Emodin influence pyroptosis-related Caspase 1-GSDMD axis alleviated cerebral ischemia-reperfusion injury in rats

Emodin influence pyroptosis-related Caspase 1-GSDMD axis alleviated cerebral ischemia-reperfusion injury in rats | 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 Article Emodin influence pyroptosis-related Caspase 1-GSDMD axis alleviated cerebral ischemia-reperfusion injury in rats Tao Liang, Guofang Zhang, Xiaolin Hu, Jun Qian, Yumei Shi, Zeng Ling, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6149634/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jun, 2025 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version Abstract Background Cerebrovascular disease encompasses a wide range of conditions characterized by cerebrovascular lesions or disruptions in blood flow. Ischemic stroke, among these conditions, is the most prevalent and is known for its substantial morbidity, disability, and mortality rates, making it a leading cause of global disability. Effective management of ischemia-reperfusion injury holds paramount importance in stroke treatment, regardless of whether thrombolytic therapy is administered. Previous studies have shown that Emodin exhibits anti-inflammatory and neuroprotective properties, providing protection against ischemia-reperfusion injury in various organs by modulating pyroptosis. However, the precise molecular mechanisms underlying the effects of Emodin in cerebral ischemia-reperfusion injury remain poorly understood. Therefore, the objective of this study was to elucidate the neuroprotective mechanisms of Emodin in the context of ischemic stroke. Methods SD rats were randomly assigned to different groups, including control group, sham operation group, model group, and Emodin intervention group with varying dosages. Cerebral ischemia-reperfusion injury was induced using the middle cerebral artery occlusion (MCAO) method. Intraperitoneal injections of 10mg/kg, 20mg/kg and 40 mg/kg Emodin were administered to assess neurological changes in the rats. The modified Neurological Severity Score (mNSS) was used to evaluate neurological deficits. The infarct volume ratio was determined through TTC staining, while HE staining was employed to observe pathomorphological changes. Using Western blotting (WB) technique and immunofluorescence, we investigated the expression levels and cellular localization of proteins associated with cell pyroptosis, including NLRP3, Caspase 1 and GSDMD. Additionally, enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of IL-1β and IL-18. The whole animal study was approved by the Affiliated Hospital of Zunyi Medical University (approval number KLLY(A)-2021-083) and all methods are reported in accordance with ARRIVE guidelines. Results Emodin exhibits significant beneficial effects in improving neurological deficits caused by cerebral ischemia-reperfusion injury. It effectively reduces the ratio of infarct volume, alleviates cytopathic damage and suppresses the expression of pyroptosis-related proteins, including NLRP3, Caspase 1 and GSDMD. Furthermore, Emodin decreases the levels of pro-inflammatory cytokines IL-1β and IL-18, thus attenuating the inflammatory response. Conclusions The expression of pyroptosis-related proteins is upregulated in rats after cerebral ischemia-reperfusion injury. Emodin demonstrates neuroprotective effects against cerebral ischemia-reperfusion injury in rats, potentially by modulating the expression of pyroptosis-related proteins mediated through the Caspase 1-GSDMD axis. Biological sciences/Drug discovery Biological sciences/Neuroscience Health sciences/Neurology Caspase 1 GSDMD Cerebral ischemia-reperfusion Emodin Pyroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Stroke is a highly prevalent disease in the field of neuroscience and ranks among the leading causes of death and disability worldwide. Acute ischemic stroke (AIS) represents the most common subtype. Cerebral ischemia-reperfusion injury serves as a significant mechanism that intensifies neurological impairments. Therefore, mitigating the detrimental effects of ischemia-reperfusion injury assumes a pivotal role in the early management of ischemic stroke. [ 1 , 2 ] . Pyroptosis, a recently identified form of programmed cell death, is primarily driven by the activation of different caspases, including Caspase 1, through the inflammasome pathway. Upon activation, these caspases cleave and polymerize various gasdermin family members, such as gasdermin D (GSDMD), leading to cell membrane permeabilization and subsequent release of multiple pro-inflammatory factors. Ultimately, this cascade of events culminates in cell death [ 3 – 8 ] . Researches have shown that suppressing the expression of pyroptosis-related proteins, including GSDMD, in rat model of cerebral ischemia-reperfusion injury can markedly enhance the viability of microglial cells [ 9 ] . Furthermore, studies conducted in rat models of renal ischemia-reperfusion injury, hepatic ischemia-reperfusion injury and myocardial ischemia-reperfusion injury have demonstrated the presence of cell pyroptosis-related protein expression. Inhibition of these protein expressions has exhibited protective effects [ 10 , 11 ] . Subsequently, it was found that suppressing the expression of GSDMD effectively reduces the levels of IL-18, Caspase 1, Caspase 11, and IL-1β, thereby inhibiting inflammation and promoting neurological recovery after cerebral ischemia-reperfusion injury. This suggests that cell pyroptosis plays a role in cerebral ischemia-reperfusion injury and blocking cell pyroptosis may contribute to the recovery from ischemic stroke [ 12 – 14 ] . In the field of neuroprotection research, an increasing number of researchers have turned their attention to traditional Chinese herbs, such as Astragaloside IV [ 15 ] , Icariside Ⅱ [ 16 ] , Astragalus [ 17 ] , Gardenoside [ 18 ] and Herba Erigerontis [ 19 ] . Emodin, also known as 1,3,8-trihydroxy-6-methylanthraquinone (C 15 H 10 O 5 ), is included in such studies. It was originally discovered in the roots and stems of plants and was first isolated in 1925. Emodin is a significant component of various traditional Chinese herbs, commonly known for its laxative effects [ 20 – 24 ] . However, with advancements in technology, numerous reports have indicated that Emodin possesses diuretic, antiviral, anticancer, hepatoprotective, immunosuppressive, and antimicrobial properties [ 25 – 29 ] . Due to its diverse therapeutic benefits, Emodin is gradually finding its place in clinical prescriptions of traditional Chinese medicine within medical institutions.Recent studies have shown that Emodin also participates in the pathogenesis of certain ischemic diseases. For instance, Emodin has demonstrated its ability to protect retinal neurons from ischemic injury [ 30 ] , improve cognitive dysfunction associated with sepsis-related encephalopathy, and inhibit pathological damage to hippocampal neurons [ 31 ] . it has been shown to alleviate lung ischemia-reperfusion injury by inhibiting the expression of GSDMD and Caspase 1 [ 32 ] . Furthermore, it exhibits protective effects in myocardial ischemia-reperfusion injury [ 33 ] . These findings highlight the protective role of Emodin in various organ ischemia-reperfusion injuries by intervening in cell pyroptosis processes. Studies have demonstrated that Emodin exhibits a certain degree of improvement on the neurological deficits observed in patients with acute ischemic stroke and in rats with cerebral ischemia-reperfusion injury, although the underlying mechanisms remain unclear [ 34 – 36 ] . Considering its protective effects on pyroptosis-mediated tissue injury in various organs such as the myocardium, lungs and retina during ischemia-reperfusion, Emodin represents a promising target for intervention in ischemic stroke treatment. However, the mechanisms by which Emodin influences cerebral ischemia-reperfusion injury in rats, particularly its involvement in the Caspase-1-GSDMD axis-mediated pyroptosis, remain unclear. In this study, we investigate the neuroprotective role of Emodin by targeting pyroptosis in a rat model of cerebral ischemia-reperfusion injury. The aim of our study is to explore the potential neuroprotective effects of Emodin through its intervention in pyroptosis in the context of cerebral ischemia-reperfusion injury. The findings may contribute to the development of neuroprotective drugs for post-ischemic stroke brain protection, providing experimental evidence to overcome current treatment challenges in clinical practice. MATERIALS AND METHOD Animals and Treatment Adult male SD rats (200-250g), provided by Tianqin Biotechnology Co., Ltd. (Changsha, China; license number: SCXK (Xiang) 2019-0004). were housed in cages under controlled conditions at a constant temperature of 24 ± 2°C. During the experimental period, the rats had access to standard laboratory food and purified drinking water. All animal procedures were conducted in accordance with the ethical guidelines for animal research and were approved by the Ethics Committee of Affiliated Hospital of Zunyi Medical University. The rats were randomly divided into the following groups: control group, sham operation group, middle cerebral artery occlusion (MCAO) group, MCAO + low-dose Emodin group (10mg/kg), MCAO + medium-dose Emodin group (20mg/kg) and MCAO + high-dose Emodin group (40mg/kg). After rats were anaesthetized with pentobarbital sodium (40 mg/kg) before sacrificed. All efforts were made to minimize the rat’s suffering. Model Establishment The MCAO model was established using the intraluminal suture method [ 37 , 38 ] . The rats were anesthetized with pentobarbital sodium (40 mg/kg) administered via intraperitoneal injection. After midline incision and tissue separation in the slightly right-sided region, the external carotid artery and common carotid artery were sequentially ligated, and the internal carotid artery was clamped with an artery clip. A small incision was made approximately 4 mm distal to the bifurcation of the internal and external carotid arteries, and the suture was inserted through this incision. The suture was gently advanced toward the internal carotid artery until resistance was felt (approximately 18–20 mm), and then it was secured in place. After 2 hours of ischemia, the suture was slowly withdrawn to initiate reperfusion. The sham operation group underwent the same surgical procedures, except for the occlusion of the internal carotid artery, which was kept patent to maintain normal cerebral blood flow. The drug intervention group received intraperitoneal injections of the respective drugs 30 minutes before the modeling procedure. Behavioral assessment Reference to the modified Neurological Severity Score (mNSS) method for neurological deficit assessment, the mNSS scores were performed by an investigator who was blinded to the experimental conditions. The scoring system ranged from 1 to 18, where scores of 1–6 indicated mild deficits, scores of 