Atractylodin attenuated hepatic ischemia reperfusion injury in mice by regulating TLR4-NLRP3-ASC signal pathway

Atractylodin attenuated hepatic ischemia reperfusion injury in mice by regulating TLR4-NLRP3-ASC signal pathway | 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 Atractylodin attenuated hepatic ischemia reperfusion injury in mice by regulating TLR4-NLRP3-ASC signal pathway Chao Li, Yan Li, Juan Zhang, Jingyuan Wan, Bin Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6193635/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Background: Hepatic ischemia-reperfusion injury (IRI), as the critical condition commonly related to shock or liver surgery, often results in extensive tissue damage and impairs liver function. Atractylodin (ATR), the main bioactive compound obtained from the Atractylodes rhizome, exhibits anti-inflammatory, antioxidant, and cytoprotective properties. Nonetheless, its effects on hepatic IRI and its associated mechanisms are largely unclear. Methods: In the present work, ATR (40 mg/kg) was administered via gavage every 12 h for 3 days prior to the induction of IRI. Meanwhile, liver function, histopathology, pyroptosis markers, oxidative stress, and inflammatory cytokine levels were assessed. Result: The results revealed that ATR pretreatment significantly improved liver function and histopathological outcomes, while reducing transaminase activity, apoptosis, inflammatory cytokine levels, and oxidative stress. Furthermore, ATR inhibited the activation of TLR4 and NOD-like receptor protein 3 (NLRP3) inflammasomes, including the NLRP3 and ASC protein, thereby attenuating downstream inflammatory factor production and maturation and reducing pyroptosis. Conclusions: ATR protects against hepatic IRI through regulating the TLR4-NLRP3-ASC signaling pathway, highlighting its potential clinical utility. Hepatic ischemia-reperfusion injury (IRI) Atractylodin (ATR) Inflammation TLR4-NLRP3-ASC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Hepatic ischemia-reperfusion injury (IRI) presents an important challenge clinically, like in liver transplantation, hepatectomy, and shock[ 1 – 2 ]. Research has demonstrated that liver ischemic damage is closely associated with both inflammation and oxidative stress[ 3 ]. In early-stage ischemia, reactive oxygen species (ROS) contribute to liver injury through mechanisms including protein oxidation, lipid peroxidation, DNA damage and mitochondrial dysfunction. Thereafter, Kupffer cells and neutrophils are recruited, further amplifying liver inflammation[ 4 – 5 ]. Consequently, inhibiting inflammation and oxidative stress has emerged as a critical therapeutic strategy for managing hepatic IRI. Simultaneously, pyroptosis represents the pro-inflammatory form of programmed cell death, which is typically associated with the robust inflammatory response[ 6 – 7 ]. Recent studies have identified a strong correlation between oxidative stress-induced tissue damage and pyroptosis pathway activation during IRI [ 8 ]. The classical pyroptosis pathway involves the caspase-1-mediated pro-inflammatory programmed cell death. The central mechanism is that Toll-like receptor 4 (TLR4) detects IRI and releases damage-associated molecular patterns (DAMPs), thereby initiating and activating the assembly of the downstream NOD-like receptor protein 3 (NLRP3) inflammasome[ 9 ]. In addition, apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC) form "pyroptosome" complexes that drive pyroptosis[ 10 ]. As demonstrated in some research, targeting the NLRP3 inflammasome and pyroptosis pathways can significantly mitigate hepatic IRI[ 11 – 14 ]. Atractylodin (ATR Fig. 1 A), as the bioactive compound obtained from the Atractylodes rhizome[ 15 ], can mitigate lipopolysaccharide (LPS)-induced acute lung injury (ALI) through suppressing inflammatory factor generation, which is primarily achieved through inhibiting NLRP3 inflammasome and TLR4 signaling pathways in the pyroptosis pathway[ 16 ]. Nonetheless, the exact mechanism of ATR in hepatic IRI is still unknown. The present work focused on exploring the effect of ATR on hepatic IRI. According to our results, ATR protects against hepatic IRI through modulating TLR4-NLRP3-ASC pathway. Materials and methods 2.1.Chemicals ATR (purity, > 98%) was provided by Chengdu Herbpurify Co. LTD (Chengdu, China), dissolved within dimethyl sulfoxide (DMSO) (Solarbio, Beijing, China) and then preserved under − 20°C. The DMSO final concentration in the solutions was maintained below 5%. 2.2.Animals The 6-8-week-old male C57/BL6 mice about 18–22 g in weight were obtained from the Animal Center of Chongqing Medical University. Mice were raised at 24 ± 1°C under the 12-h/12-h light-dark cycle, with free access to food and sterile water. The Animal Welfare and Research Ethics Committee at Chongqing Medical University approved our study. 2.3.Animal Grouping and Experimental Protocol The mice were anesthetized by intraperitoneal(IP) injection of 80 mg/kg ketamine and 10 mg/kg xylazine.Animals were randomized as Control, I/R, ATR and I/R + ATR (40 mg/kg) groups (n = 6 each). The ATR and I/R + ATR groups were given ATR through gavage every 12 h for 3 consecutive days. All mice underwent a 12-h fasting period prior to this experiment, yet they were allowed to take water freely. Blood flow in left and middle hepatic lobes was occluded using a non-traumatic arterial clamp in both I/R and I/R + ATR groups. This clamp was removed at 90 min after ischemia, then the incision was closed, and reperfusion was performed for 24 h. The Control group received identical treatment to the ATR group, but without ischemia. Mouse body temperature was kept at 37℃ with the heating blanket throughout the whole procedure. The persistent ischemia model was established. After reperfusion,Mice previously anesthetized by isofluorane anesthesia (5%) were euthanized by beheading, and venous blood was collected through the eyeball. The blood samples were put on ice at once, followed by 10 min of centrifugation at 3600 rpm and 4°C. Liver tissues from ischemic lobes were collected before fixation with 4% paraformaldehyde and embedding for histology and immunohistochemistry analyses. 2.4.Liver Function Assessment Plasma was preserved under − 20°C to determine aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. These enzymes, the important markers of liver injury, were analyzed with colorimetric kits in line with specific protocols (Nanjing Jiancheng, China). 2.5. Liver Histology Examination The paraffin-embedded blocks were prepared into 5-µm liver tissue sections, followed by hematoxylin and eosin (H&E) staining for routine histology. Besides, tissue morphology was assessed with the light microscope (Leica, Germany). 2.6.TUNEL Staining The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay kit (Roche, Switzerland) was utilized to detect and quantify hepatocellular apoptosis under light microscopy (Leica, Germany). 2.7.Analysis of Oxidative Factors in Liver Tissues Commercially available assay kits (Nanjing Jiancheng, China) were adopted to measure superoxide dismutase (SOD) and malondialdehyde (MDA) levels within liver tissues in line with specific protocols. 