vs SCII+Dio (40mg/kg) group, \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, n=5, scale bar=100μm.\u003c/p\u003e","description":"","filename":"Figure12..png","url":"https://assets-eu.researchsquare.com/files/rs-4403409/v1/84f59033c2dc7e3dfea80887.png"},{"id":57891882,"identity":"4a0f5742-b10f-4713-a0f1-1136d88d1023","added_by":"auto","created_at":"2024-06-07 06:43:50","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":1343462,"visible":true,"origin":"","legend":"\u003cp\u003eDiosmetin inhibited pyroptosis-related protein levels both in vivo and in vitro. A-E. Representative western blot results of FOXO1, NLRP3, IL-1β, and cleaved caspase-1. Vs sham group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; vs SCII group, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, n=3. F-G. Diosmetin reversed GSDMD expression caused by oxygen-glucose deprivation reoxygenation detected by immunofluorescence staining, n=3, scale bar=100μm.\u003c/p\u003e","description":"","filename":"Figure13..png","url":"https://assets-eu.researchsquare.com/files/rs-4403409/v1/3520d0802d9d0cacb96a6ed5.png"},{"id":57890914,"identity":"ca71e3a9-3ae2-4b36-be13-678262ad1537","added_by":"auto","created_at":"2024-06-07 06:27:51","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":1590479,"visible":true,"origin":"","legend":"\u003cp\u003eDiosmetin played the anti-pyroptosis role in HAPI cells via TREM2 and the following PI3K/AKT/FOXO1 pathway. A. Docking analysis parameters of the TREM2 extracellular domain structure and diosmetin structure by Autodock software. B. 3D simulated molecular docking images of TREM2 and diosmetin. C-G. Silencing TREM2 reversed the anti-pyroptosis effect of diosmetin. Vs Control group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; vs OGD/R group, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; vs OGD/R+Dio group, \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05. n=3. H-K. The application of the PI3K inhibitor LY294002 reversed the anti-pyroptosis effect of diosmetin. Vs Control group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; vs OGD/R group, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01; vs OGD/R+Dio group, \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, n=3.\u003c/p\u003e","description":"","filename":"Figure14..png","url":"https://assets-eu.researchsquare.com/files/rs-4403409/v1/ed144f0de60c61020f9c50f5.png"},{"id":58797752,"identity":"153eff39-78e4-452d-9db2-2663b66193c1","added_by":"auto","created_at":"2024-06-21 08:48:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":26764272,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4403409/v1/3a04a7e1-1da2-48a7-8fa5-b3adc1ec2a0c.pdf"},{"id":57891438,"identity":"3ffb6ff5-ee19-421b-9962-a79730634927","added_by":"auto","created_at":"2024-06-07 06:35:53","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":156661952,"visible":true,"origin":"","legend":"","description":"","filename":"Uncroppedpicture.zip","url":"https://assets-eu.researchsquare.com/files/rs-4403409/v1/7bdf3b1d7ca3798c8460b001.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"TREM2-Mediated Microglial Pyroptosis: Unveiling the Neuroprotective Role of Diosmetin in Spinal Cord Ischemia-Reperfusion Injury","fulltext":[{"header":"Background","content":"\u003cp\u003eSpinal cord ischemia-reperfusion injury (SCII) is a severe complication of thoracoabdominal aortic surgery that can cause paralysis\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e, which results in huge economic loss. Due to the lack of effective treatments, the precise mechanisms underlying the pathogenesis of SCII are poorly understood, making it urgent to identify the exact pathogenesis and implement effective prevention and treatment measures. After the ischemic episode, prolonged neuroinflammation hugely triggers a series of secondary injuries, finally leading to neuronal death. Emerging evidence suggests that neuroinflammation may predominantly contribute to the pathogenesis of SCII\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePyroptosis, a form of programmed cell death related to inflammation, is found to be involved in SCII. Gasdermin-D (GSDMD) and caspase family executed pyroptosis and finally disrupted the cell membrane barrier and the release of inflammatory factors\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Microglia, the principal cell type involved in regulating inflammatory responses and undergoing pyroptosis in the central nervous system (CNS), have been found to trigger a cascade of inflammatory and oxidative stress responses when undergoing pyroptosis\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. They release a variety of inflammatory factors and free radicals, which can directly or indirectly affect neuronal survival and function\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe role of microglial pyroptosis has been implicated in the pathophysiological processes of several diseases, including CNS ischemia-reperfusion injury\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, traumatic brain injury\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e, and neurodegenerative disease. Pyroptosis in microglia inflicts damage on neurons and accelerates disease progression. Therefore, inhibiting microglial pyroptosis can attenuate subsequent neuroinflammatory responses and may become a significant therapeutic approach for CNS inflammation-related diseases, including SCII.\u003c/p\u003e \u003cp\u003eThe triggering receptor expressed on myeloid cells 2 (TREM2), predominantly expressed on the membrane of microglia in CNS, is linked with various neuropathological diseases\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. TREM2 acts mainly through the intracellular adaptor DNAX-activation protein 12 (DAP12) and initiates downstream signaling. Previous studies of TREM2 have focused on degenerative neuropathy such as Alzheimer\u0026rsquo;s disease\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Recent studies have investigated the anti-inflammation role of TREM2 in diverse neurological disease models\u003csup\u003e[\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. TREM2 deficiency aggravated neuroinflammation in Parkinson\u0026rsquo;s disease model\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Additionally, TREM2 activation attenuates neuroinflammation and neuronal apoptosis following intracerebral hemorrhage in mice\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. TREM2 regulates the inflammatory state by modulating the phenotype of microglia and macrophages. Furthermore, TREM2 has been found to enhance pathogen clearance by inhibiting the pyroptosis of macrophages\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Specifically, a study found that TREM2 has the potential as a regulatory factor in mitigating the effects of cerebral infarction through the modulation of pyroptosis pathways\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. However, its specific mechanism is unclear and has not been reported in SCII. Considering the crucial role of TREM2 in microglia, we hypothesized that it could regulate microglial pyroptosis and inhibit neuroinflammation following SCII.\u003c/p\u003e \u003cp\u003eFOXO1, by governing the transcription of a myriad of target genes, plays a significant role in modulating the balance between cell survival and death. Recent studies have underscored the importance of FOXO1 in the context of neuroinflammation, particularly its ability to influence the pathways leading to pyroptosis. The phosphorylation status of FOXO1, often regulated by upstream signals such as the PI3K/AKT pathway, determines its nuclear translocation and subsequent gene transcription activities\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. This regulatory mechanism is crucial in controlling the expression of GSDMD\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Therefore, the modulation of FOXO1 activity presents a promising therapeutic avenue in mitigating microglial pyroptosis and, by extension, reducing the neurological sequelae of SCII. Our study reveals a novel interaction between TREM2 and FOXO1, demonstrating that TREM2inhibits FOXO1 activity. This inhibition by TREM2 may play a pivotal role in reducing microglial pyroptosis, thereby mitigating neuronal damage and enhancing recovery following SCII.\u003c/p\u003e \u003cp\u003eStudies have found that various natural plant extracts can treat or improve ischemia-reperfusion injury in many animal disease models for the antioxidant and anti-inflammatory effects. Diosmetin has been found to protect neuronal function in cerebral ischemia-reperfusion injury\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Recent studies have revealed that diosmetin can inhibit the activation of NLRP3 inflammasome in lipopolysaccharide (LPS)-induced acute lung injury model\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. In an animal model of cerebral ischemia-reperfusion injury, diosmetin protects neurons by inhibiting NLRP3 inflammasome activation\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The precise mechanism by which diosmetin inhibits NLRP3 is unclear, as well as that in TREM2. Given the common mechanism of small-molecule compounds, which often involves binding to membrane proteins\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e, we wonder whether diosmetin interact with the transmembrane protein TREM2. We initially validated this hypothesis by molecular docking of the extracellular domain structure of TREM2 and the 3D structure of diosmetin. Subsequently, we further validated our hypothesis using molecular biology techniques. This study investigates the role of diosmetin in the pathological processes of SCII both in vivo and in vitro, providing new directions for drug therapy in the prevention and treatment of SCII.