7–12 indicated moderate deficits, and scores of 13–18 indicated severe deficits. Cerebral Infarction Volume assessment The infarct volume was determined using 2,3,5-triphenyl tetrazolium chloride (TTC) staining. At 24 hours post-reperfusion, rats were anesthetized with intraperitoneal injection of pentobarbital sodium, followed by decapitation and rapid removal of the brain. The cerebellum, olfactory bulb, and lower brainstem were quickly dissected. The brain tissue was then frozen at -20°C for 20 minutes and subsequently sliced into 5 sections with a thickness of 2 mm. The brain sections were fully immersed in a 2% TTC solution and incubated at 37°C in the dark for 30 minutes. The brain sections were flipped every 5 minutes to ensure even staining. Following staining, the brain sections were transferred to a 4% paraformaldehyde solution for fixation. The infarct area was calculated using Image J software. H&E Staining Paraffin sections were prepared and subjected to hematoxylin and eosin (HE) staining. After dewaxing and hydration of the sections, they were stained, differentiated, counterstained, dehydrated, and then mounted with neutral mounting medium. The pathological changes were observed under an optical microscope and captured through photography. Immunofluorescent Detection Immunofluorescence detection was conducted on paraffin sections. The sections were dewaxed and hydrated, followed by treatment with 3% H 2 O 2 for 10 minutes. After washing three times with PBS for 5 minutes each, antigen retrieval was performed using an antigen retrieval buffer. The sections were then incubated with an appropriate amount of goat serum for 30 minutes to block non-specific binding. Primary antibodies, including Caspase 1 (1:50), NLRP3 (1:100) and GSDMD (1:50), were incubated overnight at 4℃. Following PBS washing, fluorescent secondary antibodies were applied and incubated at 37℃ for 1 hour. Finally, the sections were mounted with a mounting medium and promptly observed under a microscope. Western blotting Upon extraction, brain tissue was incubated with RIPA lysis buffer and PMSF protease inhibitor on ice for 30 minutes. The tissue was then homogenized using a tissue homogenizer, followed by centrifugation at 4°C and 12,000 rpm for 10 minutes to collect the supernatant. Protein concentration was determined using the BCA protein assay kit according to the manufacturer's instructions. Equal amounts of protein samples were separated by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with a rapid blocking solution at room temperature for 30 minutes, followed by overnight incubation at 4°C with primary antibodies against NLRP3 (ab263899, 1:1000, Abcam), Caspase 1 (22915-1-AP, 1:1000, Wuhan Sanying Biotechnology Co., Ltd.) and GSDMD (20770-1-AP, 1:2000, Wuhan Sanying Biotechnology Co., Ltd.). After removing the PVDF membranes, they were incubated with HRP-conjugated rabbit anti-mouse IgG secondary antibody (1:5000) at room temperature for 2 hours. Finally, proteins were visualized by chemiluminescence and quantitatively analyzed using ImageJ software. Enzyme-Linked Immunosorbent Assay (ELISA) Following the manufacturer’s instructions, the levels of IL-1β and IL-18 in brain tissue were determined by ELISA (IL-18 JL20882 Shanghai Jianglai Biotechnology, IL-1β JL20884, Shanghai Jianglai Biotechnology). The absorbance was measured at 450 nm using a microplate reader. Statistical Analysis. SPSS 29.0 software was used to statistical analysis of the data, and data were expressed as mean ± standard(mean ± SD)deviation. For non-normally distribut The Kruskal-Wallis rank-sum test was used for multiple group comparisons when the data were not normally distributed. For normal distribution, one-way analysis of variance (ANOVA) was employed to compare multiple groups for statistical significance. When comparing two groups, t-tests were used. P < 0.05 was considered to be statistically significant. RESULTS Pyroptosis Following Cerebral Ischemia-Reperfusion Injury Expression of Caspase 1, GSDMD and NLRP3 proteins in brain tissue detected by WB after model construction. Caspase 1, GSDMD and NLRP3 proteins were found to be expressed at basal levels in the control group and sham operation group. After cerebral ischemia-reperfusion, the expression levels of these proteins gradually increased, reaching a peak at 24 hours, and then gradually decreased, approaching normal levels by 1 week. These findings indicate that cerebral ischemia-reperfusion injury can activate the process of pyroptosis, consistent with previous literature reports. The protein expression levels in the 24-hour model group were significantly higher compared to the control group ( P < 0.05) ( Fig. 1 ). Improvement of Neurological Deficit and Cerebral Infarct Volume After ischemia-reperfusion injury, neurological deficits manifested as contralateral limb paralysis. The severity of neurological deficits was assessed using mNSS, with higher scores indicating more severe symptoms. In this study, the control group and sham operation group did not exhibit any neurological deficits. However, the model group showed significant deficits. The intervention groups treated with Emodin exhibited lower neurological function scores compared to the 24-hour model group. The middle-dose and high-dose Emodin groups showed more pronounced differences compared to the 24-hour group, and these differences were statistically significant (P < 0.05). Subsequently, infarct volume measurement was performed, and the Emodin intervention group showed a slightly smaller infarct volume compared to the 24-hour group ( P < 0.05) (Fig. 2 ). Emodin Suppressed Pathological Injury No significant pathological changes were observed in the cortical regions of the control group and sham operation group, and the cellular structure remained intact. However, severe pathological damage was observed in the cortical tissue after successful modeling, characterized by neuronal cell swelling, nuclear pyknosis, and disrupted morphological structure, along with disorganized cellular arrangement. Following intervention with Emodin medication, partial restoration of the damaged cellular structure was observed, along with improved cellular swelling and nuclear appearance. Additionally, there was an increased survival of cells in the ischemic penumbra, indicating that Emodin can ameliorate the pathological damage in the infarcted area of ischemia-reperfusion rats (Fig. 3 ). Emodin Prevented the Cell Pyroptosis by Inhibiting the expression of Caspase 1, GSDMD and NLRP3 proteins. Using immunofluorescence techniques, the cytoplasmic localization of Caspase 1, GSDMD and NLRP3 in the cerebral cortex was observed. Subsequently, the expression of Caspase 1, GSDMD and NLRP3 proteins in the ischemic penumbra region of the brain tissue after modeling was detected by WB. The results demonstrated a significant decrease in protein expression levels after intervention with Emodin compared to the control group and sham operation group, and the expression levels were dose-dependently suppressed ( P < 0.05). These findings indicate that Emodin significantly inhibits the expression of pyroptosis-related proteins in the brain tissue of the ischemia-reperfusion model ( Fig. 4 , Fig. 5 ). Emodin Reduced the Production of Pro-Inflammatory Cytokines IL-1β and IL-18 play significant roles in cellular pyroptosis and serve as downstream indicators of Caspase 1 and NLRP3 activation, leading to inflammation in the central nervous system. ELISA analysis revealed a significant increase in IL-1β and IL-18 levels following cerebral ischemia-reperfusion injury in rats. However, intervention with Emodin resulted in a remarkable suppression of IL-1β and IL-18 production, exhibiting a dose-dependent effect consistent with our WB findings. The differences were statistically significant compared to the model group ( P < 0.05). These findings indicate that Emodin effectively reduces the expression of inflammatory cytokines and significantly inhibits the occurrence of inflammation in the context of ischemia-reperfusion injury. (Fig. 6 ). Discussion Cerebrovascular disease is a significant and serious health condition that poses a major threat to human well-being, ranking second only to cardiovascular diseases as the leading global cause of mortality [ 39 , 40 ] . In recent decades, the incidence of stroke among young individuals has been increasing year by year, extending beyond the elderly population. Globally, more than 2 million young people experience stroke each year, with individuals between the ages of 18 and 45 accounting for approximately 10–15% of stroke patients [ 41 ] . Despite the gravity of cerebrovascular diseases, the current treatment outcomes remain unsatisfactory. Numerous studies have demonstrated the therapeutic potential of emodin, a natural compound, in the management of ischemic stroke [ 33 , 34 , 36 ] . However, the underlying mechanisms of its action still require further investigation. In this study, utilizing animal experimental methods, we provide the first evidence that emodin exerts neuroprotective effects by modulating the expression of cell death-related proteins through the Caspase 1-GSDMD axis, thus highlighting its potential in the treatment of cerebrovascular diseases. P revious studies have reported the occurrence of pyroptosis, in response to cerebral ischemia-reperfusion injury. These studies have highlighted the dynamic changes in the expression of pyroptosis-related proteins, peaking at 24 hours post-injury. Similarly, our research also indicate an increase in the expression of pyroptosis-related proteins, including Caspase 1, GSDMD and NLRP3, in the infarcted brain tissue of rats subjected to cerebral ischemia-reperfusion injury, as detected by WB analysis. These observations indicate the presence of pyroptosis following cerebral ischemia-reperfusion injury, with the highest protein expression observed at 24 hours of reperfusion. Additionally, histopathological alterations and increased infarct area were evident, consistent with previous research findings [ 42 ] . Therefore, based on these results, we selected 24 hours as the optimal window for drug intervention. Emodin is a natural anthraquinone derivative that can be found in various natural sources and can also be synthetically produced. It is commonly present in traditional Chinese medicinal plants, which are rich in anthraquinones, including emodin [ 20 , 23 ] . The protective effects of Emodin through its intervention in pyroptosis have been demonstrated in various organ ischemia-reperfusion injuries. However, there is currently no relevant report on whether Emodin exerts its effects through the same mechanism in cerebral ischemia-reperfusion injury. Therefore, this study proposes that Emodin's involvement in pyroptosis mediated by the Caspase 1-GSDMD axis in the context of rat cerebral ischemia-reperfusion injury is a reasonable hypothesis. In our study, Emodin was administered intraperitoneally. Statistical