2.8.Immunofluorescence analysis After 6 h of fixation within 4% paraformaldehyde, liver tissues were dehydrated in 15% and 30% sucrose phosphate-buffered saline (PBS) for a 12-h period, respectively. Afterwards, the tissues were embedded into optimal cutting temperature (OCT) compound at 4°C to prepare the 10 µm frozen sections. These sections then underwent blocking using 5% bovine serum albumin (BSA), reverse-staining using primary antibodies that targeted neutrophils, macrophages, and leukocytes, and further goat anti-rabbit secondary antibody incubation. The fluorescence microscope was adopted for observing the samples. 2.9.Immunohistochemistry Following deparaffinization and hydration of paraffin sections, 3% H₂O₂ was added to quench endogenous peroxidase activity for 30 min. After 1 h of blocking with 10% BSA under 37°C, these sections were incubated using anti-tumor nuclear factor-α (TNF-α) and anti-interleukin-6 (IL-6) primary antibodies (Thermo, China) under 4°C overnight, followed by additional 1 h of secondary antibody incubation (Goat anti-rabbit, Beijing Zhongshan, China) under 37°C, diaminobenzidine (DAB) staining, and hematoxylin counterstaining. The light microscope (Leica, Germany) was employed for examining those stained sections. 2.10.Western Blotting Radio-immunoprecipitation assay (RIPA) lysis buffer was utilized to extract total liver tissue proteins, and then protein content was measured with the bicinchoninic acid (BCA) kit. Later, protein aliquots were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for separation, followed by transfer on polyvinylidene fluoride (PVDF) membranes. The membranes were subsequently blocked using 5% defatted milk prior to overnight primary antibody incubation (TLR4, NLRP3, ASC, IL-1β, and IL-18). After being washed, the membranes were further probed using horseradish peroxidase (HRP)-conjugated secondary antibodies, and protein expression was measured with the chemiluminescence system. 2.11.Statistical Analysis Results were expressed as mean ± standard deviation (SD). Statistical analyses were performed for determining between-group significant differences, and P < 0.05 stood for statistical significance. Results 3.1.ATR improves liver function during hepatic IRI in mice ALT and AST contents in serum were assessed after hepatic IRI. As shown in (Fig. 1 B and C), The activity in both the control and ATR groups was at a low level compared to the control group, while the activity in the I/R group was significantly increased. Notably, this increase was apparently mitigated after ATR pretreatment. 3.2.ATR improves the histopathology of liver tissues Histopathological analysis was conducted to evaluate the impact of ATR on hepatic IRI. While liver tissues appeared normal in both the control and ATR groups, those in the I/R group exhibited cytoplasmic vacuolization, edema, and focal necrosis. Notably, ATR pretreatment effectively mitigated these pathological changes(Fig. 2 A and B). 3.3.ATR reduces liver cell apoptosis resulting from hepatic IRI Hepatocyte apoptosis is an important indicator of the severity of hepatic IRI. In this study, TUNEL staining was carried out to assess apoptosis in mouse liver parenchyma. According to our results, no hepatocyte apoptosis was observed in the control or ATR group, whereas extensive hepatocyte apoptosis was seen in the I/R group. Notably, ATR pretreatment significantly reduced the number of apoptotic cells(Fig. 3 A and B), and the difference in hepatocyte apoptosis rates between the I/R and ATR groups was significant. 3.4.ATR mitigates inflammatory cell infiltration into the liver Immunofluorescence analysis was conducted to evaluate inflammatory cell infiltration into the liver parenchyma during I/R, a critical marker of inflammatory response in acute liver injury. As shown in(Fig. 4 A and B), the I/R group exhibited significant leukocyte, macrophage and neutrophil infiltration, which was markedly higher than in both control and ATR groups. Notably, ATR pretreatment substantially reduced the infiltration of these inflammatory cells. 3.5.ATR reduces pro-inflammatory cytokine production induced by hepatic IRI Immunohistochemistry was performed for assessing IL-6 and TNF-α levels during hepatic IRI. As shown in Fig. 5 , both cytokines were significantly up-regulated following I/R in comparison with the control and ATR groups. But ATR preconditioning markedly reduced their expression levels. 3.6.ATR ameliorates oxidative stress in the liver Dihydroethidium (DHE) fluorescence was applied in detecting ROS levels within liver tissue. Minimal fluorescence was observed in both the control and ATR groups, while intense fluorescence was detected in the I/R group, indicating the elevated ROS levels(Fig. 6 A and B). ATR pretreatment significantly reduced the fluorescence. Additionally, ATR pretreatment also lowered the MDA content (the lipid peroxidation marker) and enhanced the SOD activity (the antioxidant enzyme), both of which were indicative of the reduced oxidative stress(Fig. 6 C and D). 3.7.ATR blocks the NLRP3 inflammasome activation within the liver For further exploring the regulatory role of ATR in liver injury and its relationship with the pyroptosis pathway, Western blotting assay was conducted to detect key proteins TLR4, NLRP3, ASC as well as their downstream cytokines IL-1β and IL-18. Our results demonstrated that the I/R group showed significant up-regulation of these proteins in comparison with the control and ATR groups, indicating the activation of the pyroptosis pathway. Notably, ATR treatment significantly inhibited the activation of this pathway, suggesting its potential role in mitigating pyroptosis during liver injury(Fig. 7 A and B). Discussion Hepatic IRI involves complex mechanisms, among which, hepatocyte death exerts an important effect on disease progression [ 1 , 17 – 18 ]. According to our results, ATR pretreatment significantly improved liver function, reduced necrotic areas, and inhibited hepatic inflammation and oxidative stress. Moreover, ATR pretreatment markedly down-regulated NLRP3, ASC, IL-1β, and IL-18 levels within the pyroptosis pathway, highlighting its potential hepatoprotective effects. Inflammation, an essential part of innate immune response aiming at defending against infection and injury, can become pathological when it is dysregulated, leading to the widespread tissue damage[ 19 – 20 ]. In the context of hepatic IRI, this pathological inflammation is primarily driven by inflammatory cell infiltration, including neutrophils and macrophages, which are recruited to the injury site by pro-inflammatory mediators[ 21 – 22 ]. These cells, through releasing factors like IL-6 and TNF-α, amplify the inflammatory response, further damaging hepatocytes and exerting an important effect on liver injury progression [ 18 , 22 – 25 ]. Our study also discovered the same inflammatory manifestations of liver ischemia, consistent with previous studies. As demonstrated by our results, ATR pretreatment remarkably decreased inflammatory cell levels and their associated cytokines within liver tissue. This suggests that ATR probably executes the anti-inflammatory