\u003c/p\u003e \u003cp\u003eIn the present study, we first identified the detrimental role of microglial pyroptosis in SCII. By investigating the influence of TREM2 expression, we explored the inhibitory effect of TREM2 on microglial pyroptosis and its neuroprotective role in SCII. Furthermore, we elucidated the neuroprotective effect of diosmetin in SCII and its close association with TREM2, suggesting its potential interaction with TREM2 as a mechanism of action.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSprague Dawley (SD) rats, 8\u0026ndash;10 weeks old, weighing 250-300g, were purchased from Beijing Vital River Laboratory. Three or four rats were housed in one standard plastic cage with free access to food and water and maintained under standard conditions with a 12 h light/dark cycle. All animal work was in accordance with the ethical committee of China Medical University on animal experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHAPI cells (Highly Aggressively Proliferating Immortalized, a rat microglia cell line) and VSC4.1 cells (the ventral spinal cord 4.1\u0026nbsp;motor neurons) were purchased from the Chinese Academy of Sciences Shanghai Cell Bank (Shanghai, China). HAPI cells were cultured in a MEM medium (HyClone, USA) with 10% FBS (Procell Life Science \u0026amp; Technology, China) and 1% penicillin/streptomycin (Solarbio Science \u0026amp; Technology, China). VSC4.1 cells were cultured in\u0026nbsp;\u003cem\u003ehigh-glucose\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eDMEM\u0026nbsp;(HyClone, USA)\u0026nbsp;with 10% FBS and 1% penicillin/streptomycin. The cells were cultured in an\u0026nbsp;incubator at 5% CO2 and a temperature of 37℃.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperiment\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e: In Vivo Investigation of Pyroptosis in SCII\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the role of pyroptosis in SCII, we conducted in vivo experiments using SD rats. The rats were divided into three groups: Sham, SCII+DMSO, and SCII+VX-765. We assessed motor neuron function and morphology using the Tarlov scoring system and Hematoxylin-eosin (HE) staining.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperiment\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003cstrong\u003e: In Vitro Investigation of Pyroptosis in SCII\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo\u0026nbsp;further\u0026nbsp;investigate the role of pyroptosis in SCII, we utilized HAPI cells in vitro. The cells were divided into three groups: Control, OGD/R+DMSO, and OGD/R+VX-765. Western blot analysis was performed to assess the expression of pyroptosis-related proteins at OGD (6h)/R (12h).\u003c/p\u003e\n\u003cp\u003eAdditionally, we examined the impact of microglial pyroptosis on neurons. Culture medium from each HAPI cell group was collected and the supernatant was used to treat VSC4.1 neuronal cells. These neuronal cells were categorized as follows: N group: Fresh complete culture medium;\u0026nbsp;N+Control group: Supernatant from Control group HAPI cells; N+VX-765 group: Supernatant from Control+VX-765 group HAPI cells; N+OGD/R group: Supernatant from OGD/R group HAPI cells; N+ OGD/R+VX-765 group: Supernatant from OGD/R+VX-765 group HAPI cells. We detected changes in the expression of the anti-apoptotic protein Bcl2 in neurons using immunofluorescence. Cell toxicity in neurons was assessed using the CCK-8 assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperiment 3: Investigating the Role of TREM2 in Microglial Pyroptosis both in vivo\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate TREM2\u0026apos;s function in SCII, we upregulated TREM2 expression in rats through Intrathecal injection of AAV particles encoding the TREM2 gene. SD rats were grouped as follows: Sham, SCII, SCII+AAV-NC\u0026nbsp;(negative control), and SCII+AAV-TREM2. Motor neuron function was assessed using the Tarlov score, while HE staining was used to evaluate neuronal morphology. Neuroinflammation was detected through immunofluorescent staining for IBA-1, and Western blotting was used to assess pyroptosis-related proteins.\u003c/p\u003e\n\u003cp\u003eTo explore the role of TREM2 in OGD/R-induced microglial pyroptosis, HAPI cells were transduced with lentiviruses overexpressing TREM2. The cells were divided into four groups: Control, OGD/R, OGD/R+OE-NC, and OGD/R+OE-TREM2. Immunofluorescence was employed to detect the expression of TREM2 and cleaved caspase-1 in each group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperiment 4: Investigating the Role of TREM2 in Modulating FOXO1 to Regulate Microglial Pyroptosis\u003c/strong\u003eWe explored the potential mechanism of TREM2 in regulating cell pyroptosis. FOXO1, an essential transcription factor regulating various cellular activities, was identified as a downstream effector of TREM2. In vitro experiments were designed to study the relationship between TREM2 and FOXO1. HAPI cells were grouped into Control, OGD/R, OGD/R+OE-NC, and OGD/R+OE-TREM2. Immunofluorescence and Western blotting were used to detect changes in FOXO1 expression among the groups.\u003c/p\u003e\n\u003cp\u003eTo further verify the relationship between TREM2 and FOXO1, HAPI cells were divided into three groups: OGD/R, OGD/R+OE-TREM2, and OGD/R+OE-TREM2+OE-FOXO1. The latter group was transfected with two lentiviruses to overexpress both TREM2 and FOXO1 before OGD/R. The expression levels of pyroptosis-related proteins were measured in these groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperiment 5: Investigating the Involvement of the PI3K/AKT Pathway in TREM2-Mediated Regulation of FOXO1 and Pyroptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether TREM2 regulates FOXO1 through the PI3K/AKT pathway, HAPI cells were grouped into Control, OGD/R, OGD/R+OE-TREM2, and OGD/R+OE-TREM2+LY294002. Western blotting was employed to assess the expression levels of PI3K, AKT, phosphorylated proteins p-PI3K and p-AKT, FOXO1, and pyroptosis-related proteins in these groups. The Gene Expression Profiling Interaction Analysis (GEPIA) database\u0026nbsp;was\u0026nbsp;used to analyze the expression correlation between FOXO1 and GSDMD. Additionally, chromatin\u0026nbsp;immunoprecipitation（CHIP）was used to investigate the transcriptional regulatory effect of FOXO1 on GSDMD.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperiment 6: Investigating the Role of Diosmetin in SCII and Its Interaction with TREM2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this experiment, we investigated the impact of diosmetin on SCII both in vivo and in vitro. For the in vivo component, SD rats were divided into\u0026nbsp;five\u0026nbsp;groups: Sham, SCII, SCII+Vehicle, SCII+Dio (40mg/kg), and SCII+Dio (80mg/kg). Behavioral assessments included Tarlov scores, and H\u0026amp;E staining was performed to evaluate different groups.\u003c/p\u003e\n\u003cp\u003eIn the in vitro part, the effects of diosmetin on microglial pyroptosis were studied. HAPI cells were divided into Control, OGD/R, and OGD/R+Dio groups. Changes in pyroptosis-related proteins were assessed in these groups.\u003c/p\u003e\n\u003cp\u003eTo further investigate the relationship between diosmetin and TREM2, molecular docking was conducted using Autodock software to predict the interaction between the extracellular domain of TREM2 and diosmetin. To validate the docking results, HAPI cells were divided into Control, OGD/R, OGD/R+Dio, and OGD/R+Dio+si-TREM2 groups. In the OGD/R+Dio+si-TREM2 group, si-TREM2 was transfected into HAPI cells, and they were subsequently exposed to complete culture medium containing diosmetin (10\u0026mu;M) for one hour before OGD/R. The expression levels of pyroptosis-related proteins were observed in these groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRat model of spinal cord ischemia-reperfusion injury and treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Ethics Committee of China Medical University. The procedures were following the guidelines set by the Institutional Animal Care and Use Committee. Rats were anesthetized with pentobarbital (Sigma, USA) by intraperitoneal injection (35 mg/kg). Following endotracheal intubation, mechanical ventilation, and aortic arch exposition, the spinal cord ischemia-reperfusion injury model was established by blocking the aortic arch for 14 min, as previously described\u003csup\u003e[28]\u003c/sup\u003e. Rats in the sham group were subjected to the same surgery without clamping. Each postoperative rat was placed in a cage alone and kept warm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReagent treatment of animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVX-765 is a specific pyroptosis inhibitor by targeting caspase-1. It was purchased from MCE company (CAS No.: 273404-37-8). According to the instructions, it was diluted\u0026nbsp;with\u0026nbsp;dimethyl sulfoxide (DMSO) to a working solution (25 mg/ml). The administration involved intraperitoneal injection at a dosage of 30 mg/kg/day for three consecutive days, with the first dose administered 30 minutes before the initiation of SCII modeling.