Targeting nucleoporin NUP85 ameliorates acetaminophen-induced liver injury by inhibiting inflammation and oxidative stress in hepatocytes

Targeting nucleoporin NUP85 ameliorates acetaminophen-induced liver injury by inhibiting inflammation and oxidative stress in hepatocytes | 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 Targeting nucleoporin NUP85 ameliorates acetaminophen-induced liver injury by inhibiting inflammation and oxidative stress in hepatocytes Mengqi Han, shanshan Ge, Guitao Zhao, Panpan Yang, Zhipan Luo, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9224000/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 Liver injury induced by excessive acetaminophen (APAP) is the main cause of drug-induced liver failure, and its pathological process is closely related to oxidative stress and inflammatory response. Nucleoporin 85 (NUP85, also known as FROUNT) plays a regulatory role in liver diseases, but its function and mechanism in APAP-induced liver injury (AILI) remain unclear. This study explored the role of NUP85 and targeted intervention strategies through in vivo and in vitro experiments. The results showed that NUP85 expression was significantly upregulated in mouse and AML-12 cells after APAP treatment. Furthermore, targeted knockdown of NUP85 in vivo reduced serum transaminase expression level, alleviated liver pathological damage, decreased macrophage infiltration and inflammatory cytokines secretion, and improved oxidative stress. In vitro experiments demonstrated that silencing NUP85 attenuates inflammation via inhibition of NF‑κB signaling pathway and mitigates oxidative stress via activation of the Nrf2–Keap1 signaling pathway, while overexpression of NUP85 showed the opposite effect. It was found through virtual screening that crocin-I can specifically bind to NUP85 and inhibit its expression. In vivo experiments confirmed that crocin-I alleviates AILI in a dose-dependent manner and exerts comparable anti-inflammatory and antioxidant effects to the clinical drug N-acetylcysteine (NAC). In conclusion, NUP85 aggravates AILI by regulating the NF-κB and Nrf2-Keap1 signaling pathways. Crocin-I can target NUP85 to exert liver-protective effects, providing new targets and candidate drugs for the clinical treatment of AILI. NUP85 AILI Inflammation Oxidative stress Crocin-I Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Acetaminophen (APAP), a commonly used over-the-counter antipyretic and analgesic drug in clinical practice, has good safety at therapeutic doses[ 1 ]. However, excessive intake APAP can lead to acute liver injury, which may progress to liver failure or even death in severe cases [ 2 ]. Notably, excessive intake APAP is one of the main causes of drug-induced liver injury (DILI) worldwide. Although N-acetylcysteine (NAC) is currently the first-line drug for the clinical treatment of APAP-induced liver injury (AILI), its therapeutic window is relatively narrow and its efficacy for severe liver injury is limited [ 3 , 4 ]. Therefore, it is of great clinical significance to deeply clarify the pathogenesis of AILI, explore new therapeutic targets and develop highly effective and low-toxicity targeted drugs. The pathophysiological process of AILI is complex, among which inflammatory response and oxidative stress are the core driving factors [ 5 , 6 ]. It is worth nothing that the reactive oxygen species (ROS) produced by excessive metabolism of APAP can directly damage liver cells, simultaneously activate innate immune cells such as kupffer cells, release a large number of inflammatory cytokines, recruit neutrophils and macrophages to infiltrate the liver, form a vicious cycle of "oxidative stress - inflammation", and aggravate liver injury [ 7 ]. Therefore, screening and identifying key targets that can regulate inflammation and oxidative stress responses is expected to provide potential intervention strategies and new ideas for the clinical treatment of AILI. Nuclear pore complexes (NPCs) are large molecular complexes of the nuclear envelope that play a role in DNA repair, the structure of chromatin, and nucleocytoplasmic transport [ 8 , 9 ]. NPCs are composed of various nuclear pore proteins (NUPs), including NUP93, NUP155, and NUP85[ 10 , 11 ]. More and more evidence indicates that NUPs are associated with oxidative stress and inflammation in liver diseases[ 12 ]. For instance, NUP93 is highly