analysis revealed that emodin intervention led to a significant reduction in the mNSS scores of the 24-hour model group. Furthermore, the scores showed a gradual decrease with increasing dosages of Emodin intervention. Compared to the model group, significant differences in scores were observed in the medium-dose and high-dose Emodin intervention groups. These findings indicate that Emodin intervention has the potential to improve neurological deficits in rats following cerebral ischemia-reperfusion injury, and this effect is dose-dependent. The results of TTC staining showed that Emodin intervention resulted in a significant decrease in infarct volume compared to the model group. This finding indicates that Emodin can reduce the infarct volume in rats following cerebral ischemia-reperfusion injury. Additionally, HE staining revealed that after Emodin intervention, there was an improvement in cellular pathological morphology and salvage of the ischemic penumbra compared to the model group. To further explore the expression of Caspase 1, GSDMD and NLRP3 proteins, WBt analysis was performed. The results demonstrated a decrease in the expression of Caspase 1, GSDMD and NLRP3 proteins after Emodin intervention. Significant differences were observed in the medium-dose and high-dose emodin intervention groups compared to the model group. However, there was no significant difference between the low-dose group and the model group. These findings are in line with the behavioral scores and TTC staining results. Previous studies have demonstrated the involvement of Caspase 1, NLRP3 and GSDMD in the process of pyroptosis. This process is initiated by the activation of Caspase 1 through the NLRP3 inflammasome, which subsequently cleaves IL-1β and IL-18 into their mature forms, promoting their pro-inflammatory activities. Meawhile, Caspase 1 activation leads to the activation of GSDMD, causing the assembly and aggregation of its N-terminal domain. GSDMD then binds to phospholipids on the cell membrane, forming pores that disrupt the membrane integrity. Consequently, active substances such as IL-1β, IL-18 and other cellular contents are released, triggering an inflammatory response [ 43 – 45 ] . Therefore, in this study, ELISA was employed to detect the expression levels of IL-1β and IL-18 in the brain tissues of different groups subjected to cerebral ischemia-reperfusion injury. The results revealed that Emodin intervention significantly reduced the expression of IL-1β and IL-18. There were statistically significant differences in the expression levels of IL-1β and IL-18 between the medium-dose and high-dose emodin intervention groups compared to the model group. This further emphasizes that Emodin can alleviate the neuroinflammatory response following cerebral ischemia-reperfusion injury by intervening in the Caspase 1-GSDMD axis-mediated pyroptosis. As a result, Emodin exerts a neuroprotective effect, providing a theoretical basis for its protective role in cerebral ischemia-reperfusion injury. In conclusion, this experiment revealed dynamic changes in the expression of Caspase 1, NLRP3 and GSDMD in the brain tissue of rats following cerebral ischemia-reperfusion injury. Emodin exhibited a certain degree of neuroprotective effect in rats subjected to cerebral ischemia-reperfusion injury, and its mechanism may be associated with the modulation of cell necrosis-related proteins through the Caspase 1-GSDMD axis. Based on the findings of this study, we report for the first time the neuroprotective effect of emodin in rats with cerebral ischemia-reperfusion injury and conducted an in-depth investigation of its mechanism. This provides experimental evidence for the clinical application of emodin and harnesses the advantages of traditional Chinese medicine in the treatment of ischemic cerebrovascular diseases, offering more therapeutic options for future clinical work. However, the investigation in this study did not explore a wider range of intervention dosages to determine the optimal dose. The limited number of experiments conducted necessitates further validation of the clinical applicability and efficacy of emodin through large-scale foundational studies, highlighting the limitations and future research directions of this study. Furthermore, cerebrovascular diseases involve complex pathological and physiological mechanisms, with several aspects still under investigation. It is plausible that emodin may exert its neuroprotective effects in cerebral ischemia-reperfusion injury through the modulation of additional mechanisms or pathways. Therefore, further in-depth research is warranted in this aspect. Conclusion In conclusion, our study further supports previous findings on the neuroprotective effects of Emodin in stroke. Emodin demonstrates its neuroprotective effects in a rat model of cerebral ischemia-reperfusion injury, possibly through the regulation of pyroptosis-related protein expression mediated by the Caspase 1-GSDMD axis.This study may provides valuable fundamental and experimental evidence, further supporting the potential therapeutic application of Emodin in ischemic stroke treatment. Declarations Acknowledgment Guofang Zhang and Tao Liang are co-first authors for this study. Zucai Xu and Jun Zhang are co- correspondance for this study . Statement of Ethics The whole animal study was approved by the Affiliated Hospital of Zunyi Medical University (approval number KLLY(A)-2021-083). CONFLICT OF INTERESTS The authors declare that there are no conflicts of interest. AUTHOR CONTRIBUTIONS Tao Liang and Jun Zhang designed the research study. Guofang Zhang, Yumei Shi and Tao Liang performed the research. Xiaolin Hu, Zeng Ling, Yingying Li, Li Li, Ping Xu, Zucai Xu, Jun Zhang provided help and advice on the experiments. Guofang Zhang and Tao Liang analyzed the data. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. Data Availability Statement The study data used to support the findings of this study are included within the article. FUNDING The study was supported by the Guizhou Administration of Traditional Chinese Medicine (No: QZYY-2021-006) and Zunshi Kehe HZ Zi (2021) No. 64.Project of Guizhou Health Commission (NO. gzwkj2023-005). References Kuriakose D, Xiao Z. Pathophysiology and Treatment of Stroke: Present Status and Future Perspectives. Int J Mol Sci. 2020. 21(20). Niu P, Li L, Zhang Y, et al. Immune regulation based on sex differences in ischemic stroke pathology. Front Immunol. 2023. 14: 1087815. Yawoot N, Chumboatong W, Sengking J, Tocharus C, Tocharus J. Chronic high-fat diet consumption exacerbates pyroptosis- and necroptosis-mediated HMGB1 signaling in the brain after ischemia and reperfusion injury. J Physiol Biochem. 2022. 78(4): 833-844. Frank D, Vince JE. Pyroptosis versus necroptosis: similarities, differences, and crosstalk. Cell Death Differ. 2019. 26(1): 99-114. Carty M, Kearney J, Shanahan KA, et al. Cell Survival and Cytokine Release after Inflammasome Activation Is Regulated by the Toll-IL-1R Protein SARM. Immunity. 2019. 50(6): 1412-1424.e6. Bibo-Verdugo B, Joglekar I, Karadi Giridhar MN, et al. Resurrection of an ancient inflammatory locus reveals switch to caspase-1 specificity on a caspase-4 scaffold. J Biol Chem. 2022. 298(6): 101931. Hu Y, Jiang Y, Li S, et al. The Gasdermin D N-terminal fragment acts as a negative feedback system to inhibit inflammasome-mediated activation of Caspase-1/11. Proc Natl Acad Sci U S A. 2022. 119(45): e2210809119. Liu Z, Wang C, Yang J, et al. Caspase-1 Engages Full-Length Gasdermin D through Two Distinct Interfaces That Mediate Caspase Recruitment and Substrate Cleavage. Immunity. 2020. 53(1): 106-114.e5. Tan LL, Li M, Feng CX, et al. [Role of microglial pyroptosis in hypoxic-ischemic brain damage]. Zhongguo Dang Dai Er Ke Za Zhi. 2020. 22(11): 1226-1232. El-Sisi A, Sokar SS, Shebl AM, Mohamed DZ, Abu-Risha SE. Octreotide and melatonin alleviate inflammasome-induced pyroptosis through inhibition of TLR4-NF-κB-NLRP3 pathway in hepatic ischemia/reperfusion injury. Toxicol Appl Pharmacol. 2021. 410: 115340. Luan F, Rao Z, Peng L, et al. Cinnamic acid preserves against myocardial ischemia/reperfusion injury via suppression of NLRP3/Caspase-1/GSDMD signaling pathway. Phytomedicine. 2022. 100: 154047. Wang K, Sun Z, Ru J, et al. Ablation of GSDMD Improves Outcome of Ischemic Stroke Through Blocking Canonical and Non-canonical Inflammasomes Dependent Pyroptosis in Microglia. Front Neurol. 2020. 11: 577927. Zhang D, Qian J, Zhang P, et al. Gasdermin D serves as a key executioner of pyroptosis in experimental cerebral ischemia and reperfusion model both in vivo and in vitro. J Neurosci Res. 2019. 97(6): 645-660. Poh L, Kang SW, Baik SH, et al. Evidence that NLRC4 inflammasome mediates apoptotic and pyroptotic microglial death following ischemic stroke. Brain Behav Immun. 2019. 75: 34-47. Zhang C, Shi Z, Xu Q, et al. Astragaloside IV alleviates stroke-triggered early brain injury by modulating neuroinflammation and ferroptosis via the Nrf2/HO-1 signaling pathway. Acta Cir Bras. 2023. 38: e380723. Kuang W, Liu T, He F, Yu L, Wang Q, Yu C. Correction to: Icariside II promotes the differentiation of human amniotic mesenchymal stem cells into dopaminergic neuron-like cells. In Vitro Cell Dev Biol Anim. 2021. 57(4): 485. Lin L, Chen W, Yao C, et al. Exploring the target and molecular mechanism of Astragalus membranaceus in the treatment of vascular cognitive impairment based on network pharmacology and molecular docking. Medicine (Baltimore). 2023. 102(12): e33063. Liu S, Zheng M, Li Y, He L, Chen T. The protective effect of Geniposide on diabetic cognitive impairment through BTK/TLR4/NF-κB pathway. Psychopharmacology (Berl). 2020. 237(2): 465-477. Lin J, Lai X, Fan X, et al. Oridonin Protects against Myocardial Ischemia-Reperfusion Injury by Inhibiting GSDMD-Mediated Pyroptosis. Genes (Basel). 2022. 13(11). Dongpeng W, He WX, Xiongjie Y, et al. Pharmacokinetics of Anthraquinones from Medicinal Plants. Front Pharmacol. 2021. 12. Neyrinck AM, Rodriguez J, Taminiau B, et al. Constipation Mitigation by Rhubarb Extract in Middle-Aged Adults Is Linked to Gut Microbiome Modulation: A Double-Blind Randomized Placebo-Controlled Trial. Int J Mol Sci. 2022. 23(23). Gao CC, Li GW, Wang TT, et al. Rhubarb extract relieves constipation by stimulating mucus production in the colon and altering the intestinal flora. Biomed Pharmacother. 2021. 138: 111479. Yang L, Wan Y, Li W, et al. Targeting intestinal flora and its metabolism to explore the laxative effects of rhubarb. Appl Microbiol Biotechnol. 2022. 106(4): 1615-1631. Takayama K, Tsutsumi H, Ishizu T, Okamura N. The influence of rhein 8-O-β-D-glucopyranoside on the purgative action of sennoside A from rhubarb in mice. Biol Pharm Bull. 2012. 35(12): 2204-8. Yim H, Lee YH, Lee CH, Lee SK. Emodin, an anthraquinone derivative isolated from the rhizomes of Rheum palmatum, selectively inhibits the activity of casein kinase II as a competitive inhibitor. Planta Med. 1999. 