\u003c/p\u003e\n\u003cp\u003eDiosmetin (C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e, molecular weight = 300.26, CAS NO.520-34-3, purity =99.8%,) was purchased from MCE. According to the instructions, the diosmetin powder was dissolved with 10% DMSO，40% PEG300，5% Tween-80, and 45% saline in turn to a concentration of 5mg/ml. Rats in the SCII+Diosmetin group were received intraperitoneal injections of diosmetin dilution separately at a dose of 40 mg/kg/day and 80 mg/kg/day for 3 continuous days before the model establishment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntrathecal injection of Adeno-associated virus and\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;si-RNAs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRats were briefly anesthetized with isoflurane and placed in a prone position with lower back elevated and flexed ventrally. A Microliter Syringes needle (Gaoge, Shanghai, China) was advanced into the subarachnoid space at L4\u0026ndash;L6 levels. A tail-flick was the mark of the success of intrathecal injection. Once this was confirmed, the solutions were injected within 1 minute.\u003c/p\u003e\n\u003cp\u003eTo increase exogenous expression of TREM2 in SD rats, The AAV-TREM2 and AAV-NC (negative control) (ObiO, Shanghai, China) were delivered by intrathecal injection. The titers of AAV particles were between 1 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e and 2 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e vg/ml and 20\u0026micro;l of AAV-TREM2 or AAV-NC was used per rat. AAV injection was performed 1 month before SCII induction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo investigate the role of FOXO1 in SCII, FOXO1 siRNA (si-FOXO1) was intrathecally injected to knock down the expression level of FOXO1. Si-FOXO1 and a control siRNA(si-NC) were purchased from RiboBio, and the sequences were as follows: si-FOXO1, 5\u0026prime;-GGACAGCAAAUCAAGUUAUtt-3\u0026prime;, si-NC, 5\u0026prime;-UUCUCCGAACGUGUCACGUT-T-3\u0026prime;. The si-FOXO1 powder was reconstituted in enzyme-free water to a concentration of 1 nmol/20 \u0026mu;l. For the 3 days preceding the modeling, si-FOXO1 or si-NC was intrathecally administered to rats at a daily dose of 1 nmol per rat for 3 consecutive days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeurological function tests\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRats were acclimated to the behavioral test environment at least 1h before testing. Tarlov scale was performed to measure the neurological function of the animals 24h following SCII induction. Modified Tarlov criteria were used as follows: 0 = no voluntary hind-limb function; 1 = only perceptible joint movement; 2 = active movement but unable to stand; 3 = able to stand but unable to walk; or 4 = completely normal hind-limb motor function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHematoxylin and eosin (H\u0026amp;E) staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHE staining was conducted on spinal cord sections. Briefly, lumbar enlargement segments of spinal cord were embedded in paraffin and sectioned at 4 um thickness. Deparaffinization and rehydration: Sections were deparaffinized in xylene (2 x 10 min) and rehydrated through a graded ethanol series (100%, 95%, 80%, and 70%; 5 min each), followed by distilled water rinse. Antigen retrieval: Sections were submerged in 10 mM sodium citrate buffer (pH 6.0) and heated in a pressure cooker for 3 min at full pressure. After cooling at room temperature for 20 min, sections were washed with PBS (2 x 5 min).Permeabilization: Sections were treated with 0.1% Triton X-100 in PBS for 10 min, followed by PBS washes (2 x 5 min).All other staining steps were performed following standard protocols.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTUNEL staining\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTUNEL staining was conducted using the Cy3 TUNEL cell apoptosis assay kit (Bioscience, China). Following\u0026nbsp;deparaffinization\u0026nbsp;and\u0026nbsp;antigen retrieval,\u0026nbsp;sections were incubated with the TUNEL reaction mixture for 60 min at 37\u0026deg;C in a humidified chamber, protected from light. Counterstaining: Sections were counterstained with DAPI (4\u0026rsquo;,6-diamidino-2-phenylindole) for 10 min, followed by a brief rinse in PBS. Fluorescence microscopy was used to visualize TUNEL-positive cells (red) and nuclei (blue).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOxygen-glucose deprivation/reperfusion model and cell cytotoxicity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA cellular model of oxygen-glucose deprivation/reperfusion was performed to mimic SCII in vitro. After normal culture for at least 24h, HAPI cells with glucose-free medium were transferred into an incubation chamber flushed with a gas mixture of 95% N2 and 5% for 15 minutes. The chamber was then sealed and placed into a humidified incubator at 37\u0026deg;C 5% CO2 for 6h. Culturing was continued for 0-24h before further measurement.\u0026nbsp;The cytotoxicity of neurons was measured by Cell Counting Kit-8 (CCK-8;\u0026nbsp;\u003ca href=\"https://www.baidu.com/link?url=VthpjBDY05XjljXDl94MjjGGOcwCzHkYGu5PyHdkNCLsnZ2ve-NrlTgfwkkTLEc7\u0026wd=\u0026eqid=822aafe9000a10510000000264b64d68\" target=\"_blank\"\u003eBeyotime Biotechnology\u003c/a\u003e, Shanghai, China) according to the instructions provided with the reagents.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVSC4.1 Neuronal Cell Culture with Conditioned Microglia Media\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo co-culture VSC4.1 neuronal cells with conditioned microglia media, HAPI cell culture media were collected following various interventions and centrifuged to obtain the supernatant. VSC4.1 neuronal cells prepared a day in advance, were washed with sterile PBS\u0026nbsp;and added with supernatant to co-culture for another 12h.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo meet experimental requirements, HAPI cells were stably transfected with lentiviral vectors (LV) for TREM2 overexpression. The TREM2 lentiviral particles were purchased from Synthbio, and the cells were stably transfected as previously described\u003csup\u003e[29]\u003c/sup\u003e. The stable cell line overexpressing TREM2 was selected by exposure to puromycin for 3 days.\u003c/p\u003e\n\u003cp\u003eAdditionally, HAPI cells were transiently transfected with TREM2 siRNA using Lipo3000 in a 12-well plate. The culture media were changed 24 hours post-transfection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDrug application in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVX-765, purchased from MCE company (CAS No.: 273404-37-8, 10 mM * 1 mL in DMSO) was diluted in a complete culture medium to a final concentration of 1 \u0026mu;M and applied before the onset of OGD/R.\u003c/p\u003e\n\u003cp\u003eLY294002 (Sigma), a competitive PI3K-AKT pathway inhibitor, were dissolved in DMSO and diluted in saline. HAPI cells were pretreated with LY294002 (20\u0026mu;M) 1 h before OGD/R.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein samples were obtained from L1-L3 spinal cord segment and HAPI cells. All the protein samples were lysed in RIPA buffer, and quantified by using a BCA protein assay kit following sonication. Equal amounts of protein (30 \u0026micro;g/lane) were separated on SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat dry milk (Nestle) and incubated at 4℃\u0026nbsp;overnight\u0026nbsp;with the following antibodies: goat anti-rabbit TREM2 (1:1000, Abmart, China), goat anti-rabbit FOXO1 (1:2000, Proteintech, China), goat anti-mouse\u0026nbsp;IL-1\u0026beta;\u0026nbsp;(1:1000, Abmart, China), goat anti-rabbit cleaved-caspase 1(1:1000, Abmart, China), GSDMD, NLRP3\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDocking and molecular dynamics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 3D structures of TREM2 ectodomains was retrieved from the PDB database (http://www.rcsb.org/pdb ), and the small-molecule ligand Diosmetin was obtained from the ZINC database (https://zinc.docking.org/). Both two 3D structures were used to perform molecular docking following hydrogenation and dehydration with Autocock Tools software. The docking results were analyzed and visualized with PyMol software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using GraphPad Prism 7.0. We tested the normality of the data using the Kolmogorov-Smirnov test. For data that followed a normal distribution, data was expressed as mean \u0026plusmn; SEM. Depending on the grouping of experiments and the characteristics of different data, the independent samples t-test was used for comparisons between two groups, and one-way ANOVA or two-way ANOVA for repeated measurements were used for comparisons among multiple groups. For non-normally distributed data, the Mann-Whitney U test was used. A p value of less than 0.05 was considered to indicate statistically significant differences.