effects through modulating chemokines and adhesion molecules that regulate leukocyte migration, and through directly inhibiting the activation of inflammatory cells. Such results conform to prior findings regarding the anti-inflammatory effects of ATR. For instance, ATR can significantly decrease liver injury and suppress inflammation in the LPS- and D-GalN-induced acute liver failure models, as well as in high-fat diet (HFD)-induced nonalcoholic fatty liver disease (NAFLD) models [ 26 – 27 ]. ATR improves organ function by inhibiting the expression of inflammatory mediators [ 28 – 30 ]. Therefore, ATR may offer protection against I/R-mediated liver injury through mitigating inflammatory cell infiltration. Oxidative stress is another key factor for hepatic IRI, where excessive ROS production overwhelms the antioxidant defense system of the liver, resulting in cell injury through lipid peroxidation, DNA damage and protein modification [ 4 , 31 ]. The antioxidant effect of ATR has been well demonstrated in the fructose-induced podocyte hypermotility model, which reduces cell protection from oxidative damage through up-regulating SOD level while reducing ROS generation. Moreover, ATR exerts an important effect on keeping redox homeostasis[ 32 ]. Similarly, ATR can inhibit oxidative stress-induced autophagy and apoptosis of MCF-7 human breast cancer cells[ 33 ], further confirming its protective effect in different tissues. Consistent with our expectations, our findings showed that ATR significantly alleviated oxidative stress in IRI liver tissues, as indicated by the decreased MDA content and increased antioxidase activities such as SOD. More importantly, oxidative stress and the secretion of these inflammatory factors are implicated in an important signaling pathway, namely, pyroptosis dominated by NLRP3 inflammasome, which is usually necessary for the development of hepatic IRI. TLR4 is widely suggested to mediate NLRP3 inflammasome activation [ 11 , 12 , 34 – 36 ]. When cells are damaged, TLR4 in the Toll-like receptor family can interact with DAMP molecules in vivo to trigger the signaling pathway, and induce the recruitment of ASC to form NLRP3 inflammasome, which can thus promote pro-inflammatory factor production and maturation (IL-1β and IL-18), thereby amplifying inflammatory response and aggravating cell death [ 37 – 38 ]. In preliminary studies, IL-1β and IL-18 have key functions in both innate and adaptive immune responses[ 39 ]. Our study revealed that ATR apparently suppressed NLRP3 inflammasome activation, which was evidenced by the decreased levels of its components and reduced production of IL-1β and IL-18. This inhibition of pyroptosis may be related to the overall liver injury mitigation observed among ATR-treated mice. Notably, the ability of ATR to inhibit pyroptosis has also been demonstrated under other inflammatory conditions, such as LPS-induced ALI where ATR effectively suppresses NLRP3 inflammasome activation, thereby alleviating pulmonary inflammation and tissue damage[ 16 ]. Other studies have reported that ATR can improve the mouse LPS-induced depression-like behavior by decreasing nerve cell injury and neuroinflammation, and the molecular mechanism is probably related to down-regulating the NLRP3 inflammasome level[ 40 ]. Conclusion In conclusion, As shown in Fig. 8 ,our study demonstrates that ATR treatment improves hepatic IRI by reducing hepatocyte damage, inflammation, and inhibiting oxidative stress responses. These beneficial effects are likely mediated by modulating TLR4-NLRP3-ASC pathway. These findings lay a certain basis for future studies on the role of ATR in disease treatment and its possible clinical applications in managing hepatic IRI. Abbreviations ALT：alanine aminotransferase；AST：aspartate aminotransferase；ASC： a caspase recruitment domain；ATR：Atractylodin；DAMPs：damage-associated molecular patterns；IRI：ischemia-reperfusion injury ；IL-6：anti-interleukin-6；NLRP3： NOD-like receptor protein 3；ROS：reactive oxygen species；SOD：superoxide dismutase ；TLR4：Toll-like receptor 4；TUNEL：transferase dUTP nick-end labeling ；TNF-α：anti-tumor nuclear factor-α；MDA：malondialdehyde Declarations Ethical statement Every animal experiment gained approval from the Animal Experiments of Chongqing Medical University and the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, and was performed strictly following the standards of the Guide for the Care and Use of Laboratory Animals. Acknowledgements Not applicable. Author contributions: C.L.: study design,execution, analysis, and interpretation of the data,manuscript writing and analysis. Y.L.,J.Z.：execution:J.Y. W: participation in the study design, critical discussion.B.W. :conceptualizations and study desigread and approved the fnal manuscript.All authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved have read and approved the manuscript. Funding The present study was financially supported by grants from the National Natural Science Foundation of China (No: 82070714) and the Chongqing Natural Science Foundation (No: CSTB2022NSCQ-MSX0061). Availability of data and materials There is no data other than the data given in the article. Ethics approval and consent to participate This experiment was performed in accordance with the National Guidelines for the Use and Care of Laboratory Animals and the study was approved by The Animal Welfare and Research Ethics Committee at Chongqing Medical University. 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Supplementary Files SupplementaryFile1.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 12 May, 2025 Reviews received at journal 03 May, 2025 Reviewers agreed at journal 30 Apr, 2025 Reviewers agreed at journal 27 Apr, 2025 Reviews received at journal 27 Apr, 2025 Reviewers agreed at journal 25 Apr, 2025 Reviewers agreed at journal 23 Apr, 2025 Reviewers agreed at journal 23 Apr, 2025 Reviewers invited by journal 04 Apr, 2025 Editor assigned by journal 04 Apr, 2025 Editor invited by journal 04 Apr, 2025 Submission checks completed at journal 03 Apr, 2025 First submitted to journal 03 Apr, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-6193635","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":446778778,"identity":"9cbbcf6f-9667-41e7-94e2-1cfcd7e2f1c2","order_by":0,"name":"Chao Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chao","middleName":"","lastName":"Li","suffix":""},{"id":446778779,"identity":"ff013684-339a-4393-825f-26a2c9e3cf9b","order_by":1,"name":"Yan Li","email":"","orcid":"","institution":"The First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Li","suffix":""},{"id":446778781,"identity":"1202bd0f-19e9-4cb5-abea-5cd514a089c0","order_by":2,"name":"Juan Zhang","email":"","orcid":"","institution":"The First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Zhang","suffix":""},{"id":446778782,"identity":"42f124d4-9c65-48e7-97fb-c4f6b77f42ea","order_by":3,"name":"Jingyuan