expressed in hepatocellular carcinoma (HCC) tissues and correlates with poor clinical prognosis. Downregulation of NUP93 inhibits tumor cell metastasis and proliferation, whereas its upregulation promotes malignant progression [ 13 ]. In recent years, it has been documented that the knockdown of NUP85 alleviates the lipid metabolism disorder, inflammatory response and oxidative stress of liver induced by the methionine-choline-deficient (MCD) diet, exerting a protective effect on the liver [ 14 , 15 ]. In the Human Protein Atlas, NUP85 is widely expressed in various organs, especially the liver [ 16 ]. The amino acid sequence of NUP85 is highly conserved between mice and humans, making it a promising therapeutic target [ 17 ]. In addition, NUP85 is presumed to play a significant role in oxidative stress and inflammatory responses related to AILI. However, NUP85 expression characteristics, biological functions, and regulatory mechanisms in AILI remain unclear at present. Therefore, this study systematically explored the role and molecular mechanism of NUP85 in AILI through in vivo and in vitro experiments, and screened natural active drugs targeting NUP85, providing new targets and strategies for the clinical treatment of AILI. 2. Materials and methods 2.1. Reagent Anti-NUP85 (15027-1-AP), anti-P65 (66535-1-Ig), anti-IкBα (66418-1-Ig), anti-IL-1β (66737-1-Ig), anti-β-actin (66009-1-Ig), anti-TNF-α (60291-1-Ig), anti-Keap1 (10503-2-AP), anti-LaminB1 (66095-1-Ig), anti-phospho-IкBα (P-IкBα, 68999-1-Ig), and anti-IL-6 (66146-1-Ig) were purchased from Proteintech (Wuhan, China). Anti-F4/80 (sc-52664) was provided by Santa Cruz Biotechnology (CA, USA). Anti-phospho-p65 (P-P65, AF2006) and anti-Nrf2 (AF0639) were purchased from Affinity (USA). The SOD (A001-2-2), MDA (A003-1-2) and GSH (A006-2-1) kits were all provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). DMEM/F12 was purchased from HyClone (Beijing, China). Fetal bovine serum and Opti-MEM were purchased from Gibco (USA).IL-1β, IL-6, and TNF-α ELISA kits were procured from ELK Biotechnology Co., Ltd. (Wuhan, China). Reverse transcription kit and SYBR Green Premix Pro Tag HS qPCR kit were purchased from Accurate Biology (Hunan, China). Cell Counting Kit-8 (CCK-8) was obtained from Biosharp (Beijing, China). APAP, crocin-I, and NAC were purchased from Med Chem Express (Shanghai, China). Nucleus and cytoplasmic protein extraction kits were bought from Beyotime (Shanghai, China). 2.2. Animal experiments Male C57BL/6J mice (6–8 weeks old, 20–22 g body weight) were obtained from Gempharmatech Co., Ltd. Mice were maintained under controlled laboratory conditions on a standard 12 h/12 h light cycle, and all animal procedures followed the ethical guidelines outlined for the Care and Use of Laboratory Animals. Animal protocols were conducted in accordance with guidelines approved by the Experimental Animal Ethics Committee of the First Affiliated Hospital of USTC (2023-N(A)-99). After a 7-day adaptation period, the mice were randomly assigned to seven experimental groups (n = 10/group): (1) control group, (2) APAP group, (3) APAP+AAV8-empty group, (4) APAP+AAV8-shRNA-NUP85 group, (5) APAP+C10 group, (6) APAP+C40 group, (7) APAP + NAC group. The APAP + AAV8-empty group and the APAP + AAV8-shRNA-NUP85 group were respectively injected with AAV8-empty plasmid or AAV8-shRNA-NUP85 plasmid via the tail vein. The APAP+C10 and APAP+C40 groups received crocin-I for one week at doses of 10 mg/kg and 40 mg/kg, respectively. APAP + NAC group gavaged daily administration of NAC (60 mg/kg) for 7 days. After the last administration of the mice, they were fasted overnight and received a single intraperitoneal injection of acetaminophen (300 mg/kg), except for the control group mice, which injected the same volume of PBS instead. Liver tissues and serum samples were collected for additional investigation after 24 h[ 18 , 19 ]. 2.3. Cell culture The AML-12 cells were obtained from the School of Pharmacy at Anhui Medical University. AML-12 cells were cultured in DMEM/F12 containing 10% fetal bovine serum in a 37℃ incubator with 5% CO 2 atmosphere. AML-12 cells were stimulated with 10 mM APAP for 6 h to establish the AILI model. 