IMRC-Exo Alleviates Limb Injury Induced by Deinagkistrodon acutus Snakebite in Rabbits Through GSDME-Dependent Pyroptosis Inhibition

IMRC-Exo Alleviates Limb Injury Induced by Deinagkistrodon acutus Snakebite in Rabbits Through GSDME-Dependent Pyroptosis Inhibition | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article IMRC-Exo Alleviates Limb Injury Induced by Deinagkistrodon acutus Snakebite in Rabbits Through GSDME-Dependent Pyroptosis Inhibition Haohao Wu, Lutao Xie, Wang Du, Linjie Lai, Peixin Shangguan, Xingzhen Wu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5901934/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Inflammation plays a critical role in the pathogenesis of limb injury caused by Deinagkistrodon acutus snakebite. Investigating its regulatory mechanisms and intervention strategies may uncover effective therapeutic approaches for this condition. Recent studies have identified pyroptosis as a key pathway exacerbating target organ damage by amplifying inflammatory responses. Immune and Matrix-Regulatory Cells (IMRC), a novel type of mesenchymal stem cell, and their derived exosomes (Exo) that have shown potential in mitigating inflammation-mediated tissue damage by suppressing pyroptosis. This study aimed to evaluate whether IMRC-Exo could alleviate Deinagkistrodon acutus venom-induced limb injury by inhibiting pyroptosis-mediated inflammation in rabbits. Methods Eighteen healthy male New Zealand white rabbits were randomly assigned to the Sham, Model, and IMRC-Exo groups. The Model group was established by intramuscular injection of Deinagkistrodon acutus venom (1.5 mg/kg), followed by intravenous infusion of anti-D. acutus venom serum (80 U/kg) after 2 hours. The IMRC-Exo group received IMRC-Exo treatment (7.5 × 10^10 particles) after model establishment. Within 24 hours post-modeling, the left thigh circumference, serum creatine kinase (CK), and myoglobin (Mb) levels were dynamically assessed. Animals were euthanized to collect muscle tissues for histopathological examination, apoptosis analysis, inflammatory cytokine quantification (HMGB1, IL-1β, IL-18), and pyroptosis-related protein detection, including caspase-3, cleaved caspase-3, gasdermin E (GSDME), and N-terminal GSDME (N-GSDME). Results Compared with the Sham group, both venom-injected groups exhibited a significant increase in left thigh circumference, elevated serum CK and Mb levels, and aggravated histopathological damage and apoptosis in muscle tissues. However, the IMRC-Exo group showed significantly reduced limb circumference, decreased muscle injury markers, and attenuated tissue damage compared with the Model group. Additionally, venom injection significantly increased HMGB1, IL-1β, IL-18 levels, and the expression of caspase-3, cleaved caspase-3, GSDME, and N-GSDME in muscle tissues of the Model and IMRC-Exo groups compared to the Sham group. Notably, these inflammatory cytokine levels and pyroptosis-related protein expressions were significantly lower in the IMRC-Exo group than in the Model group. Conclusion IMRC-Exo effectively alleviates limb wound damage induced by Deinagkistrodon acutus snakebite in rabbits. Its protective mechanism may involve the inhibition of GSDME-dependent pyroptosis-mediated inflammatory injury. Deinagkistrodon acutus snakebite immune and matrix-regulatory cell-derived exosomes pyroptosis Gasdermin E Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Snakebite envenomation is one of the most prevalent and severe forms of animal-related injuries worldwide. According to the World Health Organization (WHO), approximately 2.7 million cases of venomous snakebites occur annually, resulting in over 100,000 deaths and 300,000 permanent disabilities [ 1 ] . In China alone, there are about 300,000 cases of venomous snakebites each year, with fatality and disability rates reaching as high as 10% and 30%, respectively [ 2 ][ 3 ] . Deinagkistrodon acutus, a highly venomous snake species commonly found in China, produces hemotoxic and cytotoxic venom that is typically introduced into the bloodstream through limb bites. This venom causes severe local swelling, necrosis, and significant impairment of limb function, substantially reducing patients' quality of life. Current treatments for local wound management following Deinagkistrodon acutus bites include local debridement combined with vacuum sealing drainage (VSD) [ 4 ] , hyperbaric oxygen therapy [ 5 ] , and traditional Chinese medicine (TCM) topical applications [ 6 ] . However, these interventions are primarily applied post-injury, often with limited effectiveness, leading to delayed wound healing, secondary infections, and functional impairments. Therefore, there is an urgent need to explore effective therapeutic strategies for managing local limb injuries caused by Deinagkistrodon acutus bites. Inflammation has been identified as a primary mechanism underlying snakebite-induced limb injury [ 7 ] . However, the key regulatory mechanisms and intervention strategies remain to be elucidated. Recent studies have highlighted pyroptosis, a novel form of programmed cell death [ 8 ] , as a critical process that amplifies inflammatory responses, exacerbating muscle tissue damage caused by various factors [ 9 ][ 10 ][ 11 ] . GSDME, a key effector protein of pyroptosis, is cleaved by activated caspase-3 to release its N-terminal fragment, which inserts into the plasma membrane to form pores. This process leads to the leakage of cellular contents and triggers a robust inflammatory response [ 12 ][ 13 ][ 14 ] . Nevertheless, whether the GSDME-dependent pyroptosis pathway contributes to the inflammatory damage in snakebite-induced limb injuries has not yet been investigated. MSC-Exo have been shown to exert protective effects, including anti-inflammatory, immunomodulatory, and pro-regenerative properties, in the treatment of various diseases [ 15 ] . Notably, MSC-Exo has demonstrated significant efficacy in alleviating inflammatory injuries in wounds caused by diabetes and burns [ 16 ][ 17 ] . Furthermore, IMRC differentiated from human embryonic stem cells exhibit superior cell quality, immunoregulatory functions, and reparative capacity compared to traditional MSCs [ 18 ][ 19 ][ 20 ] . IMRC-Exo may therefore possess enhanced tissue-protective properties. However, the therapeutic potential and underlying mechanisms of MSC-Exo, including IMRC-Exo, in snakebite-induced wound management remain unexplored. In this study, we aimed to establish a rabbit model of Deinagkistrodon acutus snakebite to confirm the occurrence of pyroptosis in limb muscle tissue following envenomation and to investigate the therapeutic potential and mechanisms of IMRC-Exo in mitigating this type of injury. We hypothesized that GSDME activation mediates pyroptosis in limb muscle tissues following snakebite and that IMRC-Exo could attenuate muscle damage by inhibiting GSDME-dependent pyroptosis and associated inflammatory responses. Materials and Methods Experimental Animals Eighteen healthy male New Zealand white rabbits, with an average weight of 3.0 ± 0.1 kg, were purchased from the Fuyang Hongfeng Rabbit Breeding Farm in Hangzhou, China. All experimental procedures were performed in compliance with the guidelines of the Institutional Animal Care and Use Committee. The rabbits were housed under standard conditions: temperature controlled at 20–25°C, humidity maintained at 60–80%, with a 12-hour light/dark cycle. They were provided with free access to water and standard rabbit feed, and the housing environment was regularly disinfected. This study was approved by the Animal Ethics Committee of Lishui University (Approval No. 2023YD0113). Animal Preparation Before the experiment, the rabbits were acclimated under standard conditions for one week. On the day of the experiment, both hind limbs were shaved using a professional hair clipper to fully expose the limb areas. The right ear was marked for identification. Body weight was measured using a scale, and body temperature was recorded with a thermometer. Electrocardiographic monitoring (iM60, Shenzhen Mindray Bio-Medical Electronics Co., Ltd.) was connected to monitor and record baseline heart rate and oxygen saturation. Randomization and Interventions After animal preparation, the rabbits were randomly divided into three groups using the sealed envelope method: Sham group, Model group, and IMRC-Exo group (n = 6 per group). In the Sham group, only animal preparation was performed without model establishment. In the Model and IMRC-Exo groups, a rabbit model of Deinagkistrodon acutus envenomation-induced limb injury was established. In addition, the IMRC-Exo group received IMRC-Exo intervention during model establishment. IMRC-Exo (7.5 × 10^10 particles, purchased from Hangzhou Luyuan Biotechnology Co., Ltd.) was dissolved in 1 mL of saline and subcutaneously injected at 10 sites around the