Wan","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jingyuan","middleName":"","lastName":"Wan","suffix":""},{"id":446778784,"identity":"5943b4fe-a3ca-4df2-a547-ebd215d4b83a","order_by":4,"name":"Bin Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYBAC+/sPEh9+/PO/vp+9gVg9BxIeG0s2MDPO7DlAtJbEZxK8QC0bbiQQqYOx4XCahOQONmbJmY833mCosYkmqIWZsS3ZovAMDxu/dFqxBcOxtNwGQlrYmHkSb0iwSfBIzs4xkwDaSVgL0PgPEjxsBhIGN88QqUWChyFJgrctwcDgBg+RWgwkGJKNJc4cSJDsAfolgRi/ALUkPvxQcSCBn/3wxhsfamwIa0HVnkCKcqiNo2AUjIJRMAqwAACr0j8LuU96SwAAAABJRU5ErkJggg==","orcid":"","institution":"The First Affiliated Hospital of Chongqing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Bin","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-03-10 08:53:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6193635/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6193635/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82121013,"identity":"ce9f8087-4903-4b32-a115-eb2b7e8d613a","added_by":"auto","created_at":"2025-05-07 03:22:29","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":34114,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ATR on serum levels of ALT and AST. Mice were administered ATR at a dose of 40mg/kg via gavage every 12 h for 3 days before the induction of IRI. After 24 h reperfusion, the blood was collected and analyzed for serum levels of ALT and AST.（A）Structure of Atractylodin.（B）The level of serum ALT.（C）The level of serum AST. Data were expressed as mean ± SD, n = 6, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001 compared with I/R model group. ATR:Atractylodin; IR: ischemia reperfusion; ALT: alanine aminotransferase; AST: aspartate aminotransferase.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6193635/v1/b9ad01a0bceec0d792762f03.jpeg"},{"id":82122821,"identity":"2a7d1181-2b17-4eb4-a353-99304fcd7dc7","added_by":"auto","created_at":"2025-05-07 03:30:29","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":120747,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ATR on histopathological changes of hepatic tissues. Mice were administered ATR at a dose of 40mg/kg via gavage every 12 h for 3 days before the induction of IRI. After reperfusion for 24 h,the liver tissues were collected and stained with H\u0026amp;E.（A）H\u0026amp;E staining(200×magnification).（B）Suzuki's histological grading. Data are expressed as mean ± SD, n = 3, ** P \u0026lt; 0.01 compared with I/R model group. ATR: Atractylodin; IR: ischemia reperfusion.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6193635/v1/9914570fd044703be7eb2f91.jpeg"},{"id":82122820,"identity":"a9914b17-a7e7-4b8b-8a74-d5651fe875e9","added_by":"auto","created_at":"2025-05-07 03:30:29","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":69527,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ATR on hepatic I/R-induced cell apoptosis. Mice were administered ATR at a dose of 40mg/kg via gavage every 12 h for 3 days before the induction of IRI. After reperfusion for 24 h,the livers of the four groups of mice were selected into paraffin sections and stained by TUNEL. (A) Representative images of TUNEL (200 ×magnification). (B) Quantitative analysis of TUNEL positive cells. Data are expressed as mean ± SD, n = 3, *** P \u0026lt; 0.001 compared with I/R model group. ATR: Atractylodin; IR: Ischemia reperfusion.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6193635/v1/684ed33a6cda414a24599ce0.jpeg"},{"id":82124355,"identity":"8c684f71-0b72-4f15-9303-4c4ee8859006","added_by":"auto","created_at":"2025-05-07 03:38:29","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":134791,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ATR on the infiltration of inflammatory cells induced by HIRI. Mice were administered ATR at a dose of 40mg/kg via gavage every 12 h for 3 days before the induction of IRI. After 24 h reperfusion, the liver tissue of mice was made into frozen sections and immunofluorescence staining.（A）Results of leukocyte immunostaining with anti-CD45 antibody，macrophages immunostaining with anti-F4/80 antibody and neutrophils immunostaining with anti-neutrophil antibody in four groups (marked in green,200 ×magnification).（B）Quantitative analysis of inflammatory positive cell. Data are expressed as mean ± SD, n = 3, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001 compared with I/R model group. ATR: Atractylodin; IR: Ischemia reperfusion.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6193635/v1/e22e12237fb0fc20f236c7f2.jpeg"},{"id":82121017,"identity":"24e3d251-9c7e-4ace-9283-93641f25367e","added_by":"auto","created_at":"2025-05-07 03:22:29","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":89326,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ATR on expressions of IL-6 and TNF-α protein induced by HIRI. Mice were administered ATR at a dose of 40mg/kg via gavage every 12 h for 3 days before the induction of IRI. After reperfusion for 24 h,the livers of the four groups of mice were selected into paraffin sections and stained by immunohistochemistry.（A）The expression level of IL-6 (200 × magnification).（B）Quantitative IOD analysis of IL-6.（C）The expression level of TNF-α (200 × magnification).（D）Quantitative IOD analysis of TNF-α. Data are expressed as mean ± SD, n = 3, *** P \u0026lt; 0.001 compared with I/R model group. ATR: Atractylodin; IR: Ischemia reperfusion.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6193635/v1/67cf95853380b9eb1ef85f9d.jpeg"},{"id":82121016,"identity":"3815305a-1024-45b6-bbd1-02be138ed28c","added_by":"auto","created_at":"2025-05-07 03:22:29","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":78354,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ATR on HIR-induced oxidant stress. Mice were administered ATR at a dose of 40mg/kg via gavage every 12 h for 3 days before the induction of IRI. After 24 h reperfusion, the liver tissue of mice was made into frozen sections and immunofluorescence staining. And liver tissues were collected and homogenized.（A）ROS expression detected by DHE (marked in red, 200 × magnification).（B）Quantitative analysis of DHE positive cell.（C）The content of hepatic MDA level.（D）The content of hepatic SOD level.Data are expressed as mean ± SD, n = 3, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001 compared with I/R model group. ATR: Atractylodin; IR: Ischemia reperfusion.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6193635/v1/b36caed9fa10b6850be8a684.jpeg"},{"id":82121022,"identity":"034039a6-6131-4ed1-8f57-d4a46f16b9d3","added_by":"auto","created_at":"2025-05-07 03:22:29","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":67683,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ATR on TLR4-NLRP3-ASC signaling pathway. Mice were administered ATR at a dose of 40mg/kg via gavage every 12 h for 3 days before the induction of IRI. After 24 h reperfusion, the liver tissues were collected for analysis of TLR4-NLRP3-ASC signaling pathway by western blot.（A）The levels of proteins in TLR4-NLRP3-ASC signaling pathway.（B）Quantitative analysis of WB. Data are expressed as mean ± SD, n = 3, * P \u0026lt; 0.05, ** P \u0026lt; 0.01, *** P \u0026lt; 0.001compared with I/R model group. ATR: Atractylodin; IR: Ischemia reperfusion.