65(1): 9-13. Janeczko M, Masłyk M, Kubiński K, Golczyk H. Emodin, a natural inhibitor of protein kinase CK2, suppresses growth, hyphal development, and biofilm formation of Candida albicans. Yeast. 2017. 34(6): 253-265. Zheng Q, Li S, Li X, Liu R. Advances in the study of emodin: an update on pharmacological properties and mechanistic basis. Chin Med. 2021. 16(1): 102. Sharifi-Rad J, Herrera-Bravo J, Kamiloglu S, et al. Recent advances in the therapeutic potential of emodin for human health. Biomed Pharmacother. 2022. 154: 113555. Wang L, Zhou G, Zhao S, Yang Y. Soluble Protein Content, Bioactive Compounds and the Antioxidant Activity in Seeds of Ten Rheum tanguticum Lines from Qinghai-Tibet Plateau. Chem Biodivers. 2023 : e202200901. Chao HW, Chen YK, Liu JH, Pan HT, Lin HM, Chao HM. Emodin protected against retinal ischemia insulted neurons through the downregulation of protein overexpression of β-catenin and vascular endothelium factor. BMC Complement Med Ther. 2020. 20(1): 338. Gao LL, Wang ZH, Mu YH, Liu ZL, Pang L. Emodin Promotes Autophagy and Prevents Apoptosis in Sepsis-Associated Encephalopathy through Activating BDNF/TrkB Signaling. Pathobiology. 2022. 89(3): 135-145. Jin T, Ai F, Zhou J, et al. Emodin alleviates lung ischemia-reperfusion injury by suppressing gasdermin D-mediated pyroptosis in rats. Clin Respir J. 2023 . Zhang X, Qin Q, Dai H, Cai S, Zhou C, Guan J. Emodin protects H9c2 cells from hypoxia-induced injury by up-regulating miR-138 expression. Braz J Med Biol Res. 2019. 52(3): e7994. Lu L, Li HQ, Fu DL, Zheng GQ, Fan JP. Rhubarb root and rhizome-based Chinese herbal prescriptions for acute ischemic stroke: a systematic review and meta-analysis. Complement Ther Med. 2014. 22(6): 1060-70. Liu AJ, Song L, Li Y, et al. Active Compounds of Rhubarb Root and Rhizome in Animal Model Experiments of Focal Cerebral Ischemia. Evid Based Complement Alternat Med. 2015. 2015: 210546. Li RR, Liu XF, Feng SX, et al. Pharmacodynamics of Five Anthraquinones (Aloe-emodin, Emodin, Rhein, Chysophanol, and Physcion) and Reciprocal Pharmacokinetic Interaction in Rats with Cerebral Ischemia. Molecules. 2019. 24(10). Morris GP, Wright AL, Tan RP, et al. A Comparative Study of Variables Influencing Ischemic Injury in the Longa and Koizumi Methods of Intraluminal Filament Middle Cerebral Artery Occlusion in Mice. PLoS One. 2016 ;11(2):e0148503. [38] Zhang H, Lu M, Zhang X, et al. Isosteviol sodium protects against ischemic stroke by modulating microglia/macrophage polarization via disruption of GAS5/miR-146a-5p sponge. Sci. Rep. 2019; 9: 12221. Zheng H, Guo X, Kang S, et al. Cdh5-mediated Fpn1 deletion exerts neuroprotective effects during the acute phase and inhibitory effects during the recovery phase of ischemic stroke. Cell Death Dis. 2023. 14(2): 161. Lalu MM, Fergusson DA, Cheng W, et al. Identifying stroke therapeutics from preclinical models: A protocol for a novel application of network meta-analysis. F1000Res. 2019. 8: 11. Zhang GB, Huang HW, Guo W. [Prevention and treatment of stroke in Chinese and African young adults]. Zhonghua Yu Fang Yi Xue Za Zhi. 2022. 56(8): 1142-1149. Luheshi NM, Kovács KJ, Lopez-Castejon G, Brough D, Denes A. Interleukin-1α expression precedes IL-1β after ischemic brain injury and is localised to areas of focal neuronal loss and penumbral tissues. J Neuroinflammation. 2011. 8: 186. GLUCKSMANN A. Cell deaths in normal vertebrate ontogeny. Biol Rev Camb Philos Soc. 1951. 26(1): 59-86. Moujalled D, Strasser A, Liddell JR. Molecular mechanisms of cell death in neurological diseases. Cell Death Differ. 2021. 28(7): 2029-2044. Platnich JM, Chung H, Lau A, et al. Shiga Toxin/Lipopolysaccharide Activates Caspase-4 and Gasdermin D to Trigger Mitochondrial Reactive Oxygen Species Upstream of the NLRP3 Inflammasome. Cell Rep. 2018. 25(6): 1525-1536.e7. Additional Declarations No competing interests reported. Supplementary Files Figure1originalblots1a1c1e4a4c.4e.docx Cite Share Download PDF Status: Published Journal Publication published 03 Jun, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 31 Mar, 2025 Reviews received at journal 28 Mar, 2025 Reviews received at journal 27 Mar, 2025 Reviews received at journal 19 Mar, 2025 Reviewers agreed at journal 18 Mar, 2025 Reviewers agreed at journal 18 Mar, 2025 Reviewers agreed at journal 18 Mar, 2025 Reviewers agreed at journal 18 Mar, 2025 Reviewers invited by journal 18 Mar, 2025 Editor assigned by journal 18 Mar, 2025 Editor invited by journal 17 Mar, 2025 Submission checks completed at journal 17 Mar, 2025 First submitted to journal 03 Mar, 2025 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6149634","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":430916755,"identity":"95487894-f1e3-47ae-a4bf-3cd8adb55551","order_by":0,"name":"Tao Liang","email":"","orcid":"","institution":"Affiliated Hospital of Zunyi Medical College","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Liang","suffix":""},{"id":430916756,"identity":"6e34c586-4b83-4bc3-8c43-8e79ca7dfa3b","order_by":1,"name":"Guofang Zhang","email":"","orcid":"","institution":"Affiliated Hospital of Zunyi Medical College","correspondingAuthor":false,"prefix":"","firstName":"Guofang","middleName":"","lastName":"Zhang","suffix":""},{"id":430916757,"identity":"50a07c2b-19bd-4f7f-8340-5ba4239de458","order_by":2,"name":"Xiaolin Hu","email":"","orcid":"","institution":"Affiliated Hospital of Zunyi Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xiaolin","middleName":"","lastName":"Hu","suffix":""},{"id":430916758,"identity":"8076fff5-fbe6-478b-b9b7-2c68d6ae269f","order_by":3,"name":"Jun Qian","email":"","orcid":"","institution":"Affiliated Hospital of Zunyi Medical College","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Qian","suffix":""},{"id":430916759,"identity":"1537bc31-cf8c-408e-a97b-3ffab9f366e6","order_by":4,"name":"Yumei Shi","email":"","orcid":"","institution":"Affiliated Hospital of Zunyi Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yumei","middleName":"","lastName":"Shi","suffix":""},{"id":430916760,"identity":"517e6d4b-4d27-46d4-af58-94b7079b0fc0","order_by":5,"name":"Zeng Ling","email":"","orcid":"","institution":"Affiliated Hospital of Zunyi Medical College","correspondingAuthor":false,"prefix":"","firstName":"Zeng","middleName":"","lastName":"Ling","suffix":""},{"id":430916761,"identity":"2c0cd65f-f587-474b-9661-27c9db50b162","order_by":6,"name":"Ping Xu","email":"","orcid":"","institution":"Affiliated Hospital of Zunyi Medical College","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Xu","suffix":""},{"id":430916762,"identity":"e5748156-03f3-41c5-94f1-aaba763e6542","order_by":7,"name":"Zucai Xu","email":"","orcid":"","institution":"Affiliated Hospital of Zunyi Medical College","correspondingAuthor":false,"prefix":"","firstName":"Zucai","middleName":"","lastName":"Xu","suffix":""},{"id":430916763,"identity":"bd756802-4fb5-4fda-a32e-7eb2d1eded34","order_by":8,"name":"Jun Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIie3RsQrCMBCA4ZRAptQ6xsX6CCmBTn0TlxQhnRTExaFDQLCja/sWgotj4cCp4Ctk6tzuDtbJMRkF8833wyWHkOf9KD6+smWEMRjnJGm0EouKKO6c4FBDzp90xZzG47ow++aupACKOCqztTUJain52GW7FMLWoIfaautSTLZJTdQhhZnkgQZ7QliuBSWQ306UM6eEsg0S4RnyK3ZNGO1RUndKMJg+Wbq8Ja6Kng/H6ZQXADOUmT1Bc0m+F5TW8Y+oxcZp0PM873+9AS/4P5Uw+/+cAAAAAElFTkSuQmCC","orcid":"","institution":"Affiliated Hospital of Zunyi Medical College","correspondingAuthor":true,"prefix":"","firstName":"Jun","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-03-04 00:23:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6149634/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6149634/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-04863-y","type":"published","date":"2025-06-03T15:56:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78880831,"identity":"cb9ac188-47b4-402f-b55f-a9457fc2cc0c","added_by":"auto","created_at":"2025-03-20 08:28:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":68554,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in expression of Caspase 1, GSDMD and NLRP 3 in cerebral ischemia-repe rfusion control, sham and operation group in rats.\u003cstrong\u003e \u003c/strong\u003eA、C、E: Shows the Western Blotting band plots for different time point groups. B、D、F: Shows the bar statistics, compared with the contro group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. Original blots are presented in Supplementary Figure 1.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6149634/v1/55eb078385a2d8eae598506c.png"},{"id":78880754,"identity":"379eb61a-c54f-4c1a-8bc1-f7866edb7263","added_by":"auto","created_at":"2025-03-20 08:27:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":124998,"visible":true,"origin":"","legend":"\u003cp\u003eThe infarct volume ratio of rats in each groups. \u0026nbsp;A: The red area indicates no infarction, and the pale white area is the infarct focus. B: A bar chart with infarct volume ratio in the 24h+Emodin (high dose) group, compared with the contro group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6149634/v1/942f4e7bc31f6d48b494c2f4.png"},{"id":78880769,"identity":"2bc55844-4e9a-43ef-a12d-57416c53dfef","added_by":"auto","created_at":"2025-03-20 08:27:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":570347,"visible":true,"origin":"","legend":"\u003cp\u003eHE staining of control group, 24h group and 24h+Emodin（Medium-dose）. (20×、40×，Scale Bar 50μm)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6149634/v1/88c56c7c9c832a74b1f93e81.png"},{"id":78880770,"identity":"a255e751-e778-46d9-af38-a2658673e38d","added_by":"auto","created_at":"2025-03-20 08:27:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":91647,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of Caspase 1, GSDMD and NLRP 3 after intervention of emodin. A、C、E: Western Blotting strip plots of the groups at different time points after drug intervention. B、D、F: A a bar chart, compared with the control group, the expression level of Caspase 1, NLRP 3 and GSDMD proteins in 24h+Emodin (Medium-dose) and 24h+Emodin (High-dose) groups were increased, *\u003cem\u003eP\u003c/em\u003e＜0.05. Original blots are presented in Supplementary Figure 1.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6149634/v1/353e69243f83fec40bb9e697.png"},{"id":78880963,"identity":"d5f8a3d2-bcf7-4b92-acfd-07e18e92605e","added_by":"auto","created_at":"2025-03-20 08:35:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":170328,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence detection of Caspase 1、GSDMD、NLRP3 in rat brain tissue(40×，Scale Bar 50μm).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6149634/v1/b8268c80bea8c7f963295974.png"},{"id":78880759,"identity":"09c14223-0356-4de8-b1f2-13b026337d02","added_by":"auto","created_at":"2025-03-20 08:27:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":79026,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of IL-1β、IL-18 in each group. A: IL-18; B: IL-1β.