venom injection point. These included four sites located 0.5 cm from the center point at 0, 6, 12, and 18 o’clock positions, and six sites located 1.0 cm from the center point at 0, 4, 8, 12, 16, and 20 o’clock positions, with 7.5 × 10^9 particles at each site. Sham and Model groups received equal volumes of saline administered in the same manner. Model Establishment Based on preliminary experiments, the venom concentration for modeling was set at 1.5 mg/kg. Lyophilized Deinagkistrodon acutus venom powder (batch number 20200410) was purchased from Shanghai Seron Biotechnology Co., Ltd., and dissolved in saline to a concentration of 100 mg/mL prior to use. The venom dose was calculated based on the body weight of each rabbit and injected perpendicularly into the mid-lateral side of the left thigh at a depth of 5 mm. The injection site was pressed with a cotton swab for 1 minute to prevent leakage. Two hours post-injection, an intravenous catheter was established in the auricular vein, and 80 U/kg of anti-D. acutus venom serum diluted in 20 mL of saline (12 U/mL) was administered via an intravenous micropump. All rabbits were monitored for 6 hours post-venom injection and then returned to their cages for an 18-hour observation period. At the endpoint, rabbits were euthanized via intravenous injection of sodium pentobarbital (150 mg/kg). Observational Parameters Before venom injection, the body weight and baseline vital signs (including heart rate, body temperature, and oxygen saturation) of each rabbit were recorded. Changes in the circumference of the left thigh were measured at baseline and 6, 12, and 24 hours after venom injection. At the same time points, 2 mL of venous blood was collected and centrifuged at 3000 rpm for 10 minutes to obtain the supernatant, which was stored at − 80°C for subsequent analysis. The levels of CK and Mb were measured using Enzyme-Linked Immunosorbent Assay(ELISA) kits (Shanghai Meixuan Biotechnology Co., Ltd., China) according to the manufacturer’s instructions. Specifically, 100 µL of diluted serum samples was added to the enzyme-linked immunosorbent assay wells pre-coated with the corresponding antibodies. After incubation at 37°C for 1 hour, the plates were washed three times, followed by the addition of 100 µL of enzyme conjugate for a 30-minute incubation. The plates were washed again, and the substrate solution was added for color development. The optical density (OD) was read at 450 nm, and the concentrations of CK and Mb were calculated using standard curves. At the experimental endpoint, muscle tissue samples were collected 2 cm below the venom injection site and fixed in 10% neutral formalin for 24 hours. The tissues were then processed with routine dehydration, clearing, and paraffin embedding, followed by slicing into 4-µm-thick sections. Histopathological damage was assessed using Hematoxylin and Eosin (HE) staining. Briefly, sections were stained with hematoxylin for 5 minutes, rinsed with tap water, counterstained with eosin for 1 minute, and then dehydrated, cleared, and mounted. A light microscope (Olympus, Tokyo, Japan) at 200× magnification was used to observe three randomly selected fields for signs of muscle fiber necrosis and inflammatory cell infiltration.Additionally, apoptosis in muscle tissues was evaluated using Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling (TUNEL) staining. Tissue sections were pretreated with proteinase K and stained using a TUNEL kit (Wuhan Boster Biological Technology Co., Ltd., China) following the manufacturer’s instructions. Under a biological microscope (C×31, Olympus, Japan) at 200× magnification, three random fields were photographed, and the ratio of TUNEL-positive cells to total cells was calculated as the apoptosis rate. The expression of N-GSDME in muscle was measured by immunohistochemistry at 24 h after the model establishment. Similarly, muscle tissue samples were obtained, then fixed and embedded, and finally sliced into 4-µm-thick sections. The sections were incubated with primary anti-N-GSDME (1:200, Cell Signaling Technology, Danvers, United States), then treated with the secondary antibody, and finally reacted with diaminobenzidine (Boster Biological Technology, Wuhan, China). Three fields were randomly photographed at 200× magnification with the CX31 optical microscope. The semiquantitative analysis of the intensity of N-GSDME positive staining were performed through integrated optical density (IOD) using the Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, United States). At the experimental endpoint, muscle tissue samples were collected as described above, immediately flash-frozen in liquid nitrogen, and stored at − 80°C for subsequent analyses. During the experiments, the frozen samples were thawed and homogenized. The concentrations of HMGB1, IL-1β, and IL-18 were measured using ELISA kits (Shanghai Meixuan Biotechnology Co., Ltd., China) according to the manufacturer’s instructions. Briefly, 100 µL of tissue homogenate was added to enzyme-linked immunosorbent assay wells pre-coated with specific antibodies and incubated at 37°C for 1 hour. The plates were washed three times, followed by the addition of 100 µL of enzyme conjugate for a 30-minute incubation. After washing, substrate solution was added and incubated for 15 minutes. The reaction was terminated by adding 50 µL of stop solution, and the OD was measured at 450 nm. The concentrations of HMGB1, IL-1β, and IL-18 were calculated using standard curves. The supernatants of muscle tissue's homogenates were also used for western blotting of protein concentrations of caspase-3, cleaved caspase-3, GSDME, and N-GSDME. As follows, the samples were separated by SDS- PAGE, then transferred to a polyvinylidene fluoride membrane, and finally blocked with 5% nonfat milk. Subsequently, the membranes were incubated with primary antibodies to caspase-3 (1:1,000; Proteintech, Rosemont, IL, USA), cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), GSDME (1:1,000; Proteintech, Rosemont, IL, USA), N-GSDME (1:1,000; Abcam, Cambridge, UK), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:1,000; BBI Life Sciences Corporation, Shanghai, China). Thereafter, anti-mouse antibody (1:5,000; BBI Life Sciences Corporation, Shanghai, China) or anti-rabbit antibody (1:5,000; BBI Life Sciences Corporation, Shanghai, China) was used as the secondary antibody. Finally, the band intensities were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA), and the results were all standardized by GAPDH. Statistical Analysis Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) 20.0 software (IBM Corporation, United States). The normality of the data was assessed using the Kolmogorov-Smirnov test. Data following a normal distribution were expressed as the mean ± standard deviation (± s). Intergroup comparisons were conducted using one-way ANOVA, and post hoc pairwise comparisons were performed with the Bonferroni test. A p-value < 0.05 was considered statistically significant. Results Survival and Baseline Characteristics A total of 18 rabbits were included in this study, and all animals survived the 24-hour observation period. Before venom injection, baseline characteristics, including body weight, heart rate, body temperature, and oxygen saturation, were within normal ranges for all groups, with no significant differences between groups ( P > 0.05) (Fig. 1 ). Changes in Left Thigh Circumference and Serum CK and Mb Levels Before venom injection, there were no significant differences in left thigh circumference or serum CK and Mb concentrations between the three groups ( P > 0.05). After venom injection, both the Model and IMRC-Exo groups exhibited significant increases in left thigh circumference and serum CK and Mb levels compared with the Sham group. However, the IMRC-Exo group showed significantly reduced left thigh circumference and serum CK and Mb concentrations compared to the Model group ( P < 0.05) (Fig. 2 ). Histopathological Assessment of Muscle Tissue At 24 hours post-venom injection, HE staining in the Sham group revealed normal muscle tissue structure. In contrast, the Model group exhibited severely disorganized muscle architecture, characterized by muscle fiber rupture and necrosis, accompanied by inflammatory cell infiltration and interstitial edema. Compared to the Model group, the IMRC-Exo group demonstrated more intact muscle structure, with orderly fiber arrangement, reduced rupture and necrosis, and significantly alleviated inflammatory cell infiltration and edema (Fig. 3 ). Apoptosis in Muscle Tissue At 24 hours post-venom injection, minimal apoptosis was observed in the Sham group. As shown in Fig. 4 A, the Model group exhibited a marked increase in apoptotic cells, evidenced by a substantial number of brownish-yellow TUNEL-positive cells. However, IMRC-Exo intervention significantly reduced the number of apoptotic