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6193635/v1/dd6a5f7f0b7e43207674027c.jpeg"},{"id":82121019,"identity":"cf32ac69-496c-4357-a3cb-5ed03c5dc0d1","added_by":"auto","created_at":"2025-05-07 03:22:29","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":42773,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental hypothesis that ATR protects liver from ischemia-reperfusion injury\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6193635/v1/9b4a59520e1baa738944f3eb.jpeg"},{"id":82125259,"identity":"70a7449b-57b1-492b-a6b2-460ebc1f57d8","added_by":"auto","created_at":"2025-05-07 03:46:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1418334,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6193635/v1/364c63cb-987b-47ca-8ba7-fcf89bb08ded.pdf"},{"id":82122822,"identity":"97e0e649-4ffe-48fe-8989-5a0401f814be","added_by":"auto","created_at":"2025-05-07 03:30:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":449419,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6193635/v1/068bc1a16284e14ebff0439a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Atractylodin attenuated hepatic ischemia reperfusion injury in mice by regulating TLR4-NLRP3-ASC signal pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepatic ischemia-reperfusion injury (IRI) presents an important challenge clinically, like in liver transplantation, hepatectomy, and shock[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Research has demonstrated that liver ischemic damage is closely associated with both inflammation and oxidative stress[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In early-stage ischemia, reactive oxygen species (ROS) contribute to liver injury through mechanisms including protein oxidation, lipid peroxidation, DNA damage and mitochondrial dysfunction. Thereafter, Kupffer cells and neutrophils are recruited, further amplifying liver inflammation[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Consequently, inhibiting inflammation and oxidative stress has emerged as a critical therapeutic strategy for managing hepatic IRI.\u003c/p\u003e \u003cp\u003eSimultaneously, pyroptosis represents the pro-inflammatory form of programmed cell death, which is typically associated with the robust inflammatory response[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Recent studies have identified a strong correlation between oxidative stress-induced tissue damage and pyroptosis pathway activation during IRI [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The classical pyroptosis pathway involves the caspase-1-mediated pro-inflammatory programmed cell death. The central mechanism is that Toll-like receptor 4 (TLR4) detects IRI and releases damage-associated molecular patterns (DAMPs), thereby initiating and activating the assembly of the downstream NOD-like receptor protein 3 (NLRP3) inflammasome[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In addition, apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC) form \"pyroptosome\" complexes that drive pyroptosis[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. As demonstrated in some research, targeting the NLRP3 inflammasome and pyroptosis pathways can significantly mitigate hepatic IRI[\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAtractylodin (ATR Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), as the bioactive compound obtained from the Atractylodes rhizome[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], can mitigate lipopolysaccharide (LPS)-induced acute lung injury (ALI) through suppressing inflammatory factor generation, which is primarily achieved through inhibiting NLRP3 inflammasome and TLR4 signaling pathways in the pyroptosis pathway[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Nonetheless, the exact mechanism of ATR in hepatic IRI is still unknown. The present work focused on exploring the effect of ATR on hepatic IRI. According to our results, ATR protects against hepatic IRI through modulating TLR4-NLRP3-ASC pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1.Chemicals\u003c/h2\u003e \u003cp\u003eATR (purity, \u0026gt; 98%) was provided by Chengdu Herbpurify Co. LTD (Chengdu, China), dissolved within dimethyl sulfoxide (DMSO) (Solarbio, Beijing, China) and then preserved under \u0026minus;\u0026thinsp;20\u0026deg;C. The DMSO final concentration in the solutions was maintained below 5%.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.2.Animals\u003c/h3\u003e\n\u003cp\u003eThe 6-8-week-old male C57/BL6 mice about 18\u0026ndash;22 g in weight were obtained from the Animal Center of Chongqing Medical University. Mice were raised at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C under the 12-h/12-h light-dark cycle, with free access to food and sterile water. The Animal Welfare and Research Ethics Committee at Chongqing Medical University approved our study.\u003c/p\u003e\n\u003ch3\u003e2.3.Animal Grouping and Experimental Protocol\u003c/h3\u003e\n\u003cp\u003eThe mice were anesthetized by intraperitoneal(IP) injection of 80 mg/kg ketamine and 10 mg/kg xylazine.Animals were randomized as Control, I/R, ATR and I/R\u0026thinsp;+\u0026thinsp;ATR (40 mg/kg) groups (n\u0026thinsp;=\u0026thinsp;6 each). The ATR and I/R\u0026thinsp;+\u0026thinsp;ATR groups were given ATR through gavage every 12 h for 3 consecutive days. All mice underwent a 12-h fasting period prior to this experiment, yet they were allowed to take water freely. Blood flow in left and middle hepatic lobes was occluded using a non-traumatic arterial clamp in both I/R and I/R\u0026thinsp;+\u0026thinsp;ATR groups. This clamp was removed at 90 min after ischemia, then the incision was closed, and reperfusion was performed for 24 h. The Control group received identical treatment to the ATR group, but without ischemia. Mouse body temperature was kept at 37℃ with the heating blanket throughout the whole procedure. The persistent ischemia model was established. After reperfusion,Mice previously anesthetized by isofluorane anesthesia (5%) were euthanized by beheading, and venous blood was collected through the eyeball. The blood samples were put on ice at once, followed by 10 min of centrifugation at 3600 rpm and 4\u0026deg;C. Liver tissues from ischemic lobes were collected before fixation with 4% paraformaldehyde and embedding for histology and immunohistochemistry analyses.\u003c/p\u003e\n\u003ch3\u003e2.4.Liver Function Assessment\u003c/h3\u003e\n\u003cp\u003ePlasma was preserved under \u0026minus;\u0026thinsp;20\u0026deg;C to determine aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. These enzymes, the important markers of liver injury, were analyzed with colorimetric kits in line with specific protocols (Nanjing Jiancheng, China).\u003c/p\u003e\n\u003ch3\u003e2.5. Liver Histology Examination\u003c/h3\u003e\n\u003cp\u003eThe paraffin-embedded blocks were prepared into 5-\u0026micro;m liver tissue sections, followed by hematoxylin and eosin (H\u0026amp;E) staining for routine histology. Besides, tissue morphology was assessed with the light microscope (Leica, Germany).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6.TUNEL Staining\u003c/h2\u003e \u003cp\u003eThe terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay kit (Roche, Switzerland) was utilized to detect and quantify hepatocellular apoptosis under light microscopy (Leica, Germany).