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePyroptosis\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;occurred in SD rats following SCII\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBy searching the GEO database, we identified one experimental animal model data (GSE138966) that was relevant to our study. Using Metascape, we performed enrichment analysis on the top 500 differentially expressed genes, which revealed that the inflammatory response was the most significant pathological process in rats after spinal cord ischemia-reperfusion injury. We further examined the changes in inflammatory factors after SCII\u0026nbsp;in rats. Western blot analysis revealed an increased expression of IL-1\u0026beta;, which increased at 12h and peaked at 24h post-SCII\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). Detection of other pyroptosis-related proteins at 24h post-SCII showed elevated expression levels of NLRP3,\u0026nbsp;TNF-\u0026alpha;\u0026nbsp;and\u0026nbsp;cleaved\u0026nbsp;caspase-1\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). The above results demonstrate significant inflammation and pyroptosis occurring after SCII.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePyroptosis inhibitor VX-765 mitigated the neural damage caused by SCII\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInvestigating the impact of pyroptosis on motor neurons, we first observed behavioral changes in different groups of rats by\u0026nbsp;utilizing Tarlov scores for lower limb motor function evaluation. The Tarlov score results showed a significant decrease in the SCII+DMSO group compared to the Sham group\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), indicating impaired motor function caused by SCII. However, the rats pretreated with VX-765 exhibited an increased score\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), indicating VX-765 reversed SCII-induced motor neuronal dysfunction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHE staining of the rat spinal cord ventral horn revealed intact neurons in the different groups.\u0026nbsp;The number of intact neurons reflected the neuronal damage.\u0026nbsp;The rats in SCII group had fewer intact neurons (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), indicating neuronal damage. However, the rats with VX-765 treatment had more intact neurons\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). These findings suggest that VX-765 improved SCII-induced motor dysfunction, highlighting the role of pyroptosis in the SCII pathology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOGD/R induced microglial pyroptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirstly, through immunofluorescence staining, we evaluated the change of IL-1\u0026beta; expression levels after OGD/R. The results showed that OGD/R induced a significant increase of IL-1\u0026beta;\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), as shown in Figure 3. A-B. However, VX-765 treatment was able to inhibit the elevation of IL-1\u0026beta; triggered by OGD/R\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). Moreover, we also observed the morphological changes caused by OGD/R: the cells became swollen and round, and the cell boundaries were blurred. These changes were consistent with the characteristics of pyroptosis. We examined changes in pyroptosis-related proteins in each group. The results showed that OGD/R led to a significant increase of NLRP3, IL-1\u0026beta; and cleaved caspase-1 in microglia\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). However, VX-765 treatment was able to reverse these effects\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). The above results indicated that OGD/R induced pyroptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOGD/R-induced microglial pyroptosis enhanced neuronal damage.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe previously demonstrated that OGD/R could induce pyroptosis in microglia. To further verify its effects on neuronal cells, we treated the neuronal cells with the supernatant of microglial cells from different groups, and detected changes in the expression of the anti-apoptotic protein Bcl2 in the neuronal cells. Bcl2 expression was reduced following OGD/R (N+OGD/R vs N+Control, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). However, treatment with VX-765 appears to restore Bcl2 intensity towards normal levels (N+OGD/R+VX-75 vs N+OGD/R, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), indicating a protective effect against OGD/R-induced apoptosis.\u003c/p\u003e\n\u003cp\u003eThe CCK-8 assay was utilized to clarify the impact of OGD/R on the viability of VSC 4.1 neuronal cells (Fig. 4C). The results indicated that cell viability was impaired by OGD/R treatment and improved by VX-765 treatment. These results suggest that neuronal cells suffer damage cultured in the supernatant from microglia post-OGD/R. However, the use of the pyroptosis inhibitor VX-765 can mitigate the neuronal damage caused by OGD/R.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTREM2 improved the neurological deficit triggered by SCII\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo investigate whether TREM2 is involved in the process of SCII, we measured the expression levels\u0026nbsp;of TREM2 at different time points following SCII. The results showed that TREM2 expression increased at 12h, peaked at 24h following SCII induction\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 respectively).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe assessed TREM2\u0026apos;s potential to protect motor neurons during SCII in rats by Tarlov scores and HE staining. Based on previous experimental results, 24h post-SCII was selected as the observation time point. The upregulation of TREM2 via AAV in rat spinal cord was confirmed by Western blotting. Rats with TREM2 overexpression showed a higher Tarlov score\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), indicating improved motor function.\u003c/p\u003e\n\u003cp\u003eSimilarly, HE staining revealed that overexpression of TREM2 improved motor neuron damage induced by SCII.\u0026nbsp;When compared with Sham group,\u0026nbsp;SCII group had a lower number of intact neurons\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). Rats with TREM2 overexpression were able to reverse the loss of motor neurons induced by SCII\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). These results indicated a protective effect of TREM2 overexpression on motor neurons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTREM2 inhibited microglial activation induced by SCII\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicroglia play a crucial role in regulating inflammation and pyroptosis in the CNS. Our study examined their activation after SCII and the effect of TREM2 on this process. Findings revealed that SCII induced an increase in IBA-1 staining intensity, indicating enhanced microglial activation (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). However, the TREM2-overexpressing rats showed reduced IBA-1 staining intensity (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), as displayed in Fig 6. These findings imply that TREM2 overexpression can suppress microglia activation induced by SCII, underscoring the involvement of microglial activation in spinal cord ischemia-reperfusion injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTREM2 inhibit\u003c/strong\u003e\u003cstrong\u003eed\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethe\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003epyroptosis caused by SCII in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo discern whether TREM2\u0026apos;s neuroprotective effects against SCII was associated with pyroptosis, we examined the impact of TREM2 on pyroptosis-related proteins. Western blot analysis revealed enhanced expression of GSDMD, IL-1\u0026beta;, and cleaved caspase-1 following SCII\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). However, in the TREM2 overexpressed SCII+AAV-TREM2 group, these expression levels were reduced when compared with AAV-NC group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, we investigated the expression of apoptosis-related proteins Bcl2 and Bax. SCII caused increased pro-apoptotic Bax expression\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01)\u0026nbsp;and decreased anti-apoptotic Bcl2 expression\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05). Interestingly, the TREM2 overexpressed SCII+AAV-TREM2 group exhibited decreased expression of Bax and elevated level of Bcl2\u0026nbsp;(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). These experimental findings suggest that SCII induced pyroptosis and apoptosis in spinal cord tissue, while TREM2 overexpression reversed these changes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTREM2 overexpression inhibited cleaved caspase-1 expression caused by OGD/R in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCleaved caspase-1 serves as a critical biomarker for detecting pyroptosis. We measured the levels of cleaved caspase-1 in HAPI cells across various