cells. Quantitative analysis of the apoptosis rate (Fig. 4 B) indicated that apoptosis in muscle tissues was significantly increased in both the Model and IMRC-Exo groups compared with the Sham group ( P < 0.05). Nevertheless, the apoptosis rate was significantly lower in the IMRC-Exo group than in the Model group ( P < 0.05). Inflammatory Cytokine Levels in Muscle Tissue At 24 hours post-venom injection, ELISA results showed that the concentrations of HMGB1, IL-1β, and IL-18 in muscle tissue were significantly elevated in both the Model and IMRC-Exo groups compared with the Sham group ( P < 0.05). However, these inflammatory cytokine levels were significantly lower in the IMRC-Exo group compared with the Model group ( P < 0.05) (Fig. 5 ). Pyroptosis-Related Protein Expression Western blot analysis at 24 hours post-venom injection revealed significantly increased expression of caspase-3, cleaved caspase-3, GSDME, and N-GSDME in the Model and IMRC-Exo groups compared with the Sham group ( P < 0.05). However, the IMRC-Exo group exhibited significantly lower expression levels of these pyroptosis-related proteins compared with the Model group ( P < 0.05). Furthermore, immunohistochemical analysis demonstrated significantly higher IOD values of N-GSDME-positive staining in muscle tissues from the Model and IMRC-Exo groups compared with the Sham group. Notably, IMRC-Exo treatment significantly reduced N-GSDME-positive IOD values compared with the Model group ( P < 0.05) (Fig. 6 ). Discussion In this study, we observed the occurrence of pyroptosis in limb muscle tissues of rabbits following Deinagkistrodon acutus snakebite and evaluated the protective effects and underlying mechanisms of IMRC-Exo in mitigating snakebite-induced limb injury. Our results demonstrated that IMRC-Exo significantly reduced limb swelling, decreased levels of muscle injury biomarkers, and alleviated histopathological damage and apoptosis in muscle tissues, thereby exerting a protective effect. Furthermore, we found that GSDME activation and GSDME-mediated pyroptosis occurred in limb muscle tissues following snakebite, but IMRC-Exo markedly inhibited this process and reduced inflammatory damage, which may underlie its protective effects. MSC-Exo are membrane-bound nanovesicles enriched with proteins, nucleic acids, and lipids, and exhibit various biological effects, including anti-inflammatory, antioxidant, and pro-angiogenic activities. These properties enable MSC-Exo to protect cells and promote tissue repair in diverse settings [ 21 ] . Previous studies have shown that MSCs and their soluble components significantly attenuate ischemic injury in limb muscles through anti-inflammatory and regenerative mechanisms, highlighting their potential for muscle protection and repair [ 22 ] . In particular, MSC-Exo have been shown to inhibit pyroptosis via the circHIPK3/FOXO3a pathway, thereby promoting muscle repair and regeneration and effectively alleviating ischemic injury [ 23 ] . Additionally, MSC-Exo have demonstrated therapeutic potential in wound healing across various injury models. For instance, Yang J et al. found that umbilical cord-derived MSC-Exo combined with hydrogels enhanced wound healing and skin regeneration in diabetic rats [ 24 ] . Similarly, Li B et al. reported that MSC-Exo accelerated wound healing in a diabetic foot ulcer mouse model by promoting fibroblast proliferation and migration while inhibiting apoptosis and inflammation [ 25 ] . Moreover, Bo Y et al. demonstrated that pluripotent stem cell-derived exosomes promoted keratinocyte and endothelial cell migration through miR-762, thereby expediting burn wound repair [ 26 ] . However, the application of MSC-Exo for snakebite-induced limb injury has not yet been investigated. Based on the above evidence, this study aimed to explore the effects of MSC-Exo on snakebite-induced limb injury. We specifically utilized exosomes derived from IMRCs, as IMRCs—originating from human embryonic stem cells—have been shown to outperform MSCs from adult tissues in terms of anti-inflammatory and immunomodulatory properties [ 27 ][ 28 ] . To ensure clinical relevance, we referenced the venom dosage used in previous Deinagkistrodon acutus pig models [ 29 ] and determined an intramuscular injection dose of 1.5 mg/kg based on our preliminary experiments. Venom was injected into the mid-lateral side of the left thigh, a common site of snakebites in clinical settings, to mimic the pathological changes observed in humans. The venom injection resulted in significant limb swelling, elevated muscle injury biomarkers, histopathological damage, and apoptosis in muscle tissues, confirming successful model establishment. Notably, IMRC-Exo treatment significantly mitigated these abnormalities, demonstrating its protective effects against snakebite-induced limb injury. Pyroptosis has been implicated in the pathophysiological processes of various wounds and has emerged as a promising therapeutic target. Wang X et al. demonstrated that inhibiting pyroptosis activation promoted wound healing in diabetic foot ulcers [ 30 ] . Similarly, Hasan Maleki M et al. found that nicotinamide riboside and resveratrol aided in diabetic wound repair by suppressing pyroptosis [ 31 ] . Zhang K et al. reported that bioactive glass accelerated wound healing in soft tissue injuries by modulating connexin 43/reactive oxygen species signaling to inhibit endothelial cell pyroptosis [ 32 ] . Furthermore, MSC-Exo have been shown to regulate pyroptosis and confer protection in various organ injury models. For example, Liu P et al. demonstrated that MSC-Exo alleviated LPS-induced acute lung injury by inhibiting alveolar macrophage pyroptosis [ 33 ] . Additionally, adipose-derived MSC-Exo significantly improved liver ischemia-reperfusion injury by suppressing pyroptosis and apoptosis-related pathways, reducing systemic inflammation and liver tissue damage [ 34 ] . Exosomes from M2-polarized microglia (M2-Exos) have also been found to inhibit neuronal pyroptosis via ubiquitin-mediated degradation of TXNIP, thereby mitigating cerebral ischemia-reperfusion injury [ 35 ] . In this study, we further investigated the role of pyroptosis in snakebite-induced muscle injury and the potential mechanisms underlying the protective effects of IMRC-Exo. Our results revealed that venom injection led to significant increases in the expression of pyroptosis-related proteins, including caspase-3, cleaved caspase-3, GSDME, and N-GSDME, as well as elevated levels of inflammatory cytokines HMGB1, IL-1β, and IL-18. These findings indicate the involvement of GSDME-mediated pyroptosis in snakebite-induced muscle injury. Importantly, IMRC-Exo treatment significantly reduced the expression of these pyroptosis-related proteins and inflammatory cytokines, suggesting that IMRC-Exo attenuated inflammatory injury by inhibiting GSDME-mediated pyroptosis. This study has several limitations. First, the observation period was relatively short, which may not fully capture the dynamic changes in limb injury following snakebite. Future studies should extend the observation period to comprehensively evaluate the progression of injury and the therapeutic effects of IMRC-Exo. Second, we only used a single dose of IMRC-Exo and did not explore dose-dependent effects on treatment outcomes. Dose optimization in future studies could help identify the most effective therapeutic regimen. Finally, while this study focused on key markers of the GSDME pyroptosis pathway, further investigations are needed to elucidate the molecular mechanisms through which IMRC-Exo regulate pyroptosis in snakebite models. Conclusion This study provides the first evidence that IMRC-Exo exerts protective effects against limb wound damage induced by Deinagkistrodon acutus snakebite by inhibiting GSDME-mediated pyroptosis and the associated inflammatory injury. Our findings offer new perspectives and potential therapeutic strategies for the clinical management of snakebite-induced wounds. Abbreviations IMRC: Immune and Matrix-Regulatory Cells Exo: Exosomes CK: Creatine Kinase Mb: Myoglobin GSDME: Gasdermin E N-GSDME: N-terminal Gasdermin E WHO: World Health Organization VSD: Vacuum Sealing Drainage TCM: Traditional Chinese Medicine ELISA: Enzyme-Linked Immunosorbent Assay OD: Optical Density HE: Hematoxylin and Eosin TUNEL: Transferase dUTP Nick-End Labeling IOD: Integrated Optical Density SPSS: Statistical Package for the Social Sciences Declarations Ethics approval and consent to participate This study was approved by the Animal Ethics Committee of Lishui University (Approval No. 2023YD0113). All experimental procedures were performed in compliance with the guidelines of the Institutional Animal Care and Use Committee.Clinical trial number: not applicable. Consent for publication Not applicable. Availability of data and materials The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study was supported by the Lishui Science and Technology Project (2023SJZC073) and the Zhejiang Provincial Health Department Project (2024KY1858). Authors' contributions HW conceived and designed the study, performed the experiments, and wrote the first draft of the manuscript. LX assisted with data collection and manuscript preparation. WD and LL contributed to data collection and analysis. PS and XW provided technical support and assisted with data interpretation. JX contributed to the literature review and manuscript revision. PL supervised the entire study, provided critical revisions, and acted as the corresponding author. All authors read and approved the final manuscript. Acknowledgements Not applicable. References WHO. Snakebite envenoming. In: WHO [Internet]. 