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.7.Analysis of Oxidative Factors in Liver Tissues\u003c/h3\u003e\n\u003cp\u003eCommercially available assay kits (Nanjing Jiancheng, China) were adopted to measure superoxide dismutase (SOD) and malondialdehyde (MDA) levels within liver tissues in line with specific protocols.\u003c/p\u003e\n\u003ch3\u003e2.8.Immunofluorescence analysis\u003c/h3\u003e\n\u003cp\u003eAfter 6 h of fixation within 4% paraformaldehyde, liver tissues were dehydrated in 15% and 30% sucrose phosphate-buffered saline (PBS) for a 12-h period, respectively. Afterwards, the tissues were embedded into optimal cutting temperature (OCT) compound at 4\u0026deg;C to prepare the 10 \u0026micro;m frozen sections. These sections then underwent blocking using 5% bovine serum albumin (BSA), reverse-staining using primary antibodies that targeted neutrophils, macrophages, and leukocytes, and further goat anti-rabbit secondary antibody incubation. The fluorescence microscope was adopted for observing the samples.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9.Immunohistochemistry\u003c/h2\u003e \u003cp\u003eFollowing deparaffinization and hydration of paraffin sections, 3% H₂O₂ was added to quench endogenous peroxidase activity for 30 min. After 1 h of blocking with 10% BSA under 37\u0026deg;C, these sections were incubated using anti-tumor nuclear factor-α (TNF-α) and anti-interleukin-6 (IL-6) primary antibodies (Thermo, China) under 4\u0026deg;C overnight, followed by additional 1 h of secondary antibody incubation (Goat anti-rabbit, Beijing Zhongshan, China) under 37\u0026deg;C, diaminobenzidine (DAB) staining, and hematoxylin counterstaining. The light microscope (Leica, Germany) was employed for examining those stained sections.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10.Western Blotting\u003c/h2\u003e \u003cp\u003eRadio-immunoprecipitation assay (RIPA) lysis buffer was utilized to extract total liver tissue proteins, and then protein content was measured with the bicinchoninic acid (BCA) kit. Later, protein aliquots were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for separation, followed by transfer on polyvinylidene fluoride (PVDF) membranes. The membranes were subsequently blocked using 5% defatted milk prior to overnight primary antibody incubation (TLR4, NLRP3, ASC, IL-1β, and IL-18). After being washed, the membranes were further probed using horseradish peroxidase (HRP)-conjugated secondary antibodies, and protein expression was measured with the chemiluminescence system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11.Statistical Analysis\u003c/h2\u003e \u003cp\u003eResults were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were performed for determining between-group significant differences, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 stood for statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1.ATR improves liver function during hepatic IRI in mice\u003c/h2\u003e \u003cp\u003eALT and AST contents in serum were assessed after hepatic IRI. As shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and C), The activity in both the control and ATR groups was at a low level compared to the control group, while the activity in the I/R group was significantly increased. Notably, this increase was apparently mitigated after ATR pretreatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2.ATR improves the histopathology of liver tissues\u003c/h2\u003e \u003cp\u003eHistopathological analysis was conducted to evaluate the impact of ATR on hepatic IRI. While liver tissues appeared normal in both the control and ATR groups, those in the I/R group exhibited cytoplasmic vacuolization, edema, and focal necrosis. Notably, ATR pretreatment effectively mitigated these pathological changes(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3.ATR reduces liver cell apoptosis resulting from hepatic IRI\u003c/h2\u003e \u003cp\u003eHepatocyte apoptosis is an important indicator of the severity of hepatic IRI. In this study, TUNEL staining was carried out to assess apoptosis in mouse liver parenchyma. According to our results, no hepatocyte apoptosis was observed in the control or ATR group, whereas extensive hepatocyte apoptosis was seen in the I/R group. Notably, ATR pretreatment significantly reduced the number of apoptotic cells(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B), and the difference in hepatocyte apoptosis rates between the I/R and ATR groups was significant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.4.ATR mitigates inflammatory cell infiltration into the liver\u003c/h2\u003e \u003cp\u003eImmunofluorescence analysis was conducted to evaluate inflammatory cell infiltration into the liver parenchyma during I/R, a critical marker of inflammatory response in acute liver injury. As shown in(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B), the I/R group exhibited significant leukocyte, macrophage and neutrophil infiltration, which was markedly higher than in both control and ATR groups. Notably, ATR pretreatment substantially reduced the infiltration of these inflammatory cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.5.ATR reduces pro-inflammatory cytokine production induced by hepatic IRI\u003c/h2\u003e \u003cp\u003eImmunohistochemistry was performed for assessing IL-6 and TNF-α levels during hepatic IRI. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, both cytokines were significantly up-regulated following I/R in comparison with the control and ATR groups. But ATR preconditioning markedly reduced their expression levels.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.6.ATR ameliorates oxidative stress in the liver\u003c/h2\u003e \u003cp\u003eDihydroethidium (DHE) fluorescence was applied in detecting ROS levels within liver tissue. Minimal fluorescence was observed in both the control and ATR groups, while intense fluorescence was detected in the I/R group, indicating the elevated ROS levels(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B). ATR pretreatment significantly reduced the fluorescence. Additionally, ATR pretreatment also lowered the MDA content (the lipid peroxidation marker) and enhanced the SOD activity (the antioxidant enzyme), both of which were indicative of the reduced oxidative stress(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.7.ATR blocks the NLRP3 inflammasome activation within the liver\u003c/h2\u003e \u003cp\u003eFor further exploring the regulatory role of ATR in liver injury and its relationship with the pyroptosis pathway, Western blotting assay was conducted to detect key proteins TLR4, NLRP3, ASC as well as their downstream cytokines IL-1β and IL-18. Our results demonstrated that the I/R group showed significant up-regulation of these proteins in comparison with the control and ATR groups, indicating the activation of the pyroptosis pathway. Notably, ATR treatment significantly inhibited the activation of this pathway, suggesting its potential role in mitigating pyroptosis during liver injury(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHepatic IRI involves complex mechanisms, among which, hepatocyte death exerts an important effect on disease progression [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. According to our results, ATR pretreatment significantly improved liver function, reduced necrotic areas, and inhibited hepatic inflammation and oxidative stress. Moreover, ATR pretreatment markedly down-regulated NLRP3, ASC, IL-1β, and IL-18 levels within the pyroptosis pathway, highlighting its potential hepatoprotective effects.