2.4. SiRNA and plasmid transfection When the AML-12 cells reached 70% confluence, they were transfected with NC-siRNA, NUP85-siRNA, pcDNA3.1-3×Flag-c, and pcDNA3.1-3×Flag-c-NUP85 by Lipofectamine 2000. After culturing AML-12 cells in Opti-MEM for 6 h, the medium was replaced with DMEM/F12 supplemented with 10% FBS, and the cells were subsequently treated with 10 mM APAP for an additional 6 h. The sequences of NUP85-siRNA and the negative control siRNA are provided in Table S1 . 2.5. Cell proliferation assay Approximately 5 × 10³ AML-12 cells were plated into each well of 96-well plates. Following the standard procedure, the plates were placed in a cell culture incubator for incubation. Following a 3 h cell adherence period, the cells were incubated with crocin-I at a range of concentrations (0, 2.5, 5, 10, 20, 40, and 80 µM) for 24 h. Subsequently, they were exposed to APAP for an additional 6 h. Following this treatment, the culture medium in each well was replaced with 90 µL of fresh media supplemented with 10 µL of CCK-8 reagent. Following a 1 h incubation period, absorbance readings were taken at 450 nm using a microplate reader (SpectraMax iD3, Molecular Devices, USA). 2.6. Biochemical analysis Serum samples were obtained by centrifuging whole blood at 2000 × g for 15 min at 4°C. Serum ALT and AST activities were quantified using commercially available enzymatic assay kits following the manufacturer’s protocols. 2.7. ELISA assay ELISA assay was used to detect the expression levels of three inflammatory cytokines: IL-1β, TNF-α, and IL-6. The experimental procedures were conducted in strict accordance with the detailed instructions provided in the kit's manual. Absorbance values were subsequently measured at 450 nm using a microplate reader. The concentrations of cytokines in the test samples were quantified based on a standard curve. 2.8. Antioxidant Enzymes and Lipid Peroxidation Assay The levels of molecules related to oxidative stress in liver tissues and AML-12 cells were detected by using SOD, GSH, and MDA kits, and the experimental steps were strictly carried out in accordance with the operation guidelines of the kit instructions. 2.9. Hematoxylin and eosin (H&E) staining Mice liver tissue sections (5 µm thick) were deparaffinized in xylene and rehydrated through a graded ethanol series (100%, 95%, and 70%). Nucleus were stained with hematoxylin for 5 min, followed by rinsing in running tap water. Sections were counterstained with eosin for 2 min and dehydrated through an ascending ethanol series (70%, 95%, and 100%) [ 20 ]. Following coverslipping, tissue sections were scanned using a slide scanner (Panoramic MIDI II, 3DHISTECH, Hungary) and subsequently analyzed with CaseViewer software (version 2.4, 3DHISTECH, Hungary) for histopathological assessment. Based on previous studies, the degree of liver injury in mice is quantified and analyzed by assessing the degree of inflammatory cell infiltration and the extent of tissue necrosis [ 21 ]. 2.10. Immunohistochemistry (IHC) Liver tissue sections underwent standard dewaxing and rehydration processes. Antigen retrieval was then performed by heat-mediated epitope restoration in citrate buffer (0.01 M, pH 6.0) maintained at 95°C for 20 min. Following this, endogenous peroxidase activity was effectively blocked through 15 min incubation in 3% hydrogen peroxide at ambient temperature. To minimize nonspecific antibody binding, tissue sections were blocked with 3% bovine serum albumin for 1 h at room temperature. Primary antibodies targeting NUP85 (1:300) and F4/80 (1:100) were applied for overnight incubation at 4°C, followed by a 1 h incubation with species-matched horseradish peroxidase (HRP)-conjugated secondary antibodies at 37°C. Chromogenic visualization was performed using diaminobenzidine (DAB), and nuclei were counterstained with hematoxylin. Finally, slices were dehydrated through an ethanol series, cleared in xylene, and permanently mounted with a neutral resinous medium. 2.11. Real-time fluorescence quantitative PCR Total RNA was extracted from cells and tissue specimens using TRIzol according to the manufacturer's instructions. The RNA was reverse-transcribed into cDNA using the reverse transcription kit, and then real-time fluorescence quantitative PCR (RT-qPCR) was performed using the SYBR Green Premix Pro Tag HS qPCR kit. GAPDH served as the endogenous reference gene, and relative gene expression levels were calculated using the comparative 2 −∆∆ct method. Primer sequences are listed in Table S2. 