groups to assess the occurrence and severity of pyroptosis. To overexpress TREM2 in HAPI cells, we utilized lentiviruses, and confirmed the transfection efficiency via Western blot analysis, as shown in Figure 9A-B. Exposure to OGD/R resulted in increased expression levels of both TREM2 and cleaved caspase-1 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 respectively). The TREM2 overexpression (OE-TREM2) group displayed a substantial increase in TREM2 levels (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), confirming successful lentiviral transfection, along with a reduced level of cleaved caspase-1 compared to the control group (OE-NC) (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). The expression levels of TREM2 and cleaved caspase-1 in both the OGD/R group and the OGD/R+OE-NC group showed no significant differences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTREM2 inhibited the activity of FOXO1 caused by OGD/R\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven that TREM2 has been shown to inhibit GSDMD expression, as previously verified in Figure 8A-B, and considering earlier studies suggesting FOXO1\u0026apos;s role in regulating GSDMD transcription\u003csup\u003e[22]\u003c/sup\u003e, our research focused on whether TREM2 influences GSDMD expression through FOXO1 regulation.. To delve deeper into the relationship between TREM2 and FOXO1, we examined the impact of TREM2 overexpression on FOXO1 expression in vitro.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe results from immunofluorescence (Fig. 9A-B) revealed a significant increase in FOXO1 expression in the OGD/R group compared to the control group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). Notably, HAPI cells overexpressing TREM2 through lentiviral transfection were able to reverse the OGD/R-induced upregulation of FOXO1 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). This pattern was similarly observed in the Western blot analysis. As depicted in Fig. 9C-D, FOXO1 expression significantly rose in the OGD/R group in contrast to the Control group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). Conversely, FOXO1 expression in the OE-TREM2+OGD/R group was notably lower compared to the OGD/R group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). These findings demonstrate that TREM2 overexpression effectively counteract the OGD/R-induced increase in FOXO1 expression.\u003c/p\u003e\n\u003cp\u003eThe immunofluorescence results, as shown in Figure 9A-B, indicated a significant elevation in FOXO1 expression in the OGD/R group compared to the control group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). Remarkably, HAPI cells overexpressing TREM2 via lentiviral transfection successfully mitigated the OGD/R-induced upregulation of FOXO1 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). This trend was consistently observed in Western blot analyses; Figure 9C-D illustrates that FOXO1 expression was substantially higher in the OGD/R group than in the control group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). However, in the group overexpressing TREM2 and subjected to OGD/R (OE-TREM2+OGD/R), FOXO1 levels were significantly reduced compared to the OGD/R group alone (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). These results suggest that TREM2 overexpression effectively counteracts the OGD/R-induced increase in FOXO1 expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe rescue experiment provided further confirmation of TREM2\u0026apos;s suppression of FOXO1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo clarify the relationship between TREM2 and FOXO1 in the context of anti-pyroptosis, we co-overexpressed both genes in a single group of HAPI cells. Subsequently, we assessed the changes in inflammation and pyroptosis-associated markers across the groups. Initially, we confirmed the successful overexpression of TREM2 (Fig. 8A-B) and FOXO1 (Fig. 10A-B). As shown in Figure 10C-G, the Western blot analysis revealed that, compared to the OGD/R group, the expression levels of NLRP3, GSDMD, IL-1\u0026beta;, and cleaved caspase-1 were significantly reduced in the OGD/R+OE-TREM2 group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, respectively). Furthermore, relative to the OGD/R+OE-TREM2 group, increased expression levels of NLRP3, GSDMD, IL-1\u0026beta;, and cleaved caspase-1 were observed in the OGD/R+OE-TREM2+OE-FOXO1 group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, respectively). These findings strongly support the role of TREM2 in modulating FOXO1 activity, highlighting its influence on the regulation of inflammation and pyroptosis-related markers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTREM2 inhibited pyroptosis via PI3K/AKT/FOXO1/GSDMD pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo delve deeper into how TREM2 suppresses microglial pyroptosis and its interaction with FOXO1, we investigated the primary downstream signaling pathway of TREM2, specifically the PI3K/AKT pathway. Additionally, we utilized the PI3K inhibitor LY294002 in a rescue experiment to substantiate the underlying mechanisms further.\u003c/p\u003e\n\u003cp\u003eWe examined the impact of TREM2 overexpression in HAPI cells on the PI3K/AKT signaling pathway and its downstream effector, FOXO1, following OGD/R. The results showed that phosphorylation levels of PI3K and AKT were significantly increased in the OGD/R+OE-TREM2 group compared to the OGD/R group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), while FOXO1 expression was markedly decreased (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). Pre-treatment with LY294002 in the TREM2-overexpressing HAPI cells led to reduced phosphorylation levels of PI3K and AKT relative to the OGD/R+OE-TREM2 group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). These findings suggest that TREM2 inhibits microglial pyroptosis via the PI3K/AKT/FOXO1 signaling pathway.\u003c/p\u003e\n\u003cp\u003eMoreover, the downregulation of FOXO1 corresponded with decreased expressions of NLRP3, IL-1\u0026beta;, and particularly GSDMD (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), as depicted in Figure 11G-J. We also conducted a correlation analysis between FOXO1 and GSDMD by GEPIA database, which yielded significant P and R values (Fig. 11K). Furthermore, a CHIP assay confirmed the binding between FOXO1 and the promoter region of GSDMD (Fig. 11L). These comprehensive approaches underscore the critical role of TREM2 in modulating key inflammatory pathways and its therapeutic potential in reducing microglial pyroptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiosmetin\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003epretreatment\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;improved the neurological deficit triggered by SCII\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirstly, we explored the impact of diosmetin on SCII outcomes in vivo by administering varying concentrations of the compound. The Tarlov scores for the SCII group and the SCII+Dio (40 mg/kg) group were lower compared to the sham group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). Rats treated with diosmetin in both concentrations showed significant improvements in behavioral scores relative to those in the SCII group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). Notably, the SCII+Dio (80 mg/kg) group showed a more pronounced improvement compared to the SCII+Dio (40 mg/kg) group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). There was no statistical difference between the SCII group and the SCII+NC group.\u003c/p\u003e\n\u003cp\u003eIn line with these behavioral findings, H\u0026amp;E staining analysis indicated a significant reduction in the number of intact neurons in the SCII group compared to the sham group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). The administration of diosmetin effectively counteracted this decrease (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), with the high-concentration SCII+Dio (80 mg/kg)\u0026nbsp;group exhibiting the greater degree of amelioration(\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). The results suggest that diosmetin has a protective effect on neurons against spinal cord ischemia-reperfusion injury.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiosmetin inhibited pyroptosis-related protein changes induced by SCII\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore whether the neuroprotective effect of diosmetin is related to pyroptosis, the expression levels of pyroptosis-related proteins were detected by Western blot analysis.\u0026nbsp;Based on the results of the previous part, 80mg/kg was used as the dosage of diosmetin for subsequent experiments.\u0026nbsp;As shown in Fig. 13A-E, the results revealed that the levels of FOXO1, NLRP3, IL-1\u0026beta;, and cleaved caspase-1 were significantly increased in SCII group compared to sham group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). However, in the SCII+Dio group pretreated with diosmetin, the expression levels of FOXO1, NLRP3, IL-1\u0026beta;, and cleaved caspase-1 were significantly decreased compared to SCII group (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, respectively). Additionally, in vitro experiments also found that diosmetin could inhibit GSDMD elevation caused by OGD/R (Fig. 13F-G).