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6149634/v1/0e4f032f746a087540609d5c.png"},{"id":84242377,"identity":"4a554dbe-098f-4228-9774-383fc85f067c","added_by":"auto","created_at":"2025-06-09 16:06:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1579330,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6149634/v1/3fe98bf3-46e6-4e85-a4a5-366bf2d1ec44.pdf"},{"id":78880780,"identity":"25889e65-eee3-4d2f-961f-ba5f65c53770","added_by":"auto","created_at":"2025-03-20 08:27:45","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":210051,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1originalblots1a1c1e4a4c.4e.docx","url":"https://assets-eu.researchsquare.com/files/rs-6149634/v1/f0faca4b6ce881750c9043e2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Emodin influence pyroptosis-related Caspase 1-GSDMD axis alleviated cerebral ischemia-reperfusion injury in rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eStroke is a highly prevalent disease in the field of neuroscience and ranks among the leading causes of death and disability worldwide. Acute ischemic stroke (AIS) represents the most common subtype. Cerebral ischemia-reperfusion injury serves as a significant mechanism that intensifies neurological impairments. Therefore, mitigating the detrimental effects of ischemia-reperfusion injury assumes a pivotal role in the early management of ischemic stroke.\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Pyroptosis, a recently identified form of programmed cell death, is primarily driven by the activation of different caspases, including Caspase 1, through the inflammasome pathway. Upon activation, these caspases cleave and polymerize various gasdermin family members, such as gasdermin D (GSDMD), leading to cell membrane permeabilization and subsequent release of multiple pro-inflammatory factors. Ultimately, this cascade of events culminates in cell death\u003csup\u003e[\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Researches have shown that suppressing the expression of pyroptosis-related proteins, including GSDMD, in rat model of cerebral ischemia-reperfusion injury can markedly enhance the viability of microglial cells\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Furthermore, studies conducted in rat models of renal ischemia-reperfusion injury, hepatic ischemia-reperfusion injury and myocardial ischemia-reperfusion injury have demonstrated the presence of cell pyroptosis-related protein expression. Inhibition of these protein expressions has exhibited protective effects\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Subsequently, it was found that suppressing the expression of GSDMD effectively reduces the levels of IL-18, Caspase 1, Caspase 11, and IL-1β, thereby inhibiting inflammation and promoting neurological recovery after cerebral ischemia-reperfusion injury. This suggests that cell pyroptosis plays a role in cerebral ischemia-reperfusion injury and blocking cell pyroptosis may contribute to the recovery from ischemic stroke\u003csup\u003e[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. In the field of neuroprotection research, an increasing number of researchers have turned their attention to traditional Chinese herbs, such as Astragaloside IV\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e, Icariside Ⅱ \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e, Astragalus\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e, Gardenoside \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e and Herba Erigerontis \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Emodin, also known as 1,3,8-trihydroxy-6-methylanthraquinone (C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e), is included in such studies. It was originally discovered in the roots and stems of plants and was first isolated in 1925. Emodin is a significant component of various traditional Chinese herbs, commonly known for its laxative effects \u003csup\u003e[\u003cspan additionalcitationids=\"CR21 CR22 CR23\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHowever, with advancements in technology, numerous reports have indicated that Emodin possesses diuretic, antiviral, anticancer, hepatoprotective, immunosuppressive, and antimicrobial properties\u003csup\u003e[\u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Due to its diverse therapeutic benefits, Emodin is gradually finding its place in clinical prescriptions of traditional Chinese medicine within medical institutions.Recent studies have shown that Emodin also participates in the pathogenesis of certain ischemic diseases. For instance, Emodin has demonstrated its ability to protect retinal neurons from ischemic injury\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, improve cognitive dysfunction associated with sepsis-related encephalopathy, and inhibit pathological damage to hippocampal neurons \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. it has been shown to alleviate lung ischemia-reperfusion injury by inhibiting the expression of GSDMD and Caspase 1 \u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Furthermore, it exhibits protective effects in myocardial ischemia-reperfusion injury \u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. These findings highlight the protective role of Emodin in various organ ischemia-reperfusion injuries by intervening in cell pyroptosis processes.\u003c/p\u003e \u003cp\u003eStudies have demonstrated that Emodin exhibits a certain degree of improvement on the neurological deficits observed in patients with acute ischemic stroke and in rats with cerebral ischemia-reperfusion injury, although the underlying mechanisms remain unclear\u003csup\u003e[\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Considering its protective effects on pyroptosis-mediated tissue injury in various organs such as the myocardium, lungs and retina during ischemia-reperfusion, Emodin represents a promising target for intervention in ischemic stroke treatment. However, the mechanisms by which Emodin influences cerebral ischemia-reperfusion injury in rats, particularly its involvement in the Caspase-1-GSDMD axis-mediated pyroptosis, remain unclear. In this study, we investigate the neuroprotective role of Emodin by targeting pyroptosis in a rat model of cerebral ischemia-reperfusion injury. The aim of our study is to explore the potential neuroprotective effects of Emodin through its intervention in pyroptosis in the context of cerebral ischemia-reperfusion injury. The findings may contribute to the development of neuroprotective drugs for post-ischemic stroke brain protection, providing experimental evidence to overcome current treatment challenges in clinical practice.\u003c/p\u003e"},{"header":"MATERIALS AND METHOD","content":"\u003cp\u003eAnimals and Treatment\u003c/p\u003e \u003cp\u003eAdult male SD rats (200-250g), provided by Tianqin Biotechnology Co., Ltd. (Changsha, China; license number: SCXK (Xiang) 2019-0004). were housed in cages under controlled conditions at a constant temperature of 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. During the experimental period, the rats had access to standard laboratory food and purified drinking water. All animal procedures were conducted in accordance with the ethical guidelines for animal research and were approved by the Ethics Committee of Affiliated Hospital of Zunyi Medical University. The rats were randomly divided into the following groups: control group, sham operation group, middle cerebral artery occlusion (MCAO) group, MCAO\u0026thinsp;+\u0026thinsp;low-dose Emodin group (10mg/kg), MCAO\u0026thinsp;+\u0026thinsp;medium-dose Emodin group (20mg/kg) and MCAO\u0026thinsp;+\u0026thinsp;high-dose Emodin group (40mg/kg). After rats were anaesthetized with pentobarbital sodium (40 mg/kg) before sacrificed. All efforts were made to minimize the rat\u0026rsquo;s suffering.\u003c/p\u003e \u003cp\u003eModel Establishment\u003c/p\u003e \u003cp\u003eThe MCAO model was established using the intraluminal suture method\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. The rats were anesthetized with pentobarbital sodium (40 mg/kg) administered via intraperitoneal injection. After midline incision and tissue separation in the slightly right-sided region, the external carotid artery and common carotid artery were sequentially ligated, and the internal carotid artery was clamped with an artery clip. A small incision was made approximately 4 mm distal to the bifurcation of the internal and external carotid arteries, and the suture was inserted through this incision. The suture was gently advanced toward the internal carotid artery until resistance was felt (approximately 18\u0026ndash;20 mm), and then it was secured in place. After 2 hours of ischemia, the suture was slowly withdrawn to initiate reperfusion. The sham operation group underwent the same surgical procedures, except for the occlusion of the internal carotid artery, which was kept patent to maintain normal cerebral blood flow. The drug intervention group received intraperitoneal injections of the respective drugs 30 minutes before the modeling procedure.\u003c/p\u003e \u003cp\u003eBehavioral assessment\u003c/p\u003e \u003cp\u003eReference to the modified Neurological Severity Score (mNSS) method for neurological deficit assessment, the mNSS scores were performed by an investigator who was blinded to the experimental conditions. The scoring system ranged from 1 to 18, where scores of 1\u0026ndash;6 indicated mild deficits, scores of 7\u0026ndash;12 indicated moderate deficits, and scores of 13\u0026ndash;18 indicated severe deficits.\u003c/p\u003e \u003cp\u003eCerebral Infarction Volume assessment\u003c/p\u003e \u003cp\u003eThe infarct volume was determined using 2,3,5-triphenyl tetrazolium chloride (TTC) staining. At 24 hours post-reperfusion, rats were anesthetized with intraperitoneal injection of pentobarbital sodium, followed by decapitation and rapid removal of the brain. The cerebellum, olfactory bulb, and lower brainstem were quickly dissected. The brain tissue was then frozen at -20\u0026deg;C for 20 minutes and subsequently sliced into 5 sections with a thickness of 2 mm. The brain sections were fully immersed in a 2% TTC solution and incubated at 37\u0026deg;C in the dark for 30 minutes. The brain sections were flipped every 5 minutes to ensure even staining. Following staining, the brain sections were transferred to a 4% paraformaldehyde solution for fixation. The infarct area was calculated using Image J software.\u003c/p\u003e \u003cp\u003eH\u0026amp;E Staining\u003c/p\u003e \u003cp\u003eParaffin sections were prepared and subjected to hematoxylin and eosin (HE) staining. After dewaxing and hydration of the sections, they were stained, differentiated, counterstained, dehydrated, and then mounted with neutral mounting medium. The pathological changes were observed under an optical microscope and captured through photography.