12 Sep 2023 [cited 12 Sep 2023].Available:https://www.who.int/zh/news-room/fact-sheets/detail/snakebite-envenoming. 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Cytotherapy. 2023 Apr;25(4):375-386. doi: 10.1016/j.jcyt.2022.11.010. Yan B, Zhang Y, Liang C, Liu B, Ding F, Wang Y, et al. Stem cell-derived exosomes prevent pyroptosis and repair ischemic muscle injury through a novel exosome/circHIPK3/ FOXO3a pathway. Theranostics. 2020 May 18;10(15):6728-6742. doi: 10.7150/thno.42259. Yang J, Chen Z, Pan D, Li H, Shen J. Umbilical Cord-Derived Mesenchymal Stem Cell-Derived Exosomes Combined Pluronic F127 Hydrogel Promote Chronic Diabetic Wound Healing and Complete Skin Regeneration. Int J Nanomedicine. 2020 Aug 11;15:5911-5926. doi: 10.2147/IJN.S249129. Li B, Luan S, Chen J, Zhou Y, Wang T, Li Z, et al. The MSC-Derived Exosomal lncRNA H19 Promotes Wound Healing in Diabetic Foot Ulcers by Upregulating PTEN via MicroRNA-152-3p. Mol Ther Nucleic Acids. 2020 Mar 6;19:814-826. doi: 10.2147/IJN.S249129. Bo Y, Yang L, Liu B, Tian G, Li C, Zhang L, et al. Exosomes from human induced pluripotent stem cells-derived keratinocytes accelerate burn wound healing through miR-762 mediated promotion of keratinocytes and endothelial cells migration. J Nanobiotechnology. 2022 Jun 21;20(1):291. doi: 10.1186/s12951-022-01504-8. Zhou HS, Cui Z, Wang H, Gao TT, Wang L, Wu J, et al. The therapeutic effects of human embryonic stem cells-derived immunity-and-matrix regulatory cells on membranous nephropathy. Stem Cell Res Ther. 2022 Jun 7;13(1):240. doi: 10.1186/s13287-022-02917-w. Hu W, Yang J, Xue J, Ma J, Wu S, Wang J, et al. Secretome of hESC-Derived MSC-like Immune and Matrix Regulatory Cells Mitigate Pulmonary Fibrosis through Antioxidant and Anti-Inflammatory Effects. Biomedicines. 2023 Feb 5;11(2):463. doi: 10.3390/biomedicines11020463. Lai L, Xie L, Chen Y, Du W, Yang X, Liu W, et al. The establishment and evaluation of a swine model of deinagkistrodon acutus snakebite envenomation. Toxicon. 2024 Apr;241:107683. doi: 10.1016/j.toxicon.2024.107683. Wang X, Li X, Liu J, Tao Y, Wang T, Li L. Lactobacillus Plantarum Promotes Wound Healing by Inhibiting the NLRP3 Inflammasome and Pyroptosis Activation in Diabetic Foot Wounds. J Inflamm Res. 2024 Mar 16;17:1707-1720. doi: 10.2147/JIR.S449565. Hasan Maleki M, Siri M, Jafarabadi A, Rajabi M, Amirhossein Mazhari S, Noori Z, et al. Boosting wound healing in diabetic rats: The role of nicotinamide riboside and resveratrol in UPR modulation and pyroptosis inhibition. Int Immunopharmacol. 2024 May 10;132:112013. doi: 10.1016/j.intimp.2024.112013. Zhang K, Chai B, Ji H, Chen L, Ma Y, Zhu L, et al. Bioglass promotes wound healing by inhibiting endothelial cell pyroptosis through regulation of the connexin 43/reactive oxygen species (ROS) signaling pathway. Lab Invest. 2022 Jan;102(1):90-101. doi: 10.1038/s41374-021-00675-6. Liu P, Yang S, Shao X, Li C, Wang Z, Dai H, et al. Mesenchymal Stem Cells-Derived Exosomes Alleviate Acute Lung Injury by Inhibiting Alveolar Macrophage Pyroptosis. Stem Cells Transl Med. 2024 Apr 15;13(4):371-386. doi: 10.1093/stcltm/szad094. Wang Y, Piao C, Liu T, Lu X, Ma Y, Zhang J, et al. Effects of the exosomes of adipose-derived mesenchymal stem cells on apoptosis and pyroptosis of injured liver in miniature pigs. Biomed Pharmacother. 2023 Dec 31;169:115873. doi: 10.1016/j.biopha.2023.115873. Li Z, Pang Y, Hou L, Xing X, Yu F, Gao M, et al. Exosomal OIP5-AS1 attenuates cerebral ischemia-reperfusion injury by negatively regulating TXNIP protein stability and inhibiting neuronal pyroptosis. Int Immunopharmacol. 2024 Jan 25;127:111310. doi: 10.1016/j.intimp.2023.111310. Additional Declarations No competing interests reported. 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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-5901934","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":408434384,"identity":"a9bffd40-523e-4f6a-bd8e-7bfe08dc8adf","order_by":0,"name":"Haohao Wu","email":"","orcid":"","institution":"The Fifth Affiliated Hospital of Wenzhou Medical University, Lishui Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Haohao","middleName":"","lastName":"Wu","suffix":""},{"id":408434385,"identity":"2fc9baf6-c4ce-49d9-b7b0-79fc6e60db9f","order_by":1,"name":"Lutao Xie","email":"","orcid":"","institution":"The Fifth Affiliated Hospital of Wenzhou Medical University, Lishui Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lutao","middleName":"","lastName":"Xie","suffix":""},{"id":408434386,"identity":"745139a2-c345-497f-ac7d-47eefc58917d","order_by":2,"name":"Wang Du","email":"","orcid":"","institution":"The Fifth Affiliated Hospital of Wenzhou Medical University, Lishui Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Wang","middleName":"","lastName":"Du","suffix":""},{"id":408434387,"identity":"0c98ffb6-adb0-46c4-bd7c-e52ea0c96c17","order_by":3,"name":"Linjie Lai","email":"","orcid":"","institution":"The Fifth Affiliated Hospital of Wenzhou Medical University, Lishui Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Linjie","middleName":"","lastName":"Lai","suffix":""},{"id":408434388,"identity":"f2a82f54-6d05-451c-95a1-7f9647cf5106","order_by":4,"name":"Peixin Shangguan","email":"","orcid":"","institution":"The Fifth Affiliated Hospital of Wenzhou Medical University, Lishui Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Peixin","middleName":"","lastName":"Shangguan","suffix":""},{"id":408434389,"identity":"aaf82fd0-9464-48aa-a4ca-d465edae10ca","order_by":5,"name":"Xingzhen Wu","email":"","orcid":"","institution":"The Fifth Affiliated Hospital of Wenzhou Medical University, Lishui Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xingzhen","middleName":"","lastName":"Wu","suffix":""},{"id":408434390,"identity":"c84a3c94-d27d-4465-88c6-5359072eafec","order_by":6,"name":"Jiefeng Xu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jiefeng","middleName":"","lastName":"Xu","suffix":""},{"id":408434391,"identity":"6e94bec0-fe03-4b0b-ad4a-d7956af63cbf","order_by":7,"name":"Pin Lan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYBACPhDxgMEGwuMhRgsbiEhgSCNdy2FStPAffyaRuON84toZCYwP3rYxyJsT1CKRkGyQeOZ24rYbCcyGc9sYDHc2ENTCcPBBYtvtXKAWNmneNoYEgwMEHXaw4UBi2zmQFvbfxGlhSGYE2nIAbAszcVok0piBfkmu33bmYbPknHMShhsIaeEHhdjHHXbGZseTD354U2YjT9AWMGBsgJMSxKiHaxkFo2AUjIJRgAMAAPGJQEBO35/mAAAAAElFTkSuQmCC","orcid":"","institution":"The Fifth Affiliated Hospital of Wenzhou Medical University, Lishui Central Hospital","correspondingAuthor":true,"prefix":"","firstName":"Pin","middleName":"","lastName":"Lan","suffix":""}],"badges":[],"createdAt":"2025-01-25 13:38:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5901934/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5901934/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75108652,"identity":"2090c337-2204-4fb3-b793-6cd5cbc39f99","added_by":"auto","created_at":"2025-01-30 14:57:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":209366,"visible":true,"origin":"","legend":"\u003cp\u003eInitial body weight and baseline vital signs. (A) Initial body weight; (B) Heart rate; (C) Oxygen saturation; (D) Body temperature.\u003c/p\u003e\n\u003cp\u003eIMRC-Exo, Immune and Matrix-Regulatory Cell-derived Exosomes.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5901934/v1/d891e795dac867c712e153ac.png"},{"id":75108653,"identity":"09e44002-3e2d-41fb-acd1-78cbe8f9c238","added_by":"auto","created_at":"2025-01-30 14:57:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":264677,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in left thigh circumference and muscle injury markers. (A) Left thigh circumference; (B) Creatine kinase (CK); (C) Myoglobin (Mb). *\u003cem\u003e P\u003c/em\u003e\u0026lt;0.05 compared to the Sham group; #\u003cem\u003e P\u003c/em\u003e\u0026lt;0.05 compared to the Model group. BL: Baseline.\u003c/p\u003e\n\u003cp\u003eIMRC-Exo, Immune and Matrix-Regulatory Cell-derived Exosomes.