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiosmetin played the anti-pyroptosis role via TREM2\u003c/strong\u003e\u003cstrong\u003e/PI3K/AKT/FOXO1 pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore whether the anti-pyroptosis effect of diosmetin was related to its binding to TREM2, we first performed molecular docking analysis between them. We obtained the 3D structure of the extracellular domain of TREM2 (PDB: 6YYE) from the Protein Data Bank (PDB) and the 3D structure of the small molecule compound Diosmetin (ZINC5733652) from the ZINC database (zinc.docking.org/). Subsequently, we performed dehydration and hydrogenation treatments on the protein and the small molecule compound separately using AutoDock software. We then conducted docking simulations and selected the binding site with the highest negative binding energy for analysis. The results indicated that the extracellular domain structure of TREM2 and Diosmetin formed hydrogen bonds at the GLU-220 and ILE-246 residues, with a binding energy of -5.46 kJ/mol. To further investigate whether diosmetin\u0026apos;s anti-pyroptosis effect is related to TREM2, we utilized siRNA technology to silence TREM2 expression in HAPI cells. Expression levels of GSDMD, IL-1\u0026beta;, and cleaved caspase-1 in different experimental groups were detected. As shown in Fig. 14C-G, compared to the OGD/R group, the OGD/R+Dio group exhibited a reduction in the expression levels of GSDMD, FOXO1, IL-1\u0026beta;, and cleaved caspase-1 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05), consistent with our previous results.\u003c/p\u003e\n\u003cp\u003eTo elucidate the specific mechanism of diosmetin, we used a PI3K inhibitor to detect the changes in phosphorylation and expression levels of PI3K/AKT/FOXO1 proteins in different groups. The results showed that compared with OGD/R group, the phosphorylation levels of PI3K and AKT were increased (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), while the expression level of FOXO1 was decreased (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01) in OGD/R+Dio group. Compared with OGD/R+Dio group, the phosphorylation levels of PI3K and AKT were decreased (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05), while the expression level of FOXO1 was increased (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) in OGD/R+Dio+LY294002 group. These results suggested that diosmetin regulated pyroptosis through the PI3K/AKT/FOXO1 pathway.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the current study of SCII, we underscore the significant role of microglial pyroptosis in contributing to neuronal damage and functional impairments. Our findings suggest that modulating microglial behavior can reduce pyroptosis, thereby improving neuronal function. Additionally, we have identified a natural compound that acts as a modulator, offering neuroprotection by reducing microglial pyroptosis. These discoveries open new pharmacological research directions for the treatment and prevention of SCII.\u003c/p\u003e \u003cp\u003eMicroglia pyroptosis has been identified as a critical factor in neuroinflammation, leading to significant neuronal damage and impairment of neuronal survival参考文献. Numerous studies have demonstrated that pyroptosis in microglia plays a pivotal role in central nervous system ischemia-reperfusion injury and oxygen-glucose deprivation-reoxygenation models\u003csup\u003e[\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. It has been shown that inhibiting microglial pyroptosis can ameliorate neuronal cell damage\u003csup\u003e[\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. In this study, we focused on the role of microglial pyroptosis in SCII and found that SCII induces microglial activation characterized by the upregulation of NLRP3, caspase-1, and IL-1β, which are crucial in exacerbating neuronal damage. The significant finding of our research is that inhibition of microglial pyroptosis through VX-765, a caspase-1 inhibitor, markedly reduces SCII-induced motor neuron injury and neurological dysfunction. VX-765 served effectively to establish a benchmark for the inhibition of microglial pyroptosis, confirming the pathway's critical role in neuronal injury and the potential for therapeutic intervention. We chose VX-765 for its proven effectiveness and tolerability in animal studies, and its unique ability to reduce inflammation without the side effects typical of similar compounds\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. These outcomes imply that microglial pyroptosis plays a role in the pathogenesis of SCII and may represent a promising therapeutic target.\u003c/p\u003e \u003cp\u003eTREM2 has been implicated in the process of pyroptosis\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Enhanced TREM2 signaling has been shown to reduce microglial activation, decrease neuroinflammation, and subsequently reduce neuronal damage and neurodegeneration \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. Several reports have highlighted the role of TREM2 in mitigating neuroinflammation by inhibiting the NRLP3 inflammasome in the CNS \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e.. We observed an increase in TREM2 expression following SCII, which was time-dependent and consistent with increases noted in other disease models\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. The change of TREM2 expression implies that it is involved in the pathophysiological process of SCII. Additionally, we found that TREM2 overexpression could inhibit microglia activation and improve motor neuron function damage caused by SCII. Further research has revealed that TREM2 can suppress the expression of proteins associated with pyroptosis. In vitro experiments utilizing immunofluorescence assays have demonstrated that microglia overexpressing TREM2 exhibit reduced levels of cleaved-caspase-1 following OGD/R, further confirming the inhibitory effect of TREM2 on microglial pyroptosis. Prior studies have indicated that TREM2 enhances corneal resistance to damage by inhibiting caspase-1-dependent pyroptosis\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e and promotes pathogen clearance by preventing macrophage pyroptosis \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Moreover, macrophages with TREM2 deficiency are more vulnerable to pyroptotic death resulting in increased inflammation\u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Taken together, these findings, including ours, suggest that TREM2 can regulate its host cells to inhibit pyroptosis, providing a multi-faceted approach to controlling inflammatory processes in neurodegenerative conditions.\u003c/p\u003e \u003cp\u003eFOXO1 is involved in the anti-pyroptosis effect of TREM2. FOXO1 plays a critical role in regulating genes associated with metabolic disorders, autophagy, and oxidative stress. It has been observed to alleviate myocardial and renal ischemia-reperfusion injuries by dampening inflammatory responses and protecting mitochondrial function\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. However, in cases of hepatic ischemia-reperfusion injury, FOXO1 triggers inflammation and cell death through the XBP1-Foxo1 axis\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. Additionally, one study found that neutrophils expressing high levels of FOXO1 infiltrate the brain in both acute and chronic phase after traumatic brain injury (TBI), exacerbating acute inflammatory brain injury and promoting depression caused by late TBI\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. Hence, the role of FOXO1 in ischemia-reperfusion injuries can vary depending on the tissue, organ, signaling pathways, and regulatory factors involved. FOXO1\u0026rsquo;s activity and function are modulated by various post-translational modifications, with phosphorylation being a predominant modification that affects its subcellular localization and transcriptional activity. Our study unveiled that TREM2 suppressed FOXO1 activity, which was verified by co-overexpressing both TREM2 and FOXO1 genes to rescue. We subsequently verified that TREM2 phosphorylates FOXO1 via the PI3K/AKT pathway by utilizing the PI3K inhibitor LY294002. This led to FOXO's nuclear translocation and the inhibition of its transcriptional functions. Additionally, our research confirmed that FOXO1 is capable of regulating the transcription of GSDMD, a pivotal executor of pyroptosis, as has been validated in other disease models\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. These observations strongly indicate that FOXO1\u0026rsquo;s role in the transcriptional regulation of GSDMD is both consistent and widespread across different disease states.