\u003c/p\u003e \u003cp\u003eImmunofluorescent Detection\u003c/p\u003e \u003cp\u003eImmunofluorescence detection was conducted on paraffin sections. The sections were dewaxed and hydrated, followed by treatment with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 10 minutes. After washing three times with PBS for 5 minutes each, antigen retrieval was performed using an antigen retrieval buffer. The sections were then incubated with an appropriate amount of goat serum for 30 minutes to block non-specific binding. Primary antibodies, including Caspase 1 (1:50), NLRP3 (1:100) and GSDMD (1:50), were incubated overnight at 4℃. Following PBS washing, fluorescent secondary antibodies were applied and incubated at 37℃ for 1 hour. Finally, the sections were mounted with a mounting medium and promptly observed under a microscope.\u003c/p\u003e \u003cp\u003eWestern blotting\u003c/p\u003e \u003cp\u003eUpon extraction, brain tissue was incubated with RIPA lysis buffer and PMSF protease inhibitor on ice for 30 minutes. The tissue was then homogenized using a tissue homogenizer, followed by centrifugation at 4\u0026deg;C and 12,000 rpm for 10 minutes to collect the supernatant. Protein concentration was determined using the BCA protein assay kit according to the manufacturer's instructions. Equal amounts of protein samples were separated by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with a rapid blocking solution at room temperature for 30 minutes, followed by overnight incubation at 4\u0026deg;C with primary antibodies against NLRP3 (ab263899, 1:1000, Abcam), Caspase 1 (22915-1-AP, 1:1000, Wuhan Sanying Biotechnology Co., Ltd.) and GSDMD (20770-1-AP, 1:2000, Wuhan Sanying Biotechnology Co., Ltd.). After removing the PVDF membranes, they were incubated with HRP-conjugated rabbit anti-mouse IgG secondary antibody (1:5000) at room temperature for 2 hours. Finally, proteins were visualized by chemiluminescence and quantitatively analyzed using ImageJ software.\u003c/p\u003e \u003cp\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/p\u003e \u003cp\u003eFollowing the manufacturer\u0026rsquo;s instructions, the levels of IL-1β and IL-18 in brain tissue were determined by ELISA (IL-18 JL20882 Shanghai Jianglai Biotechnology, IL-1β JL20884, Shanghai Jianglai Biotechnology). The absorbance was measured at 450 nm using a microplate reader.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis.\u003c/h2\u003e \u003cp\u003eSPSS 29.0 software was used to statistical analysis of the data, and data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard(mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)deviation. For non-normally distribut The Kruskal-Wallis rank-sum test was used for multiple group comparisons when the data were not normally distributed. For normal distribution, one-way analysis of variance (ANOVA) was employed to compare multiple groups for statistical significance. When comparing two groups, t-tests were used. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003ePyroptosis Following Cerebral Ischemia-Reperfusion Injury\u003c/p\u003e \u003cp\u003eExpression of Caspase 1, GSDMD and NLRP3 proteins in brain tissue detected by WB after model construction. Caspase 1, GSDMD and NLRP3 proteins were found to be expressed at basal levels in the control group and sham operation group. After cerebral ischemia-reperfusion, the expression levels of these proteins gradually increased, reaching a peak at 24 hours, and then gradually decreased, approaching normal levels by 1 week. These findings indicate that cerebral ischemia-reperfusion injury can activate the process of pyroptosis, consistent with previous literature reports. The protein expression levels in the 24-hour model group were significantly higher compared to the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) ( Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImprovement of Neurological Deficit and Cerebral Infarct Volume\u003c/p\u003e \u003cp\u003eAfter ischemia-reperfusion injury, neurological deficits manifested as contralateral limb paralysis. The severity of neurological deficits was assessed using mNSS, with higher scores indicating more severe symptoms. In this study, the control group and sham operation group did not exhibit any neurological deficits. However, the model group showed significant deficits. The intervention groups treated with Emodin exhibited lower neurological function scores compared to the 24-hour model group. The middle-dose and high-dose Emodin groups showed more pronounced differences compared to the 24-hour group, and these differences were statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Subsequently, infarct volume measurement was performed, and the Emodin intervention group showed a slightly smaller infarct volume compared to the 24-hour group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEmodin Suppressed Pathological Injury\u003c/p\u003e \u003cp\u003eNo significant pathological changes were observed in the cortical regions of the control group and sham operation group, and the cellular structure remained intact. However, severe pathological damage was observed in the cortical tissue after successful modeling, characterized by neuronal cell swelling, nuclear pyknosis, and disrupted morphological structure, along with disorganized cellular arrangement. Following intervention with Emodin medication, partial restoration of the damaged cellular structure was observed, along with improved cellular swelling and nuclear appearance. Additionally, there was an increased survival of cells in the ischemic penumbra, indicating that Emodin can ameliorate the pathological damage in the infarcted area of ischemia-reperfusion rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEmodin Prevented the Cell Pyroptosis by Inhibiting the expression of Caspase 1, GSDMD and NLRP3 proteins.\u003c/p\u003e \u003cp\u003eUsing immunofluorescence techniques, the cytoplasmic localization of Caspase 1, GSDMD and NLRP3 in the cerebral cortex was observed. Subsequently, the expression of Caspase 1, GSDMD and NLRP3 proteins in the ischemic penumbra region of the brain tissue after modeling was detected by WB. The results demonstrated a significant decrease in protein expression levels after intervention with Emodin compared to the control group and sham operation group, and the expression levels were dose-dependently suppressed (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings indicate that Emodin significantly inhibits the expression of pyroptosis-related proteins in the brain tissue of the ischemia-reperfusion model ( Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEmodin Reduced the Production of Pro-Inflammatory Cytokines\u003c/p\u003e \u003cp\u003eIL-1β and IL-18 play significant roles in cellular pyroptosis and serve as downstream indicators of Caspase 1 and NLRP3 activation, leading to inflammation in the central nervous system. ELISA analysis revealed a significant increase in IL-1β and IL-18 levels following cerebral ischemia-reperfusion injury in rats. However, intervention with Emodin resulted in a remarkable suppression of IL-1β and IL-18 production, exhibiting a dose-dependent effect consistent with our WB findings. The differences were statistically significant compared to the model group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings indicate that Emodin effectively reduces the expression of inflammatory cytokines and significantly inhibits the occurrence of inflammation in the context of ischemia-reperfusion injury. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCerebrovascular disease is a significant and serious health condition that poses a major threat to human well-being, ranking second only to cardiovascular diseases as the leading global cause of mortality\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. In recent decades, the incidence of stroke among young individuals has been increasing year by year, extending beyond the elderly population. Globally, more than 2\u0026nbsp;million young people experience stroke each year, with individuals between the ages of 18 and 45 accounting for approximately 10\u0026ndash;15% of stroke patients\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e. Despite the gravity of cerebrovascular diseases, the current treatment outcomes remain unsatisfactory. Numerous studies have demonstrated the therapeutic potential of emodin, a natural compound, in the management of ischemic stroke\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. However, the underlying mechanisms of its action still require further investigation. In this study, utilizing animal experimental methods, we provide the first evidence that emodin exerts neuroprotective effects by modulating the expression of cell death-related proteins through the Caspase 1-GSDMD axis, thus highlighting its potential in the treatment of cerebrovascular diseases.\u003c/p\u003e \u003cp\u003e \u003cb\u003eP\u003c/b\u003erevious studies have reported the occurrence of pyroptosis, in response to cerebral ischemia-reperfusion injury. These studies have highlighted the dynamic changes in the expression of pyroptosis-related proteins, peaking at 24 hours post-injury. Similarly, our research also indicate an increase in the expression of pyroptosis-related proteins, including Caspase 1, GSDMD and NLRP3, in the infarcted brain tissue of rats subjected to cerebral ischemia-reperfusion injury, as detected by WB analysis. These observations indicate the presence of pyroptosis following cerebral ischemia-reperfusion injury, with the highest protein expression observed at 24 hours of reperfusion. Additionally, histopathological alterations and increased infarct area were evident, consistent with previous research findings \u003csup\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. Therefore, based on these results, we selected 24 hours as the optimal window for drug intervention.\u003c/p\u003e \u003cp\u003eEmodin is a natural anthraquinone derivative that can be found in various natural sources and can also be synthetically produced. It is commonly present in traditional Chinese medicinal plants, which are rich in anthraquinones, including emodin\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The protective effects of Emodin through its intervention in pyroptosis have been demonstrated in various organ ischemia-reperfusion injuries. However, there is currently no relevant report on whether Emodin exerts its effects through the same mechanism in cerebral ischemia-reperfusion injury. Therefore, this study proposes that Emodin's involvement in pyroptosis mediated by the Caspase 1-GSDMD axis in the context of rat cerebral ischemia-reperfusion injury is a reasonable hypothesis.\u003c/p\u003e \u003cp\u003eIn our study, Emodin was administered intraperitoneally. Statistical analysis revealed that emodin intervention led to a significant reduction in the mNSS scores of the 24-hour model group. Furthermore, the scores showed a gradual decrease with increasing dosages of Emodin intervention. Compared to the model group, significant differences in scores were observed in the medium-dose and high-dose Emodin intervention groups. These findings indicate that Emodin intervention has the potential to improve neurological deficits in rats following cerebral ischemia-reperfusion injury, and this effect is dose-dependent.