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5901934/v1/f873b2b0c6c302b2573dfcd3.png"},{"id":75110361,"identity":"172c901f-f45f-42f6-b917-cb870d20128e","added_by":"auto","created_at":"2025-01-30 15:13:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":800953,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of pathological damage of skeletal muscle tissue in left thigh (representative micrographs of HE staining, ×200 magnification).\u003c/p\u003e\n\u003cp\u003eIMRC-Exo, Immune and Matrix-Regulatory Cell-derived Exosomes.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5901934/v1/a59e7437d470e43da205345e.png"},{"id":75108663,"identity":"97d1e5de-5ce3-42e4-a2dd-c28a17da6c18","added_by":"auto","created_at":"2025-01-30 14:57:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":952686,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of apoptosis in skeletal muscle tissue of the left thigh. (A) Representative images of TUNEL staining (×200 magnification); (B) Analysis of apoptosis rates. *\u003cem\u003e P\u003c/em\u003e\u0026lt;0.05 compared to the Sham group; #\u003cem\u003e P\u003c/em\u003e\u0026lt;0.05 compared to the Model group.\u003c/p\u003e\n\u003cp\u003eIMRC-Exo, immune and matrix-regulatory cell-derived exosomes.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5901934/v1/c9b574eaade35946265f568d.png"},{"id":75108842,"identity":"b6d62e37-cef1-4c92-9455-74922828aefe","added_by":"auto","created_at":"2025-01-30 15:05:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":136187,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in inflammatory cytokines in skeletal muscle tissue of the left thigh. (A–C) ELISA analysis of High Mobility Group Box 1 (HMGB1), Interleukin-1β (IL-1β), and Interleukin-18 (IL-18) levels. *\u003cem\u003e P\u003c/em\u003e\u0026lt;0.05 compared to the Sham group; #\u003cem\u003e P\u003c/em\u003e\u0026lt;0.05 compared to the Model group.\u003c/p\u003e\n\u003cp\u003eIMRC-Exo, Immune and Matrix-Regulatory Cell-derived Exosomes.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5901934/v1/5f92edda287a73f2f5665116.png"},{"id":75108838,"identity":"78e4f88f-1b7d-49ad-95d8-4ffdb5e44453","added_by":"auto","created_at":"2025-01-30 15:05:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1440812,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in pyroptosis-related proteins in skeletal muscle tissue of the left thigh. (A) Western blot analysis of caspase-3, cleaved caspase-3, Gasdermin E (GSDME), and N-terminal GSDME (N-GSDME) protein bands; (B–E) Quantitative analysis of caspase-3, cleaved caspase-3, GSDME, and N-GSDME expression levels; (F) Representative immunohistochemical staining images of N-GSDME (×200 magnification); (G) Analysis of IOD values for N-GSDME-positive staining. *\u003cem\u003e P\u003c/em\u003e\u0026lt;0.05 compared to the Sham group; #\u003cem\u003e P\u003c/em\u003e\u0026lt;0.05 compared to the Model group.\u003c/p\u003e\n\u003cp\u003eIMRC-Exo, Immune and Matrix-Regulatory Cell-derived Exosomes.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5901934/v1/5eada4470dbae96ecc41118d.png"},{"id":76190416,"identity":"af5389b4-7732-4f85-b370-87205a99778b","added_by":"auto","created_at":"2025-02-13 09:32:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6355141,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5901934/v1/c20579db-64b9-4674-9cf7-86844ea33617.pdf"},{"id":75108660,"identity":"affdedf9-c7b3-4340-9d37-51ec3f206e07","added_by":"auto","created_at":"2025-01-30 14:57:20","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":374370,"visible":true,"origin":"","legend":"","description":"","filename":"UncroppedWesternBlotGels.docx","url":"https://assets-eu.researchsquare.com/files/rs-5901934/v1/69614c7cbf4c8afe1706e9f3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"IMRC-Exo Alleviates Limb Injury Induced by Deinagkistrodon acutus Snakebite in Rabbits Through GSDME-Dependent Pyroptosis Inhibition","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSnakebite envenomation is one of the most prevalent and severe forms of animal-related injuries worldwide. According to the World Health Organization (WHO), approximately 2.7\u0026nbsp;million cases of venomous snakebites occur annually, resulting in over 100,000 deaths and 300,000 permanent disabilities\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. In China alone, there are about 300,000 cases of venomous snakebites each year, with fatality and disability rates reaching as high as 10% and 30%, respectively\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e][\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Deinagkistrodon acutus, a highly venomous snake species commonly found in China, produces hemotoxic and cytotoxic venom that is typically introduced into the bloodstream through limb bites. This venom causes severe local swelling, necrosis, and significant impairment of limb function, substantially reducing patients' quality of life. Current treatments for local wound management following Deinagkistrodon acutus bites include local debridement combined with vacuum sealing drainage (VSD)\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, hyperbaric oxygen therapy\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, and traditional Chinese medicine (TCM) topical applications\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. However, these interventions are primarily applied post-injury, often with limited effectiveness, leading to delayed wound healing, secondary infections, and functional impairments. Therefore, there is an urgent need to explore effective therapeutic strategies for managing local limb injuries caused by Deinagkistrodon acutus bites.\u003c/p\u003e \u003cp\u003eInflammation has been identified as a primary mechanism underlying snakebite-induced limb injury\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. However, the key regulatory mechanisms and intervention strategies remain to be elucidated. Recent studies have highlighted pyroptosis, a novel form of programmed cell death\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, as a critical process that amplifies inflammatory responses, exacerbating muscle tissue damage caused by various factors\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e][\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e][\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. GSDME, a key effector protein of pyroptosis, is cleaved by activated caspase-3 to release its N-terminal fragment, which inserts into the plasma membrane to form pores. This process leads to the leakage of cellular contents and triggers a robust inflammatory response\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e][\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e][\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Nevertheless, whether the GSDME-dependent pyroptosis pathway contributes to the inflammatory damage in snakebite-induced limb injuries has not yet been investigated.\u003c/p\u003e \u003cp\u003eMSC-Exo have been shown to exert protective effects, including anti-inflammatory, immunomodulatory, and pro-regenerative properties, in the treatment of various diseases\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Notably, MSC-Exo has demonstrated significant efficacy in alleviating inflammatory injuries in wounds caused by diabetes and burns\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e][\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Furthermore, IMRC differentiated from human embryonic stem cells exhibit superior cell quality, immunoregulatory functions, and reparative capacity compared to traditional MSCs\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e][\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e][\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. IMRC-Exo may therefore possess enhanced tissue-protective properties. However, the therapeutic potential and underlying mechanisms of MSC-Exo, including IMRC-Exo, in snakebite-induced wound management remain unexplored.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to establish a rabbit model of Deinagkistrodon acutus snakebite to confirm the occurrence of pyroptosis in limb muscle tissue following envenomation and to investigate the therapeutic potential and mechanisms of IMRC-Exo in mitigating this type of injury. We hypothesized that GSDME activation mediates pyroptosis in limb muscle tissues following snakebite and that IMRC-Exo could attenuate muscle damage by inhibiting GSDME-dependent pyroptosis and associated inflammatory responses.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Animals\u003c/h2\u003e \u003cp\u003eEighteen healthy male New Zealand white rabbits, with an average weight of 3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 kg, were purchased from the Fuyang Hongfeng Rabbit Breeding Farm in Hangzhou, China. All experimental procedures were performed in compliance with the guidelines of the Institutional Animal Care and Use Committee. The rabbits were housed under standard conditions: temperature controlled at 20\u0026ndash;25\u0026deg;C, humidity maintained at 60\u0026ndash;80%, with a 12-hour light/dark cycle. They were provided with free access to water and standard rabbit feed, and the housing environment was regularly disinfected. This study was approved by the Animal Ethics Committee of Lishui University (Approval No. 2023YD0113).