\u003c/p\u003e \u003cp\u003eDiosmetin exhibits anti-pyroptotic properties and is associated with TREM2 Our study revealed that pre-treatment with diosmetin significantly ameliorated SCII-induced motor neuron deficits, aligning with the findings of previous research that showcased diosmetin's ability to protect neurons in cases of cerebral ischemia-reperfusion injury\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Furthermore, our in vitro experiments demonstrated that diosmetin mitigated OGD/R-induced pyroptosis of microglia. These outcomes introduce new prospects for the prevention and treatment of SCII in clinical practice. To elucidate the working mechanism of diosmetin, we explored its interaction with TREM2. It is known that TREM2 is a membrane protein exclusively expressed on the surface of microglia in the CNS, where it plays a role in inhibiting pyroptosis. Our investigation indicated that diosmetin may bind to TREM2, thereby exerting its anti-pyroptotic and neuroprotective effects through this pathway. By using Autodock software for molecular docking, we identified a potential binding relationship between diosmetin and the extracellular domain structure of TREM2, with a binding energy of -5.46 kJ/mol. Recent studies have suggested that a binding energy of less than \u0026minus;\u0026thinsp;5 kJ/mol signifies a strong interaction between small molecule compounds and proteins\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e. Additionally, when we knocked down TREM2 in HAPI cells in vitro, the neuroprotective impact of diosmetin significantly diminished, further substantiating the hypothesis that diosmetin exerts its effects through binding to TREM2.\u003c/p\u003e \u003cp\u003eWhile these findings offer new insights into the roles of diosmetin and TREM2 in SCII, our study does come with certain limitations. One limitation pertains to the utilization of cell lines instead of primary cells, which may not entirely capture the characteristics of microglial cells. Nevertheless, the use of cell lines is well-documented in prior studies and can still provide valuable insights into microglial biology and function. Additionally, we require more experimental evidence to definitively confirm the precise binding relationship between diosmetin and TREM2. Furthermore, the pharmacological properties, side effects, and safety profile of diosmetin warrant further investigation. More clinical studies are also essential in the future to validate our findings and explore the potential application of diosmetin in the clinical prevention and treatment of SCII.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur results suggest that the TREM2-mediated PI3K/AKT/FOXO1/GSDMD pathway holds promise as a therapeutic target for SCII. Furthermore, diosmetin emerges as a promising candidate for the treatment of spinal cord I/R injury, with its anti-pyroptosis actions offering potential therapeutic benefits. Subsequent studies are necessary to validate these findings and fully unlock the therapeutic potential of diosmetin in SCII treatment.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAAV: Adeno-associated virus\u003c/p\u003e\n\u003cp\u003eAKT:\u0026nbsp;/protein kinase B\u003c/p\u003e\n\u003cp\u003eCNS:\u0026nbsp;central nervous system\u003c/p\u003e\n\u003cp\u003eDMSO: Dimethyl sulfoxide\u003c/p\u003e\n\u003cp\u003eFOXO1: forkhead box protein O1\u003c/p\u003e\n\u003cp\u003eGSDMD: gasdermin D\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLV: Lentivirus\u003c/p\u003e\n\u003cp\u003eOGD/R: Oxygen-glucose deprivation/reoxygenation\u003c/p\u003e\n\u003cp\u003ePI3K:\u0026nbsp;phosphatidylinositol 3-kinase\u003c/p\u003e\n\u003cp\u003eSCII: Spinal cord ischemia-reperfusion injury\u003c/p\u003e\n\u003cp\u003eTREM2: Triggering receptor expressed on myeloid cells 2\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the National Institutes of Health\u0026apos;s Guide for the Care and Use of Laboratory Animals and received approval from the Institutional Animal Care and Use Committee of China Medical University\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors are consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China [grant numbers 82371287].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHM and SDL designed the experiment. TF and SDL drafted the main manuscript, and HM revised and proofread the manuscript. SDL and XYZ performed the experiments, \u0026nbsp;YD prepared all the figures. KXW and YJZ analyzed the data and organized the original experimental materials. YD and TF provided technical guidance for the experiments. \u0026nbsp;All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003eThe authors declare that there is no conflict of interest on the article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHIRAOKA T, KOMIYA T, TSUNEYOSHI H, et al. Risk factors for spinal cord ischaemia after thoracic endovascular aortic repair [J]. Interact Cardiovasc Thorac Surg, 2018, 27(1): 54-9.\u003c/li\u003e\n\u003cli\u003eZHU P, LI J X, FUJINO M, et al. Development and treatments of inflammatory cells and cytokines in spinal cord ischemia-reperfusion injury [J]. Mediators Inflamm, 2013, 2013: 701970.\u003c/li\u003e\n\u003cli\u003eMI J, YANG Y, YAO H, et al. Inhibition of heat shock protein family A member 8 attenuates spinal cord ischemia-reperfusion injury via astrocyte NF-kappaB/NLRP3 inflammasome pathway : HSPA8 inhibition protects spinal ischemia-reperfusion injury [J]. J Neuroinflammation, 2021, 18(1): 170.\u003c/li\u003e\n\u003cli\u003eFU J, SUN H, WEI H, et al. Astaxanthin alleviates spinal cord ischemia-reperfusion injury via activation of PI3K/Akt/GSK-3beta pathway in rats [J]. J Orthop Surg Res, 2020, 15(1): 275.\u003c/li\u003e\n\u003cli\u003eHSU S K, LI C Y, LIN I L, et al. Inflammation-related pyroptosis, a novel programmed cell death pathway, and its crosstalk with immune therapy in cancer treatment [J]. Theranostics, 2021, 11(18): 8813-35.\u003c/li\u003e\n\u003cli\u003eGU L, SUN M, LI R, et al. Microglial pyroptosis: Therapeutic target in secondary brain injury following intracerebral hemorrhage [J]. Front Cell Neurosci, 2022, 16: 971469.\u003c/li\u003e\n\u003cli\u003eGU L, SUN M, LI R, et al. Didymin Suppresses Microglia Pyroptosis and Neuroinflammation Through the Asc/Caspase-1/GSDMD Pathway Following Experimental Intracerebral Hemorrhage [J]. Front Immunol, 2022, 13: 810582.\u003c/li\u003e\n\u003cli\u003eLI Y, SONG W, TONG Y, et al. Isoliquiritin ameliorates depression by suppressing NLRP3-mediated pyroptosis via miRNA-27a/SYK/NF-kappaB axis [J]. J Neuroinflammation, 2021, 18(1): 1.\u003c/li\u003e\n\u003cli\u003eLIU X, ZHANG M, LIU H, et al. Bone marrow mesenchymal stem cell-derived exosomes attenuate cerebral ischemia-reperfusion injury-induced neuroinflammation and pyroptosis by modulating microglia M1/M2 phenotypes [J]. Exp Neurol, 2021, 341: 113700.\u003c/li\u003e\n\u003cli\u003eKOBER D L, BRETT T J. TREM2-Ligand Interactions in Health and Disease [J]. J Mol Biol, 2017, 429(11): 1607-29.\u003c/li\u003e\n\u003cli\u003eJAY T R, VON SAUCKEN V E, LANDRETH G E. TREM2 in Neurodegenerative Diseases [J]. Mol Neurodegener, 2017, 12(1): 56.\u003c/li\u003e\n\u003cli\u003eQIN Q, TENG Z, LIU C, et al. TREM2, microglia, and Alzheimer\u0026apos;s disease [J]. Mech Ageing Dev, 2021, 195: 111438.\u003c/li\u003e\n\u003cli\u003eDEL-AGUILA J L, BENITEZ B A, LI Z, et al. TREM2 brain transcript-specific studies in AD and TREM2 mutation carriers [J]. Mol Neurodegener, 2019, 14(1): 18.\u003c/li\u003e\n\u003cli\u003eCIGNARELLA F, FILIPELLO F, BOLLMAN B, et al. TREM2 activation on microglia promotes myelin debris clearance and remyelination in a model of multiple sclerosis [J]. Acta Neuropathol, 2020, 140(4): 513-34.\u003c/li\u003e\n\u003cli\u003eZHANG Y, FENG S, NIE K, et al. TREM2 modulates microglia phenotypes in the neuroinflammation of Parkinson\u0026apos;s disease [J]. Biochem Biophys Res Commun, 2018, 499(4): 797-802.\u003c/li\u003e\n\u003cli\u003eTREM2 inhibits inflammatory responses in mouse microglia by suppressing the PI3K/NF-\u0026kappa;B signaling [J]. Cell Biology International.\u003c/li\u003e\n\u003cli\u003eGUO Y, WEI X, YAN H, et al. TREM2 deficiency aggravates alpha-synuclein-induced neurodegeneration and neuroinflammation in Parkinson\u0026apos;s disease models [J]. FASEB J, 2019, 33(11): 12164-74.\u003c/li\u003e\n\u003cli\u003eCHEN S, PENG J, SHERCHAN P, et al. TREM2 activation attenuates neuroinflammation and neuronal apoptosis via PI3K/Akt pathway after intracerebral hemorrhage in mice [J]. J Neuroinflammation, 2020, 17(1): 168.\u003c/li\u003e\n\u003cli\u003eWANG Y, CAO C, ZHU Y, et al. TREM2/beta-catenin attenuates NLRP3 inflammasome-mediated macrophage pyroptosis to promote bacterial clearance of pyogenic bacteria [J]. Cell Death Dis, 2022, 13(9): 771.\u003c/li\u003e\n\u003cli\u003eLIANG S, XU L, XIN X, et al. Study on pyroptosis-related genes Casp8, Gsdmd and Trem2 in mice with cerebral infarction [J]. PeerJ, 2024, 12: e16818.\u003c/li\u003e\n\u003cli\u003eHUANG L K, ZENG X S, JIANG Z W, et al. Echinacoside alleviates glucocorticoid induce osteonecrosis of femoral head in rats through PI3K/AKT/FOXO1 pathway [J]. Chem Biol Interact, 2024, 391: 110893.\u003c/li\u003e\n\u003cli\u003eXU S, WANG J, ZHONG J, et al. CD73 alleviates GSDMD-mediated microglia pyroptosis in spinal cord injury through PI3K/AKT/Foxo1 signaling [J]. Clin Transl Med, 2021, 11(1): e269.\u003c/li\u003e\n\u003cli\u003eSHI M, WANG J, BI F, et al. Diosmetin alleviates cerebral ischemia-reperfusion injury through Keap1-mediated Nrf2/ARE signaling pathway activation and NLRP3 inflammasome inhibition [J]. Environ Toxicol, 2022, 37(6): 1529-42.