\u003c/p\u003e \u003cp\u003eThe results of TTC staining showed that Emodin intervention resulted in a significant decrease in infarct volume compared to the model group. This finding indicates that Emodin can reduce the infarct volume in rats following cerebral ischemia-reperfusion injury. Additionally, HE staining revealed that after Emodin intervention, there was an improvement in cellular pathological morphology and salvage of the ischemic penumbra compared to the model group.\u003c/p\u003e \u003cp\u003eTo further explore the expression of Caspase 1, GSDMD and NLRP3 proteins, WBt analysis was performed. The results demonstrated a decrease in the expression of Caspase 1, GSDMD and NLRP3 proteins after Emodin intervention. Significant differences were observed in the medium-dose and high-dose emodin intervention groups compared to the model group. However, there was no significant difference between the low-dose group and the model group. These findings are in line with the behavioral scores and TTC staining results.\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated the involvement of Caspase 1, NLRP3 and GSDMD in the process of pyroptosis. This process is initiated by the activation of Caspase 1 through the NLRP3 inflammasome, which subsequently cleaves IL-1β and IL-18 into their mature forms, promoting their pro-inflammatory activities. Meawhile, Caspase 1 activation leads to the activation of GSDMD, causing the assembly and aggregation of its N-terminal domain. GSDMD then binds to phospholipids on the cell membrane, forming pores that disrupt the membrane integrity. Consequently, active substances such as IL-1β, IL-18 and other cellular contents are released, triggering an inflammatory response \u003csup\u003e[\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e. Therefore, in this study, ELISA was employed to detect the expression levels of IL-1β and IL-18 in the brain tissues of different groups subjected to cerebral ischemia-reperfusion injury. The results revealed that Emodin intervention significantly reduced the expression of IL-1β and IL-18. There were statistically significant differences in the expression levels of IL-1β and IL-18 between the medium-dose and high-dose emodin intervention groups compared to the model group. This further emphasizes that Emodin can alleviate the neuroinflammatory response following cerebral ischemia-reperfusion injury by intervening in the Caspase 1-GSDMD axis-mediated pyroptosis. As a result, Emodin exerts a neuroprotective effect, providing a theoretical basis for its protective role in cerebral ischemia-reperfusion injury.\u003c/p\u003e \u003cp\u003eIn conclusion, this experiment revealed dynamic changes in the expression of Caspase 1, NLRP3 and GSDMD in the brain tissue of rats following cerebral ischemia-reperfusion injury. Emodin exhibited a certain degree of neuroprotective effect in rats subjected to cerebral ischemia-reperfusion injury, and its mechanism may be associated with the modulation of cell necrosis-related proteins through the Caspase 1-GSDMD axis. Based on the findings of this study, we report for the first time the neuroprotective effect of emodin in rats with cerebral ischemia-reperfusion injury and conducted an in-depth investigation of its mechanism. This provides experimental evidence for the clinical application of emodin and harnesses the advantages of traditional Chinese medicine in the treatment of ischemic cerebrovascular diseases, offering more therapeutic options for future clinical work. However, the investigation in this study did not explore a wider range of intervention dosages to determine the optimal dose. The limited number of experiments conducted necessitates further validation of the clinical applicability and efficacy of emodin through large-scale foundational studies, highlighting the limitations and future research directions of this study. Furthermore, cerebrovascular diseases involve complex pathological and physiological mechanisms, with several aspects still under investigation. It is plausible that emodin may exert its neuroprotective effects in cerebral ischemia-reperfusion injury through the modulation of additional mechanisms or pathways. Therefore, further in-depth research is warranted in this aspect.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our study further supports previous findings on the neuroprotective effects of Emodin in stroke. Emodin demonstrates its neuroprotective effects in a rat model of cerebral ischemia-reperfusion injury, possibly through the regulation of pyroptosis-related protein expression mediated by the Caspase 1-GSDMD axis.This study may provides valuable fundamental and experimental evidence, further supporting the potential therapeutic application of Emodin in ischemic stroke treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgment\u003c/p\u003e\n\u003cp\u003eGuofang \u0026nbsp;Zhang and Tao Liang are co-first authors for this study. Zucai Xu and Jun Zhang are co- correspondance for this study .\u003c/p\u003e\n\u003cp\u003eStatement of Ethics\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe whole animal study was approved by the Affiliated Hospital of Zunyi Medical University (approval number KLLY(A)-2021-083).\u003c/p\u003e\n\u003cp\u003eCONFLICT OF INTERESTS\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003eAUTHOR CONTRIBUTIONS\u003c/p\u003e\n\u003cp\u003eTao Liang and Jun Zhang designed the research study. Guofang \u0026nbsp; Zhang, Yumei Shi and Tao Liang performed the research. Xiaolin Hu, Zeng Ling, Yingying Li, Li Li, Ping Xu, Zucai Xu,\u0026nbsp;Jun Zhang\u0026nbsp;provided help and advice on the experiments. Guofang \u0026nbsp; Zhang and Tao Liang analyzed the data. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eData Availability Statement\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe study data used to support the findings of this study are included within the article.\u003c/p\u003e\n\u003cp\u003eFUNDING\u003c/p\u003e\n\u003cp\u003eThe study was supported by the Guizhou Administration of Traditional Chinese Medicine (No: QZYY-2021-006) and Zunshi Kehe HZ Zi (2021) No. 64.Project of Guizhou Health Commission (NO. gzwkj2023-005).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKuriakose D, Xiao Z. Pathophysiology and Treatment of Stroke: Present Status and Future Perspectives. Int J Mol Sci. 2020. 21(20).\u003c/li\u003e\n\u003cli\u003eNiu P, Li L, Zhang Y, et al. Immune regulation based on sex differences in ischemic stroke pathology. Front Immunol. 2023. 14: 1087815.\u003c/li\u003e\n\u003cli\u003eYawoot N, Chumboatong W, Sengking J, Tocharus C, Tocharus J. Chronic high-fat diet consumption exacerbates pyroptosis- and necroptosis-mediated HMGB1 signaling in the brain after ischemia and reperfusion injury. J Physiol Biochem. 2022. 78(4): 833-844.\u003c/li\u003e\n\u003cli\u003eFrank D, Vince JE. Pyroptosis versus necroptosis: similarities, differences, and crosstalk. Cell Death Differ. 2019. 26(1): 99-114.\u003c/li\u003e\n\u003cli\u003eCarty M, Kearney J, Shanahan KA, et al. Cell Survival and Cytokine Release after Inflammasome Activation Is Regulated by the Toll-IL-1R Protein SARM. Immunity. 2019. 50(6): 1412-1424.e6.\u003c/li\u003e\n\u003cli\u003eBibo-Verdugo B, Joglekar I, Karadi Giridhar MN, et al. Resurrection of an ancient inflammatory locus reveals switch to caspase-1 specificity on a caspase-4 scaffold. J Biol Chem. 2022. 298(6): 101931.\u003c/li\u003e\n\u003cli\u003eHu Y, Jiang Y, Li S, et al. The Gasdermin D N-terminal fragment acts as a negative feedback system to inhibit inflammasome-mediated activation of Caspase-1/11. Proc Natl Acad Sci U S A. 2022. 119(45): e2210809119.\u003c/li\u003e\n\u003cli\u003eLiu Z, Wang C, Yang J, et al. Caspase-1 Engages Full-Length Gasdermin D through Two Distinct Interfaces That Mediate Caspase Recruitment and Substrate Cleavage. Immunity. 2020. 53(1): 106-114.e5.\u003c/li\u003e\n\u003cli\u003eTan LL, Li M, Feng CX, et al. [Role of microglial pyroptosis in hypoxic-ischemic brain damage]. Zhongguo Dang Dai Er Ke Za Zhi. 2020. 22(11): 1226-1232.\u003c/li\u003e\n\u003cli\u003eEl-Sisi A, Sokar SS, Shebl AM, Mohamed DZ, Abu-Risha SE. Octreotide and melatonin alleviate inflammasome-induced pyroptosis through inhibition of TLR4-NF-\u0026kappa;B-NLRP3 pathway in hepatic ischemia/reperfusion injury. Toxicol Appl Pharmacol. 2021. 410: 115340.\u003c/li\u003e\n\u003cli\u003eLuan F, Rao Z, Peng L, et al. Cinnamic acid preserves against myocardial ischemia/reperfusion injury via suppression of NLRP3/Caspase-1/GSDMD signaling pathway. Phytomedicine. 2022. 100: 154047.\u003c/li\u003e\n\u003cli\u003eWang K, Sun Z, Ru J, et al. Ablation of GSDMD Improves Outcome of Ischemic Stroke Through Blocking Canonical and Non-canonical Inflammasomes Dependent Pyroptosis in Microglia. Front Neurol. 2020. 11: 577927.\u003c/li\u003e\n\u003cli\u003eZhang D, Qian J, Zhang P, et al. Gasdermin D serves as a key executioner of pyroptosis in experimental cerebral ischemia and reperfusion model both in vivo and in vitro. J Neurosci Res. 2019. 97(6): 645-660.\u003c/li\u003e\n\u003cli\u003ePoh L, Kang SW, Baik SH, et al. Evidence that NLRC4 inflammasome mediates apoptotic and pyroptotic microglial death following ischemic stroke. Brain Behav Immun. 2019. 75: 34-47.\u003c/li\u003e\n\u003cli\u003eZhang C, Shi Z, Xu Q, et al. Astragaloside IV alleviates stroke-triggered early brain injury by modulating neuroinflammation and ferroptosis via the Nrf2/HO-1 signaling pathway. Acta Cir Bras. 2023. 38: e380723.\u003c/li\u003e\n\u003cli\u003eKuang W, Liu T, He F, Yu L, Wang Q, Yu C. Correction to: Icariside II promotes the differentiation of human amniotic mesenchymal stem cells into dopaminergic neuron-like cells. In Vitro Cell Dev Biol Anim. 2021. 57(4): 485.\u003c/li\u003e\n\u003cli\u003eLin L, Chen W, Yao C, et al. Exploring the target and molecular mechanism of Astragalus membranaceus in the treatment of vascular cognitive impairment based on network pharmacology and molecular docking. Medicine (Baltimore). 2023. 102(12): e33063.\u003c/li\u003e\n\u003cli\u003eLiu S, Zheng M, Li Y, He L, Chen T. The protective effect of Geniposide on diabetic cognitive impairment through BTK/TLR4/NF-\u0026kappa;B pathway. Psychopharmacology (Berl). 2020. 237(2): 465-477.\u003c/li\u003e\n\u003cli\u003eLin J, Lai X, Fan X, et al. Oridonin Protects against Myocardial Ischemia-Reperfusion Injury by Inhibiting GSDMD-Mediated Pyroptosis. Genes (Basel). 2022. 13(11).\u003c/li\u003e\n\u003cli\u003eDongpeng W, He WX, Xiongjie Y, et al. Pharmacokinetics of Anthraquinones from Medicinal Plants. Front Pharmacol. 2021. 12.