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal Preparation\u003c/h3\u003e\n\u003cp\u003eBefore the experiment, the rabbits were acclimated under standard conditions for one week. On the day of the experiment, both hind limbs were shaved using a professional hair clipper to fully expose the limb areas. The right ear was marked for identification. Body weight was measured using a scale, and body temperature was recorded with a thermometer. Electrocardiographic monitoring (iM60, Shenzhen Mindray Bio-Medical Electronics Co., Ltd.) was connected to monitor and record baseline heart rate and oxygen saturation.\u003c/p\u003e\n\u003ch3\u003eRandomization and Interventions\u003c/h3\u003e\n\u003cp\u003eAfter animal preparation, the rabbits were randomly divided into three groups using the sealed envelope method: Sham group, Model group, and IMRC-Exo group (n\u0026thinsp;=\u0026thinsp;6 per group). In the Sham group, only animal preparation was performed without model establishment. In the Model and IMRC-Exo groups, a rabbit model of Deinagkistrodon acutus envenomation-induced limb injury was established. In addition, the IMRC-Exo group received IMRC-Exo intervention during model establishment. IMRC-Exo (7.5 \u0026times; 10^10 particles, purchased from Hangzhou Luyuan Biotechnology Co., Ltd.) was dissolved in 1 mL of saline and subcutaneously injected at 10 sites around the venom injection point. These included four sites located 0.5 cm from the center point at 0, 6, 12, and 18 o\u0026rsquo;clock positions, and six sites located 1.0 cm from the center point at 0, 4, 8, 12, 16, and 20 o\u0026rsquo;clock positions, with 7.5 \u0026times; 10^9 particles at each site. Sham and Model groups received equal volumes of saline administered in the same manner.\u003c/p\u003e\n\u003ch3\u003eModel Establishment\u003c/h3\u003e\n\u003cp\u003eBased on preliminary experiments, the venom concentration for modeling was set at 1.5 mg/kg. Lyophilized Deinagkistrodon acutus venom powder (batch number 20200410) was purchased from Shanghai Seron Biotechnology Co., Ltd., and dissolved in saline to a concentration of 100 mg/mL prior to use. The venom dose was calculated based on the body weight of each rabbit and injected perpendicularly into the mid-lateral side of the left thigh at a depth of 5 mm. The injection site was pressed with a cotton swab for 1 minute to prevent leakage. Two hours post-injection, an intravenous catheter was established in the auricular vein, and 80 U/kg of anti-D. acutus venom serum diluted in 20 mL of saline (12 U/mL) was administered via an intravenous micropump. All rabbits were monitored for 6 hours post-venom injection and then returned to their cages for an 18-hour observation period. At the endpoint, rabbits were euthanized via intravenous injection of sodium pentobarbital (150 mg/kg).\u003c/p\u003e\n\u003ch3\u003eObservational Parameters\u003c/h3\u003e\n\u003cp\u003eBefore venom injection, the body weight and baseline vital signs (including heart rate, body temperature, and oxygen saturation) of each rabbit were recorded. Changes in the circumference of the left thigh were measured at baseline and 6, 12, and 24 hours after venom injection. At the same time points, 2 mL of venous blood was collected and centrifuged at 3000 rpm for 10 minutes to obtain the supernatant, which was stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent analysis. The levels of CK and Mb were measured using Enzyme-Linked Immunosorbent Assay(ELISA) kits (Shanghai Meixuan Biotechnology Co., Ltd., China) according to the manufacturer\u0026rsquo;s instructions. Specifically, 100 \u0026micro;L of diluted serum samples was added to the enzyme-linked immunosorbent assay wells pre-coated with the corresponding antibodies. After incubation at 37\u0026deg;C for 1 hour, the plates were washed three times, followed by the addition of 100 \u0026micro;L of enzyme conjugate for a 30-minute incubation. The plates were washed again, and the substrate solution was added for color development. The optical density (OD) was read at 450 nm, and the concentrations of CK and Mb were calculated using standard curves.\u003c/p\u003e \u003cp\u003eAt the experimental endpoint, muscle tissue samples were collected 2 cm below the venom injection site and fixed in 10% neutral formalin for 24 hours. The tissues were then processed with routine dehydration, clearing, and paraffin embedding, followed by slicing into 4-\u0026micro;m-thick sections. Histopathological damage was assessed using Hematoxylin and Eosin (HE) staining. Briefly, sections were stained with hematoxylin for 5 minutes, rinsed with tap water, counterstained with eosin for 1 minute, and then dehydrated, cleared, and mounted. A light microscope (Olympus, Tokyo, Japan) at 200\u0026times; magnification was used to observe three randomly selected fields for signs of muscle fiber necrosis and inflammatory cell infiltration.Additionally, apoptosis in muscle tissues was evaluated using Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling (TUNEL) staining. Tissue sections were pretreated with proteinase K and stained using a TUNEL kit (Wuhan Boster Biological Technology Co., Ltd., China) following the manufacturer\u0026rsquo;s instructions. Under a biological microscope (C\u0026times;31, Olympus, Japan) at 200\u0026times; magnification, three random fields were photographed, and the ratio of TUNEL-positive cells to total cells was calculated as the apoptosis rate. The expression of N-GSDME in muscle was measured by immunohistochemistry at 24 h after the model establishment. Similarly, muscle tissue samples were obtained, then fixed and embedded, and finally sliced into 4-\u0026micro;m-thick sections. The sections were incubated with primary anti-N-GSDME (1:200, Cell Signaling Technology, Danvers, United States), then treated with the secondary antibody, and finally reacted with diaminobenzidine (Boster Biological Technology, Wuhan, China). Three fields were randomly photographed at 200\u0026times; magnification with the CX31 optical microscope. The semiquantitative analysis of the intensity of N-GSDME positive staining were performed through integrated optical density (IOD) using the Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, United States).\u003c/p\u003e \u003cp\u003eAt the experimental endpoint, muscle tissue samples were collected as described above, immediately flash-frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent analyses. During the experiments, the frozen samples were thawed and homogenized. The concentrations of HMGB1, IL-1β, and IL-18 were measured using ELISA kits (Shanghai Meixuan Biotechnology Co., Ltd., China) according to the manufacturer\u0026rsquo;s instructions. Briefly, 100 \u0026micro;L of tissue homogenate was added to enzyme-linked immunosorbent assay wells pre-coated with specific antibodies and incubated at 37\u0026deg;C for 1 hour. The plates were washed three times, followed by the addition of 100 \u0026micro;L of enzyme conjugate for a 30-minute incubation. After washing, substrate solution was added and incubated for 15 minutes. The reaction was terminated by adding 50 \u0026micro;L of stop solution, and the OD was measured at 450 nm. The concentrations of HMGB1, IL-1β, and IL-18 were calculated using standard curves. The supernatants of muscle tissue's homogenates were also used for western blotting of protein concentrations of caspase-3, cleaved caspase-3, GSDME, and N-GSDME. As follows, the samples were separated by SDS- PAGE, then transferred to a polyvinylidene fluoride membrane, and finally blocked with 5% nonfat milk. Subsequently, the membranes were incubated with primary antibodies to caspase-3 (1:1,000; Proteintech, Rosemont, IL, USA), cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), GSDME (1:1,000; Proteintech, Rosemont, IL, USA), N-GSDME (1:1,000; Abcam, Cambridge, UK), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:1,000; BBI Life Sciences Corporation, Shanghai, China). Thereafter, anti-mouse antibody (1:5,000; BBI Life Sciences Corporation, Shanghai, China) or anti-rabbit antibody (1:5,000; BBI Life Sciences Corporation, Shanghai, China) was used as the secondary antibody. Finally, the band intensities were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA), and the results were all standardized by GAPDH.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using Statistical Package for the Social Sciences (SPSS) 20.0 software (IBM Corporation, United States). The normality of the data was assessed using the Kolmogorov-Smirnov test. Data following a normal distribution were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (\u0026plusmn;\u0026thinsp;s). Intergroup comparisons were conducted using one-way ANOVA, and post hoc pairwise comparisons were performed with the Bonferroni test. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSurvival and Baseline Characteristics\u003c/h2\u003e \u003cp\u003eA total of 18 rabbits were included in this study, and all animals survived the 24-hour observation period. Before venom injection, baseline characteristics, including body weight, heart rate, body temperature, and oxygen saturation, were within normal ranges for all groups, with no significant differences between groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChanges in Left Thigh Circumference and Serum CK and Mb Levels\u003c/h2\u003e \u003cp\u003eBefore venom injection, there were no significant differences in left thigh circumference or serum CK and Mb concentrations between the three groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). After venom injection, both the Model and IMRC-Exo groups exhibited significant increases in left thigh circumference and serum CK and Mb levels compared with the Sham group. However, the IMRC-Exo group showed significantly reduced left thigh circumference and serum CK and Mb concentrations compared to the Model 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 \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological Assessment of Muscle Tissue\u003c/h2\u003e \u003cp\u003eAt 24 hours post-venom injection, HE staining in the Sham group revealed normal muscle tissue structure. In contrast, the Model group exhibited severely disorganized muscle architecture, characterized by muscle fiber rupture and necrosis, accompanied by inflammatory cell infiltration and interstitial edema. Compared to the Model group, the IMRC-Exo group demonstrated more intact muscle structure, with orderly fiber arrangement, reduced rupture and necrosis, and significantly alleviated inflammatory cell infiltration and edema (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis in Muscle Tissue\u003c/h2\u003e \u003cp\u003eAt 24 hours post-venom injection, minimal apoptosis was observed in the Sham group. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, the Model group exhibited a marked increase in apoptotic cells, evidenced by a substantial number of brownish-yellow TUNEL-positive cells. However, IMRC-Exo intervention significantly reduced the number of apoptotic cells. Quantitative analysis of the apoptosis rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) indicated that apoptosis in muscle tissues was significantly increased in both the Model and IMRC-Exo groups compared with the Sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Nevertheless, the apoptosis rate was significantly lower in the IMRC-Exo group than in the Model group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eInflammatory Cytokine Levels in Muscle Tissue\u003c/h2\u003e \u003cp\u003eAt 24 hours post-venom injection, ELISA results showed that the concentrations of HMGB1, IL-1β, and IL-18 in muscle tissue were significantly elevated in both the Model and IMRC-Exo groups compared with the Sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, these inflammatory cytokine levels were significantly lower in the IMRC-Exo group compared with the Model group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePyroptosis-Related Protein Expression\u003c/h2\u003e \u003cp\u003eWestern blot analysis at 24 hours post-venom injection revealed significantly increased expression of caspase-3, cleaved caspase-3, GSDME, and N-GSDME in the Model and IMRC-Exo groups compared with the Sham group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, the IMRC-Exo group exhibited significantly lower expression levels of these pyroptosis-related proteins compared with the Model group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, immunohistochemical analysis demonstrated significantly higher IOD values of N-GSDME-positive staining in muscle tissues from the Model and IMRC-Exo groups compared with the Sham group. Notably, IMRC-Exo treatment significantly reduced N-GSDME-positive IOD values compared with the Model group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we observed the occurrence of pyroptosis in limb muscle tissues of rabbits following Deinagkistrodon acutus snakebite and evaluated the protective effects and underlying mechanisms of IMRC-Exo in mitigating snakebite-induced limb injury. Our results demonstrated that IMRC-Exo significantly reduced limb swelling, decreased levels of muscle injury biomarkers, and alleviated histopathological damage and apoptosis in muscle tissues, thereby exerting a protective effect. Furthermore, we found that GSDME activation and GSDME-mediated pyroptosis occurred in limb muscle tissues following snakebite, but IMRC-Exo markedly inhibited this process and reduced inflammatory damage, which may underlie its protective effects.\u003c/p\u003e \u003cp\u003eMSC-Exo are membrane-bound nanovesicles enriched with proteins, nucleic acids, and lipids, and exhibit various biological effects, including anti-inflammatory, antioxidant, and pro-angiogenic activities. These properties enable MSC-Exo to protect cells and promote tissue repair in diverse settings\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Previous studies have shown that MSCs and their soluble components significantly attenuate ischemic injury in limb muscles through anti-inflammatory and regenerative mechanisms, highlighting their potential for muscle protection and repair\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. In particular, MSC-Exo have been shown to inhibit pyroptosis via the circHIPK3/FOXO3a pathway, thereby promoting muscle repair and regeneration and effectively alleviating ischemic injury\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Additionally, MSC-Exo have demonstrated therapeutic potential in wound healing across various injury models. For instance, Yang J et al. found that umbilical cord-derived MSC-Exo combined with hydrogels enhanced wound healing and skin regeneration in diabetic rats\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Similarly, Li B et al. reported that MSC-Exo accelerated wound healing in a diabetic foot ulcer mouse model by promoting fibroblast proliferation and migration while inhibiting apoptosis and inflammation\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Moreover, Bo Y et al. demonstrated that pluripotent stem cell-derived exosomes promoted keratinocyte and endothelial cell migration through miR-762, thereby expediting burn wound repair\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. However, the application of MSC-Exo for snakebite-induced limb injury has not yet been investigated.\u003c/p\u003e \u003cp\u003eBased on the above evidence, this study aimed to explore the effects of MSC-Exo on snakebite-induced limb injury. We specifically utilized exosomes derived from IMRCs, as IMRCs\u0026mdash;originating from human embryonic stem cells\u0026mdash;have been shown to outperform MSCs from adult tissues in terms of anti-inflammatory and immunomodulatory properties\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e][\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. To ensure clinical relevance, we referenced the venom dosage used in previous Deinagkistrodon acutus pig models\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e and determined an intramuscular injection dose of 1.5 mg/kg based on our preliminary experiments. Venom was injected into the mid-lateral side of the left thigh, a common site of snakebites in clinical settings, to mimic the pathological changes observed in humans. The venom injection resulted in significant limb swelling, elevated muscle injury biomarkers, histopathological damage, and apoptosis in muscle tissues, confirming successful model establishment. Notably, IMRC-Exo treatment significantly mitigated these abnormalities, demonstrating its protective effects against snakebite-induced limb injury.