\u003c/p\u003e \u003cp\u003eInflammation, an essential part of innate immune response aiming at defending against infection and injury, can become pathological when it is dysregulated, leading to the widespread tissue damage[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In the context of hepatic IRI, this pathological inflammation is primarily driven by inflammatory cell infiltration, including neutrophils and macrophages, which are recruited to the injury site by pro-inflammatory mediators[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These cells, through releasing factors like IL-6 and TNF-α, amplify the inflammatory response, further damaging hepatocytes and exerting an important effect on liver injury progression [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Our study also discovered the same inflammatory manifestations of liver ischemia, consistent with previous studies.\u003c/p\u003e \u003cp\u003eAs demonstrated by our results, ATR pretreatment remarkably decreased inflammatory cell levels and their associated cytokines within liver tissue. This suggests that ATR probably executes the anti-inflammatory effects through modulating chemokines and adhesion molecules that regulate leukocyte migration, and through directly inhibiting the activation of inflammatory cells. Such results conform to prior findings regarding the anti-inflammatory effects of ATR. For instance, ATR can significantly decrease liver injury and suppress inflammation in the LPS- and D-GalN-induced acute liver failure models, as well as in high-fat diet (HFD)-induced nonalcoholic fatty liver disease (NAFLD) models [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. ATR improves organ function by inhibiting the expression of inflammatory mediators [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Therefore, ATR may offer protection against I/R-mediated liver injury through mitigating inflammatory cell infiltration.\u003c/p\u003e \u003cp\u003eOxidative stress is another key factor for hepatic IRI, where excessive ROS production overwhelms the antioxidant defense system of the liver, resulting in cell injury through lipid peroxidation, DNA damage and protein modification [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The antioxidant effect of ATR has been well demonstrated in the fructose-induced podocyte hypermotility model, which reduces cell protection from oxidative damage through up-regulating SOD level while reducing ROS generation. Moreover, ATR exerts an important effect on keeping redox homeostasis[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Similarly, ATR can inhibit oxidative stress-induced autophagy and apoptosis of MCF-7 human breast cancer cells[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], further confirming its protective effect in different tissues. Consistent with our expectations, our findings showed that ATR significantly alleviated oxidative stress in IRI liver tissues, as indicated by the decreased MDA content and increased antioxidase activities such as SOD.\u003c/p\u003e \u003cp\u003eMore importantly, oxidative stress and the secretion of these inflammatory factors are implicated in an important signaling pathway, namely, pyroptosis dominated by NLRP3 inflammasome, which is usually necessary for the development of hepatic IRI. TLR4 is widely suggested to mediate NLRP3 inflammasome activation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. When cells are damaged, TLR4 in the Toll-like receptor family can interact with DAMP molecules in vivo to trigger the signaling pathway, and induce the recruitment of ASC to form NLRP3 inflammasome, which can thus promote pro-inflammatory factor production and maturation (IL-1β and IL-18), thereby amplifying inflammatory response and aggravating cell death [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In preliminary studies, IL-1β and IL-18 have key functions in both innate and adaptive immune responses[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study revealed that ATR apparently suppressed NLRP3 inflammasome activation, which was evidenced by the decreased levels of its components and reduced production of IL-1β and IL-18. This inhibition of pyroptosis may be related to the overall liver injury mitigation observed among ATR-treated mice. Notably, the ability of ATR to inhibit pyroptosis has also been demonstrated under other inflammatory conditions, such as LPS-induced ALI where ATR effectively suppresses NLRP3 inflammasome activation, thereby alleviating pulmonary inflammation and tissue damage[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Other studies have reported that ATR can improve the mouse LPS-induced depression-like behavior by decreasing nerve cell injury and neuroinflammation, and the molecular mechanism is probably related to down-regulating the NLRP3 inflammasome level[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e,our study demonstrates that ATR treatment improves hepatic IRI by reducing hepatocyte damage, inflammation, and inhibiting oxidative stress responses. These beneficial effects are likely mediated by modulating TLR4-NLRP3-ASC pathway. These findings lay a certain basis for future studies on the role of ATR in disease treatment and its possible clinical applications in managing hepatic IRI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eALT：alanine aminotransferase；AST：aspartate aminotransferase；ASC： a caspase recruitment domain；ATR：Atractylodin；DAMPs：damage-associated molecular patterns；IRI：ischemia-reperfusion injury ；IL-6：anti-interleukin-6；NLRP3： NOD-like receptor protein 3；ROS：reactive oxygen species；SOD：superoxide dismutase ；TLR4：Toll-like receptor 4；TUNEL：transferase dUTP nick-end labeling ；TNF-\u0026alpha;：anti-tumor nuclear factor-\u0026alpha;；MDA：malondialdehyde\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEvery animal experiment gained approval from the Animal Experiments of Chongqing Medical University and the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, and was performed strictly following the standards of the Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions: \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.L.: study design,execution, analysis, and interpretation of the data,manuscript writing and analysis. Y.L.,J.Z.：execution:J.Y. W: participation in the study design,\u003c/p\u003e\n\u003cp\u003ecritical discussion.B.W. :conceptualizations and study desigread and approved the fnal manuscript.All authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study was financially supported by grants from the National Natural Science Foundation of China (No: 82070714) and the Chongqing Natural Science Foundation (No: CSTB2022NSCQ-MSX0061).