\u003c/li\u003e\n\u003cli\u003eNeyrinck AM, Rodriguez J, Taminiau B, et al. Constipation Mitigation by Rhubarb Extract in Middle-Aged Adults Is Linked to Gut Microbiome Modulation: A Double-Blind Randomized Placebo-Controlled Trial. Int J Mol Sci. 2022. 23(23).\u003c/li\u003e\n\u003cli\u003eGao CC, Li GW, Wang TT, et al. Rhubarb extract relieves constipation by stimulating mucus production in the colon and altering the intestinal flora. Biomed Pharmacother. 2021. 138: 111479.\u003c/li\u003e\n\u003cli\u003eYang L, Wan Y, Li W, et al. Targeting intestinal flora and its metabolism to explore the laxative effects of rhubarb. Appl Microbiol Biotechnol. 2022. 106(4): 1615-1631.\u003c/li\u003e\n\u003cli\u003eTakayama K, Tsutsumi H, Ishizu T, Okamura N. The influence of rhein 8-O-\u0026beta;-D-glucopyranoside on the purgative action of sennoside A from rhubarb in mice. Biol Pharm Bull. 2012. 35(12): 2204-8.\u003c/li\u003e\n\u003cli\u003eYim H, Lee YH, Lee CH, Lee SK. Emodin, an anthraquinone derivative isolated from the rhizomes of Rheum palmatum, selectively inhibits the activity of casein kinase II as a competitive inhibitor. Planta Med. 1999. 65(1): 9-13.\u003c/li\u003e\n\u003cli\u003eJaneczko M, Masłyk M, Kubiński K, Golczyk H. Emodin, a natural inhibitor of protein kinase CK2, suppresses growth, hyphal development, and biofilm formation of Candida albicans. Yeast. 2017. 34(6): 253-265.\u003c/li\u003e\n\u003cli\u003eZheng Q, Li S, Li X, Liu R. Advances in the study of emodin: an update on pharmacological properties and mechanistic basis. Chin Med. 2021. 16(1): 102.\u003c/li\u003e\n\u003cli\u003eSharifi-Rad J, Herrera-Bravo J, Kamiloglu S, et al. Recent advances in the therapeutic potential of emodin for human health. Biomed Pharmacother. 2022. 154: 113555.\u003c/li\u003e\n\u003cli\u003eWang L, Zhou G, Zhao S, Yang Y. Soluble Protein Content, Bioactive Compounds and the Antioxidant Activity in Seeds of Ten Rheum tanguticum Lines from Qinghai-Tibet Plateau. Chem Biodivers. 2023 : e202200901.\u003c/li\u003e\n\u003cli\u003eChao HW, Chen YK, Liu JH, Pan HT, Lin HM, Chao HM. Emodin protected against retinal ischemia insulted neurons through the downregulation of protein overexpression of \u0026beta;-catenin and vascular endothelium factor. BMC Complement Med Ther. 2020. 20(1): 338.\u003c/li\u003e\n\u003cli\u003eGao LL, Wang ZH, Mu YH, Liu ZL, Pang L. Emodin Promotes Autophagy and Prevents Apoptosis in Sepsis-Associated Encephalopathy through Activating BDNF/TrkB Signaling. Pathobiology. 2022. 89(3): 135-145.\u003c/li\u003e\n\u003cli\u003eJin T, Ai F, Zhou J, et al. Emodin alleviates lung ischemia-reperfusion injury by suppressing gasdermin D-mediated pyroptosis in rats. Clin Respir J. 2023 .\u003c/li\u003e\n\u003cli\u003eZhang X, Qin Q, Dai H, Cai S, Zhou C, Guan J. Emodin protects H9c2 cells from hypoxia-induced injury by up-regulating miR-138 expression. Braz J Med Biol Res. 2019. 52(3): e7994.\u003c/li\u003e\n\u003cli\u003eLu L, Li HQ, Fu DL, Zheng GQ, Fan JP. Rhubarb root and rhizome-based Chinese herbal prescriptions for acute ischemic stroke: a systematic review and meta-analysis. Complement Ther Med. 2014. 22(6): 1060-70.\u003c/li\u003e\n\u003cli\u003eLiu AJ, Song L, Li Y, et al. Active Compounds of Rhubarb Root and Rhizome in Animal Model Experiments of Focal Cerebral Ischemia. Evid Based Complement Alternat Med. 2015. 2015: 210546.\u003c/li\u003e\n\u003cli\u003eLi RR, Liu XF, Feng SX, et al. Pharmacodynamics of Five Anthraquinones (Aloe-emodin, Emodin, Rhein, Chysophanol, and Physcion) and Reciprocal Pharmacokinetic Interaction in Rats with Cerebral Ischemia. Molecules. 2019. 24(10).\u003c/li\u003e\n\u003cli\u003eMorris GP, Wright AL, Tan RP, et al. A Comparative Study of Variables Influencing Ischemic Injury in the Longa and Koizumi Methods of Intraluminal Filament Middle Cerebral Artery Occlusion in Mice. PLoS One. 2016 ;11(2):e0148503. \u003c/li\u003e\n\u003cli\u003e[38] Zhang H, Lu M, Zhang X, et al. Isosteviol sodium protects against ischemic stroke by modulating microglia/macrophage polarization via disruption of GAS5/miR-146a-5p sponge. Sci. Rep. 2019; 9: 12221.\u003c/li\u003e\n\u003cli\u003eZheng H, Guo X, Kang S, et al. Cdh5-mediated Fpn1 deletion exerts neuroprotective effects during the acute phase and inhibitory effects during the recovery phase of ischemic stroke. Cell Death Dis. 2023. 14(2): 161.\u003c/li\u003e\n\u003cli\u003eLalu MM, Fergusson DA, Cheng W, et al. Identifying stroke therapeutics from preclinical models: A protocol for a novel application of network meta-analysis. F1000Res. 2019. 8: 11.\u003c/li\u003e\n\u003cli\u003eZhang GB, Huang HW, Guo W. [Prevention and treatment of stroke in Chinese and African young adults]. Zhonghua Yu Fang Yi Xue Za Zhi. 2022. 56(8): 1142-1149.\u003c/li\u003e\n\u003cli\u003eLuheshi NM, Kov\u0026aacute;cs KJ, Lopez-Castejon G, Brough D, Denes A. Interleukin-1\u0026alpha; expression precedes IL-1\u0026beta; after ischemic brain injury and is localised to areas of focal neuronal loss and penumbral tissues. J Neuroinflammation. 2011. 8: 186.\u003c/li\u003e\n\u003cli\u003eGLUCKSMANN A. Cell deaths in normal vertebrate ontogeny. Biol Rev Camb Philos Soc. 1951. 26(1): 59-86.\u003c/li\u003e\n\u003cli\u003eMoujalled D, Strasser A, Liddell JR. Molecular mechanisms of cell death in neurological diseases. Cell Death Differ. 2021. 28(7): 2029-2044.\u003c/li\u003e\n\u003cli\u003ePlatnich JM, Chung H, Lau A, et al. Shiga Toxin/Lipopolysaccharide Activates Caspase-4 and Gasdermin D to Trigger Mitochondrial Reactive Oxygen Species Upstream of the NLRP3 Inflammasome. Cell Rep. 2018. 25(6): 1525-1536.e7.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Caspase 1, GSDMD, Cerebral ischemia-reperfusion, Emodin, Pyroptosis","lastPublishedDoi":"10.21203/rs.3.rs-6149634/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6149634/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCerebrovascular disease encompasses a wide range of conditions characterized by cerebrovascular lesions or disruptions in blood flow. Ischemic stroke, among these conditions, is the most prevalent and is known for its substantial morbidity, disability, and mortality rates, making it a leading cause of global disability. Effective management of ischemia-reperfusion injury holds paramount importance in stroke treatment, regardless of whether thrombolytic therapy is administered. Previous studies have shown that Emodin exhibits anti-inflammatory and neuroprotective properties, providing protection against ischemia-reperfusion injury in various organs by modulating pyroptosis. However, the precise molecular mechanisms underlying the effects of Emodin in cerebral ischemia-reperfusion injury remain poorly understood. Therefore, the objective of this study was to elucidate the neuroprotective mechanisms of Emodin in the context of ischemic stroke.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSD rats were randomly assigned to different groups, including control group, sham operation group, model group, and Emodin intervention group with varying dosages. Cerebral ischemia-reperfusion injury was induced using the middle cerebral artery occlusion (MCAO) method. Intraperitoneal injections of 10mg/kg, 20mg/kg and 40 mg/kg Emodin were administered to assess neurological changes in the rats. The modified Neurological Severity Score (mNSS) was used to evaluate neurological deficits. The infarct volume ratio was determined through TTC staining, while HE staining was employed to observe pathomorphological changes. Using Western blotting (WB) technique and immunofluorescence, we investigated the expression levels and cellular localization of proteins associated with cell pyroptosis, including NLRP3, Caspase 1 and GSDMD. Additionally, enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of IL-1β and IL-18. The whole animal study was approved by the Affiliated Hospital of Zunyi Medical University (approval number KLLY(A)-2021-083) and all methods are reported in accordance with ARRIVE guidelines.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eEmodin exhibits significant beneficial effects in improving neurological deficits caused by cerebral ischemia-reperfusion injury. It effectively reduces the ratio of infarct volume, alleviates cytopathic damage and suppresses the expression of pyroptosis-related proteins, including NLRP3, Caspase 1 and GSDMD. Furthermore, Emodin decreases the levels of pro-inflammatory cytokines IL-1β and IL-18, thus attenuating the inflammatory response.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe expression of pyroptosis-related proteins is upregulated in rats after cerebral ischemia-reperfusion injury. Emodin demonstrates neuroprotective effects against cerebral ischemia-reperfusion injury in rats, potentially by modulating the expression of pyroptosis-related proteins mediated through the Caspase 1-GSDMD axis.\u003c/p\u003e","manuscriptTitle":"Emodin influence pyroptosis-related Caspase 1-GSDMD axis alleviated cerebral ischemia-reperfusion injury in rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-20 08:27:36","doi":"10.21203/rs.3.rs-6149634/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-31T04:55:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-28T09:19:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-27T19:00:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-19T08:27:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"79124577953782184717983533128497186562","date":"2025-03-18T11:35:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"39437795345570357663342725690800193431","date":"2025-03-18T07:54:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87686356008780741973156960628299350484","date":"2025-03-18T05:49:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"245808541537730686990153966433127635588","date":"2025-03-18T05:47:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-18T05:41:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-18T05:38:27+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-17T14:46:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-17T05:29:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-04T00:19:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dddebf75-6986-4bd1-bc3c-e4e6b9d94974","owner":[],"postedDate":"March 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45894723,"name":"Biological sciences/Drug discovery"},{"id":45894724,"name":"Biological sciences/Neuroscience"},{"id":45894725,"name":"Health sciences/Neurology"}],"tags":[],"updatedAt":"2025-06-09T15:59:04+00:00","versionOfRecord":{"articleIdentity":"rs-6149634","link":"https://doi.org/10.1038/s41598-025-04863-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-06-03 15:56:50","publishedOnDateReadable":"June 3rd, 2025"},"versionCreatedAt":"2025-03-20 08:27:36","video":"","vorDoi":"10.1038/s41598-025-04863-y","vorDoiUrl":"https://doi.org/10.1038/s41598-025-04863-y","workflowStages":[]},"version":"v1","identity":"rs-6149634","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6149634","identity":"rs-6149634","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}