\u003c/p\u003e \u003cp\u003ePyroptosis has been implicated in the pathophysiological processes of various wounds and has emerged as a promising therapeutic target. Wang X et al. demonstrated that inhibiting pyroptosis activation promoted wound healing in diabetic foot ulcers\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Similarly, Hasan Maleki M et al. found that nicotinamide riboside and resveratrol aided in diabetic wound repair by suppressing pyroptosis\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Zhang K et al. reported that bioactive glass accelerated wound healing in soft tissue injuries by modulating connexin 43/reactive oxygen species signaling to inhibit endothelial cell pyroptosis\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Furthermore, MSC-Exo have been shown to regulate pyroptosis and confer protection in various organ injury models. For example, Liu P et al. demonstrated that MSC-Exo alleviated LPS-induced acute lung injury by inhibiting alveolar macrophage pyroptosis\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Additionally, adipose-derived MSC-Exo significantly improved liver ischemia-reperfusion injury by suppressing pyroptosis and apoptosis-related pathways, reducing systemic inflammation and liver tissue damage\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Exosomes from M2-polarized microglia (M2-Exos) have also been found to inhibit neuronal pyroptosis via ubiquitin-mediated degradation of TXNIP, thereby mitigating cerebral ischemia-reperfusion injury\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we further investigated the role of pyroptosis in snakebite-induced muscle injury and the potential mechanisms underlying the protective effects of IMRC-Exo. Our results revealed that venom injection led to significant increases in the expression of pyroptosis-related proteins, including caspase-3, cleaved caspase-3, GSDME, and N-GSDME, as well as elevated levels of inflammatory cytokines HMGB1, IL-1β, and IL-18. These findings indicate the involvement of GSDME-mediated pyroptosis in snakebite-induced muscle injury. Importantly, IMRC-Exo treatment significantly reduced the expression of these pyroptosis-related proteins and inflammatory cytokines, suggesting that IMRC-Exo attenuated inflammatory injury by inhibiting GSDME-mediated pyroptosis.\u003c/p\u003e \u003cp\u003eThis study has several limitations. First, the observation period was relatively short, which may not fully capture the dynamic changes in limb injury following snakebite. Future studies should extend the observation period to comprehensively evaluate the progression of injury and the therapeutic effects of IMRC-Exo. Second, we only used a single dose of IMRC-Exo and did not explore dose-dependent effects on treatment outcomes. Dose optimization in future studies could help identify the most effective therapeutic regimen. Finally, while this study focused on key markers of the GSDME pyroptosis pathway, further investigations are needed to elucidate the molecular mechanisms through which IMRC-Exo regulate pyroptosis in snakebite models.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides the first evidence that IMRC-Exo exerts protective effects against limb wound damage induced by Deinagkistrodon acutus snakebite by inhibiting GSDME-mediated pyroptosis and the associated inflammatory injury. Our findings offer new perspectives and potential therapeutic strategies for the clinical management of snakebite-induced wounds.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eIMRC: Immune and Matrix-Regulatory Cells\u003c/p\u003e\n\u003cp\u003eExo: Exosomes\u003c/p\u003e\n\u003cp\u003eCK: Creatine Kinase\u003c/p\u003e\n\u003cp\u003eMb: Myoglobin\u003c/p\u003e\n\u003cp\u003eGSDME: Gasdermin E\u003c/p\u003e\n\u003cp\u003eN-GSDME: N-terminal Gasdermin E\u003c/p\u003e\n\u003cp\u003eWHO: World Health Organization\u003c/p\u003e\n\u003cp\u003eVSD: Vacuum Sealing Drainage\u003c/p\u003e\n\u003cp\u003eTCM: Traditional Chinese Medicine\u003c/p\u003e\n\u003cp\u003eELISA: Enzyme-Linked Immunosorbent Assay\u003c/p\u003e\n\u003cp\u003eOD: Optical Density\u003c/p\u003e\n\u003cp\u003eHE: Hematoxylin and Eosin\u003c/p\u003e\n\u003cp\u003eTUNEL: Transferase dUTP Nick-End Labeling\u003c/p\u003e\n\u003cp\u003eIOD: Integrated Optical Density\u003c/p\u003e\n\u003cp\u003eSPSS: Statistical Package for the Social Sciences\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Animal Ethics Committee of Lishui University (Approval No. 2023YD0113). All experimental procedures were performed in compliance with the guidelines of the Institutional Animal Care and Use Committee.Clinical trial number: not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Lishui Science and Technology Project (2023SJZC073) and the Zhejiang Provincial Health Department Project (2024KY1858).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHW conceived and designed the study, performed the experiments, and wrote the first draft of the manuscript. LX assisted with data collection and manuscript preparation. WD and LL contributed to data collection and analysis. PS and XW provided technical support and assisted with data interpretation. JX contributed to the literature review and manuscript revision. PL supervised the entire study, provided critical revisions, and acted as the corresponding author. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWHO. Snakebite envenoming. In: WHO [Internet]. 12 Sep 2023 [cited 12 Sep 2023].Available:https://www.who.int/zh/news-room/fact-sheets/detail/snakebite-envenoming.\u003c/li\u003e\n\u003cli\u003eQin Gongping. Toxicology of Snakes in China [M]. 2nd ed. Nanning: Guangxi Science and Technology Press; 1998. p. 46-772. [in Chinese]\u003c/li\u003e\n\u003cli\u003eGong Xuchu, Yang Wanfu. 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Int Immunopharmacol. 2024 Jan 25;127:111310. doi: 10.1016/j.intimp.2023.111310.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Deinagkistrodon acutus, snakebite, immune and matrix-regulatory cell-derived exosomes, pyroptosis, Gasdermin E","lastPublishedDoi":"10.21203/rs.3.rs-5901934/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5901934/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eInflammation plays a critical role in the pathogenesis of limb injury caused by Deinagkistrodon acutus snakebite. Investigating its regulatory mechanisms and intervention strategies may uncover effective therapeutic approaches for this condition. Recent studies have identified pyroptosis as a key pathway exacerbating target organ damage by amplifying inflammatory responses. Immune and Matrix-Regulatory Cells (IMRC), a novel type of mesenchymal stem cell, and their derived exosomes (Exo) that have shown potential in mitigating inflammation-mediated tissue damage by suppressing pyroptosis. This study aimed to evaluate whether IMRC-Exo could alleviate Deinagkistrodon acutus venom-induced limb injury by inhibiting pyroptosis-mediated inflammation in rabbits.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eEighteen healthy male New Zealand white rabbits were randomly assigned to the Sham, Model, and IMRC-Exo groups. The Model group was established by intramuscular injection of Deinagkistrodon acutus venom (1.5 mg/kg), followed by intravenous infusion of anti-D. acutus venom serum (80 U/kg) after 2 hours. The IMRC-Exo group received IMRC-Exo treatment (7.5 \u0026times; 10^10 particles) after model establishment. Within 24 hours post-modeling, the left thigh circumference, serum creatine kinase (CK), and myoglobin (Mb) levels were dynamically assessed. Animals were euthanized to collect muscle tissues for histopathological examination, apoptosis analysis, inflammatory cytokine quantification (HMGB1, IL-1β, IL-18), and pyroptosis-related protein detection, including caspase-3, cleaved caspase-3, gasdermin E (GSDME), and N-terminal GSDME (N-GSDME).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCompared with the Sham group, both venom-injected groups exhibited a significant increase in left thigh circumference, elevated serum CK and Mb levels, and aggravated histopathological damage and apoptosis in muscle tissues. However, the IMRC-Exo group showed significantly reduced limb circumference, decreased muscle injury markers, and attenuated tissue damage compared with the Model group. Additionally, venom injection significantly increased HMGB1, IL-1β, IL-18 levels, and the expression of caspase-3, cleaved caspase-3, GSDME, and N-GSDME in muscle tissues of the Model and IMRC-Exo groups compared to the Sham group. Notably, these inflammatory cytokine levels and pyroptosis-related protein expressions were significantly lower in the IMRC-Exo group than in the Model group.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIMRC-Exo effectively alleviates limb wound damage induced by Deinagkistrodon acutus snakebite in rabbits. Its protective mechanism may involve the inhibition of GSDME-dependent pyroptosis-mediated inflammatory injury.\u003c/p\u003e","manuscriptTitle":"IMRC-Exo Alleviates Limb Injury Induced by Deinagkistrodon acutus Snakebite in Rabbits Through GSDME-Dependent Pyroptosis Inhibition","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-30 14:57:15","doi":"10.21203/rs.3.rs-5901934/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b127eae8-d4bc-4499-8b9f-5e9fd85810f0","owner":[],"postedDate":"January 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-13T09:23:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-30 14:57:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5901934","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5901934","identity":"rs-5901934","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}