\u003c/li\u003e\n\u003cli\u003eMEI Z, DU L, LIU X, et al. Diosmetin alleviated cerebral ischemia/reperfusion injury in vivo and in vitro by inhibiting oxidative stress via the SIRT1/Nrf2 signaling pathway [J]. Food Funct, 2022, 13(1): 198-212.\u003c/li\u003e\n\u003cli\u003eXIA J, LI J, DENG M, et al. Diosmetin alleviates acute lung injury caused by lipopolysaccharide by targeting barrier function [J]. Inflammopharmacology, 2023: 1-11.\u003c/li\u003e\n\u003cli\u003eYIN H, FLYNN A D. Drugging Membrane Protein Interactions [J]. Annu Rev Biomed Eng, 2016, 18: 51-76.\u003c/li\u003e\n\u003cli\u003eGONG J, CHEN Y, PU F, et al. Understanding Membrane Protein Drug Targets in Computational Perspective [J]. Curr Drug Targets, 2019, 20(5): 551-64.\u003c/li\u003e\n\u003cli\u003eLI X Q, YU Q, FANG B, et al. Knockdown of the AIM2 molecule attenuates ischemia-reperfusion-induced spinal neuronal pyroptosis by inhibiting AIM2 inflammasome activation and subsequent release of cleaved caspase-1 and IL-1beta [J]. Neuropharmacology, 2019, 160: 107661.\u003c/li\u003e\n\u003cli\u003eLIU S, CAO X, WU Z, et al. TREM2 improves neurological dysfunction and attenuates neuroinflammation, TLR signaling and neuronal apoptosis in the acute phase of intracerebral hemorrhage [J]. Frontiers in Aging Neuroscience, 2022, 14.\u003c/li\u003e\n\u003cli\u003eYU P, ZHANG X, LIU N, et al. Pyroptosis: mechanisms and diseases [J]. Signal Transduct Target Ther, 2021, 6(1): 128.\u003c/li\u003e\n\u003cli\u003eRAN Y, SU W, GAO F, et al. Curcumin Ameliorates White Matter Injury after Ischemic Stroke by Inhibiting Microglia/Macrophage Pyroptosis through NF-kappaB Suppression and NLRP3 Inflammasome Inhibition [J]. Oxid Med Cell Longev, 2021, 2021: 1552127.\u003c/li\u003e\n\u003cli\u003eDING R, LI H, LIU Y, et al. Activating cGAS-STING axis contributes to neuroinflammation in CVST mouse model and induces inflammasome activation and microglia pyroptosis [J]. J Neuroinflammation, 2022, 19(1): 137.\u003c/li\u003e\n\u003cli\u003eWAN P, SU W, ZHANG Y, et al. LncRNA H19 initiates microglial pyroptosis and neuronal death in retinal ischemia/reperfusion injury [J]. Cell Death Differ, 2020, 27(1): 176-91.\u003c/li\u003e\n\u003cli\u003eHU Z, YUAN Y, ZHANG X, et al. Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Attenuate Oxygen-Glucose Deprivation/Reperfusion-Induced Microglial Pyroptosis by Promoting FOXO3a-Dependent Mitophagy [J]. Oxid Med Cell Longev, 2021, 2021: 6219715.\u003c/li\u003e\n\u003cli\u003eCHANG Y, ZHU J, WANG D, et al. NLRP3 inflammasome-mediated microglial pyroptosis is critically involved in the development of post-cardiac arrest brain injury [J]. J Neuroinflammation, 2020, 17(1): 219.\u003c/li\u003e\n\u003cli\u003eZHANG X, ZHANG Y, WANG B, et al. Pyroptosis-mediator GSDMD promotes Parkinson\u0026apos;s disease pathology via microglial activation and dopaminergic neuronal death [J]. Brain Behav Immun, 2024, 119: 129-45.\u003c/li\u003e\n\u003cli\u003eWEN S, DENG F, LI L, et al. VX-765 ameliorates renal injury and fibrosis in diabetes by regulating caspase-1-mediated pyroptosis and inflammation [J]. J Diabetes Investig, 2022, 13(1): 22-33.\u003c/li\u003e\n\u003cli\u003eLI N, WANG Y, WANG X, et al. Pathway network of pyroptosis and its potential inhibitors in acute kidney injury [J]. Pharmacol Res, 2022, 175: 106033.\u003c/li\u003e\n\u003cli\u003eWANG Y, LIN Y, WANG L, et al. TREM2 ameliorates neuroinflammatory response and cognitive impairment via PI3K/AKT/FoxO3a signaling pathway in Alzheimer\u0026apos;s disease mice [J]. Aging (Albany NY), 2020, 12(20): 20862-79.\u003c/li\u003e\n\u003cli\u003eWU R, LI X, XU P, et al. TREM2 protects against cerebral ischemia/reperfusion injury [J]. Mol Brain, 2017, 10(1): 20.\u003c/li\u003e\n\u003cli\u003eLI Y, LONG W, GAO M, et al. TREM2 Regulates High Glucose-Induced Microglial Inflammation via the NLRP3 Signaling Pathway [J]. Brain Sci, 2021, 11(7).\u003c/li\u003e\n\u003cli\u003eYANG S, YANG Y, WANG F, et al. TREM2 Dictates Antibacterial Defense and Viability of Bone Marrow-derived Macrophages during Bacterial Infection [J]. Am J Respir Cell Mol Biol, 2021, 65(2): 176-88.\u003c/li\u003e\n\u003cli\u003eJIANG W, LIU F, LI H, et al. TREM2 ameliorates anesthesia and surgery-induced cognitive impairment by regulating mitophagy and NLRP3 inflammasome in aged C57/BL6 mice [J]. Neurotoxicology, 2022, 90: 216-27.\u003c/li\u003e\n\u003cli\u003eCAO C, DING J, CAO D, et al. TREM2 modulates neuroinflammation with elevated IRAK3 expression and plays a neuroprotective role after experimental SAH in rats [J]. Neurobiol Dis, 2022, 171: 105809.\u003c/li\u003e\n\u003cli\u003eQU W, WANG Y, WU Y, et al. Triggering Receptors Expressed on Myeloid Cells 2 Promotes Corneal Resistance Against Pseudomonas aeruginosa by Inhibiting Caspase-1-Dependent Pyroptosis [J]. Front Immunol, 2018, 9: 1121.\u003c/li\u003e\n\u003cli\u003eZHANG B, SUN C, LIU Y, et al. Exosomal miR-27b-3p Derived from Hypoxic Cardiac Microvascular Endothelial Cells Alleviates Rat Myocardial Ischemia/Reperfusion Injury through Inhibiting Oxidative Stress-Induced Pyroptosis via Foxo1/GSDMD Signaling [J]. Oxid Med Cell Longev, 2022, 2022: 8215842.\u003c/li\u003e\n\u003cli\u003eWANG D, WANG Y, ZOU X, et al. FOXO1 inhibition prevents renal ischemia-reperfusion injury via cAMP-response element binding protein/PPAR-gamma coactivator-1alpha-mediated mitochondrial biogenesis [J]. Br J Pharmacol, 2020, 177(2): 432-48.\u003c/li\u003e\n\u003cli\u003eZHOU M, LIU Y-W-Y, HE Y-H, et al. FOXO1 reshapes neutrophils to aggravate acute brain damage and promote late depression after traumatic brain injury [J]. Military Medical Research, 2024, 11(1).\u003c/li\u003e\n\u003cli\u003eQU X, YANG T, WANG X, et al. Macrophage RIPK3 triggers inflammation and cell death via the XBP1\u0026ndash;Foxo1 axis in liver ischaemia\u0026ndash;reperfusion injury [J]. JHEP Reports, 2023, 5(11).\u003c/li\u003e\n\u003cli\u003eZAHRA N, ZESHAN B, ISHAQ M. Carbapenem resistance gene crisis in A. baumannii: a computational analysis [J]. BMC Microbiol, 2022, 22(1): 290.\u003c/li\u003e\n\u003cli\u003eISMAIL S, ABBASI S W, YOUSAF M, et al. Design of a Multi-Epitopes Vaccine against Hantaviruses: An Immunoinformatics and Molecular Modelling Approach [J]. Vaccines (Basel), 2022, 10(3).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Spinal cord ischemia-reperfusion injury, TREM2, Diosmetin, FOXO1, GSDMD","lastPublishedDoi":"10.21203/rs.3.rs-4403409/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4403409/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSpinal cord ischemia-reperfusion injury (SCII) is a severe neurological condition marked by neuronal damage and functional impairments. The contribution of microglial pyroptosis, an inflammatory form of cell death, to SCII's development is increasingly acknowledged. Yet, the complex molecular mechanisms and potential therapeutic strategies targeting microglial pyroptosis in SCII are not fully understood.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eOur research utilized both in vivo and in vitro models to evaluate the influence of TREM2 modulation on microglial pyroptosis and neuronal function in SCII. Principal methods included Tarlov scoring, Western blot analysis, Chromatin Immunoprecipitation (CHIP) and histological techniques, with an emphasis on proteins such as Forkhead Box O1 (FOXO1) and pyroptosis-related proteins to decipher the underlying mechanisms. Molecular docking was employed to investigate the interaction between the small molecule diosmetin and TREM2.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe observed a marked increase in TREM2 expression following SCII, and demonstrated that TREM2 overexpression mitigated microglial pyroptosis and enhanced motor neuron functionality. Further investigation revealed that TREM2 engagement leads to the activation of Forkhead Box O1 (FOXO1) phosphorylation through the Phosphatidylinositol 3-Kinase (PI3K)/Protein Kinase B (AKT) signaling pathway. This activation sequence culminates in the downregulation of Gasdermin D (GSDMD), the primary effector of pyroptosis. Additionally, we identified diosmetin, a natural compound known for its anti-inflammatory and antioxidant effects, as a potent modulator of TREM2-mediated microglial pyroptosis. Experimental data demonstrate diosmetin's binding affinity to TREM2, conferring neuroprotection by impeding microglial pyroptosis through the TREM2/PI3K/AKT/FOXO1/GSDMD axis.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur findings underscore the pivotal role of TREM2 in microglial pyroptosis and its therapeutic potential in SCII, positioning diosmetin as a viable pharmacological candidate for SCII prevention and therapy.\u003c/p\u003e","manuscriptTitle":"TREM2-Mediated Microglial Pyroptosis: Unveiling the Neuroprotective Role of Diosmetin in Spinal Cord Ischemia-Reperfusion Injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-07 06:27:45","doi":"10.21203/rs.3.rs-4403409/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5b20fc7a-2c83-4772-b2bc-d002f3e254ce","owner":[],"postedDate":"June 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-21T08:39:38+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-07 06:27:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4403409","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4403409","identity":"rs-4403409","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}