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no data other than the data given in the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis experiment was performed in accordance with the National Guidelines\u003c/p\u003e\n\u003cp\u003efor the Use and Care of Laboratory Animals and the study was approved by\u003c/p\u003e\n\u003cp\u003eThe Animal Welfare and Research Ethics Committee at Chongqing Medical University.\u003c/p\u003e\n\u003cp\u003e(Ethics Committee Number: IACUC-CQMU-2023-0069.Dated:04.27.2023).\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\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\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Anesthesiology,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China.\u003csup\u003e2\u003c/sup\u003eGaoping District People's Hospital of Nanchong,Sichuan Province,Nanchong 637100,China.\u003csup\u003e3\u003c/sup\u003eKey Laboratory of Biochemistry and Molecular Pharmacology, Chongqing Medical University, Chongqing 400016, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSerracino-Inglott F, ∙ Habib NA, ∙, Mathie. 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Atractylodin inhibits fructose-induced human podocyte hypermotility via anti-oxidant to down-regulate TRPC6/p-CaMK4 signaling. Eur J Pharmacol. 2021;913:174616.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z, Song Y, Hou W, et al. Atractylodin induces oxidative stress-mediated apoptosis and autophagy in human breast cancer MCF-7 cells through inhibition of the P13K/Akt/mTOR pathway. J Biochem Mol Toxicol. 2022;36(8):e23081.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl-Sisi AEE, 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.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJuliana C, Fernandes-Alnemri T, Kang S, Farias A, Qin F, Alnemri ES. Non-transcriptional priming and deubiquitination regulate NLRP3 inflammasome activation. J Biol Chem. 2012;287(43):36617\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe D, Guo Z, Pu JL, et al. Resveratrol preconditioning protects hepatocytes against hepatic ischemia reperfusion injury via Toll-like receptor 4/nuclear factor-κB signaling pathway in vitro and in vivo. Int Immunopharmacol. 2016;35:201\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLv X, Chen J, He J, et al. Gasdermin D-mediated pyroptosis suppresses liver regeneration after 70% partial hepatectomy. Hepatol Commun. 2022;6(9):2340\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKelley N, Jeltema D, Duan Y, He Y. The NLRP3 Inflammasome: An Overview of Mechanisms of Activation and Regulation. Int J Mol Sci. 2019;20(13):3328.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDinarello CA. Overview of the IL-1 family in innate inflammation and acquired immunity. Immunol Rev. 2018;281(1):8\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu F, Wang Y, Li D, Yang T. Atractylodin ameliorates lipopolysaccharide-induced depressive-like behaviors in mice through reducing neuroinflammation and neuronal damage. J Neuroimmunol. 2024;390:578349.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"bmc-pharmacology-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"phat","sideBox":"Learn more about [BMC Pharmacology and Toxicology](http://bmcpharmacoltoxicol.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/phat/Default.aspx","title":"BMC Pharmacology and Toxicology","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hepatic ischemia-reperfusion injury (IRI), Atractylodin (ATR), Inflammation, TLR4-NLRP3-ASC","lastPublishedDoi":"10.21203/rs.3.rs-6193635/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6193635/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eHepatic ischemia-reperfusion injury (IRI), as the critical condition commonly related to shock or liver surgery, often results in extensive tissue damage and impairs liver function. Atractylodin (ATR), the main bioactive compound obtained from the Atractylodes rhizome, exhibits anti-inflammatory, antioxidant, and cytoprotective properties. Nonetheless, its effects on hepatic IRI and its associated mechanisms are largely unclear.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eIn the present work, ATR (40 mg/kg) was administered via gavage every 12 h for 3 days prior to the induction of IRI. Meanwhile, liver function, histopathology, pyroptosis markers, oxidative stress, and inflammatory cytokine levels were assessed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResult: \u003c/strong\u003eThe results revealed that ATR pretreatment significantly improved liver function and histopathological outcomes, while reducing transaminase activity, apoptosis, inflammatory cytokine levels, and oxidative stress. Furthermore, ATR inhibited the activation of TLR4 and NOD-like receptor protein 3 (NLRP3) inflammasomes, including the NLRP3 and ASC protein, thereby attenuating downstream inflammatory factor production and maturation and reducing pyroptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eATR protects against hepatic IRI through regulating the TLR4-NLRP3-ASC signaling pathway, highlighting its potential clinical utility.\u003c/p\u003e","manuscriptTitle":"Atractylodin attenuated hepatic ischemia reperfusion injury in mice by regulating TLR4-NLRP3-ASC signal pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 03:22:24","doi":"10.21203/rs.3.rs-6193635/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-12T09:33:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-03T19:22:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"248035407432089606154343068043347182180","date":"2025-04-30T08:34:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292167429296481267829813917856108839152","date":"2025-04-27T21:04:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-27T13:48:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"248480109888114053130777235562210510874","date":"2025-04-25T13:23:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"124802189667223621045923840326530460604","date":"2025-04-23T06:25:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"302262722000228506454595509806213335032","date":"2025-04-23T04:15:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-04T10:08:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-04T10:05:37+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-04T09:00:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-03T13:48:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pharmacology and Toxicology","date":"2025-04-03T13:47:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pharmacology-and-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"phat","sideBox":"Learn more about [BMC Pharmacology and Toxicology](http://bmcpharmacoltoxicol.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/phat/Default.aspx","title":"BMC Pharmacology and Toxicology","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b72ff2f5-8c54-4758-a37e-7eaf41fb4fe0","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-05-22T01:38:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-07 03:22:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6193635","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6193635","identity":"rs-6193635","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}