2.12. Western blotting Liver tissues or cells were homogenized in RIPA buffer containing a mixture of protease and phosphatase inhibitors. Protein extracts were loaded onto 10% acrylamide gels, electrophoresed and transferred to polyvinylidenedifluoride (PVDF) membranes. Membranes were incubated with specific antibodies, followed by secondary antibodies conjugated to horseradish peroxidase. The primary antibody included NUP85 (1:1000), p-P65 (1:1000), P65 (1:1000), TNF-α (1:1000), IкBα (1:6000), P-IкBα (1:3000), IL-6 (1:1000), IL-1β (1:1000), Keap1 (1:1000), Nrf2 (1:10000), LaminB1 (1:6000), and β-actin (1:6000). Finally, the grayscale values of the protein bands were analyzed using ImageJ software (version 1.8.0; National Institutes of Health) to determine the relative expression levels of the target proteins. 2.13. Molecular docking Molecular docking of crocin-I (Pub Chem CID: 5281233) to protein NUP85 (Uniprot ID: Q8R480) using Auto Dock Vina 1.1.2 software. Protein pre-processing was accomplished using PyMol 2.4. Auto Dock Tools 1.5.6 was used to generate PDBQT files for the docking simulation. The docking conformation with the lowest binding energy and highest clustering frequency is considered to be the most potent binding mode between ligand and protein. Finally, the docking results were visualized using PLIP and PyMOL 2.4 software. 2.14. Cellular thermal shift assay (CETSA) AML-12 cells were divided into two groups for the experiment: one group received 20 µM crocin-I treatment for 6 h, and the other acted as a control with no crocin-I administration. Following this, the cells were collected and subjected to three freeze-thaw cycles using liquid nitrogen. The cell lysates were then centrifuged at 20 000 × g for 30 min to obtain the supernatants. Each set of supernatants was divided into six aliquots and heated at different temperatures (37, 45, 50, 55, 60, and 65°C) for 3 min. Finally, the expression levels of NUP85 in the samples were analyzed by western blotting[ 22 ]. 2.15. Statistical analysis At least three replicate experiments verified all findings in this study. Data are expressed as mean ± standard error of the mean (SEM). One-way ANOVA was employed to assess inter-group differences. Statistical significance was analyzed using GraphPad Prism version 9.0, with significance levels defined as * P < 0.05, ** P < 0.01, and *** P < 0.001. 3. Results 3.1. NUP85 is upregulated in AILI mice To assess the effects of APAP overdose in mice, a dose of 300 mg/kg was administered intraperitoneally for 24 h. Macroscopic examination revealed that livers in the APAP group exhibited increased volume and a paler colouration compared to those in the control group, indicating significant morphological changes (Fig. 1 A). Additionally, serum levels of ALT and AST were significantly elevated in the APAP group (Fig. 1 B). Histological examination via H&E staining revealed that the liver tissues in the control group exhibited intact lobular architecture with no evidence of pathological alterations. In contrast, the liver of APAP-treated mice demonstrated marked inflammatory cell infiltration and hepatocellular necrosis (Fig. 1 C and 1 E). The above results indicated that the AILI model has been successfully established. To explore the correlation between NUP85 and AILI, NUP85 expression in liver tissues from APAP-treated mice was examined. NUP85 was highly upregulated in the APAP group, as shown by IHC staining, western blotting, and RT-qPCR analysis (Fig. 1 D and 1 F-H). All of these results suggested that NUP85 expression level was significantly elevated in mice administered with APAP, which may exacerbate liver injury. 3.2. NUP85 knockdown alleviates liver injury in AILI mice To investigate the biological role of NUP85 in AILI, we established a mouse model of NUP85 gene silencing via tail vein injection of AAV8-shRNA-NUP85. The results of western blotting, RT-qPCR, and immunohistochemical analysis revealed that this intervention significantly downregulated the protein and mRNA expression levels of NUP85 in the mouse liver (Fig. 2 A-E). Specifically, the expression levels of serum ALT and AST were significantly decreased (Fig. 2 F), and the degree of liver injury was significantly alleviated as shown by H&E staining of liver tissue (Fig. 2 D and 2 G). Moreover, immunohistochemical analysis revealed a significant reduction in the number of F4/80-positive macrophage infiltrations in the liver (Fig. 2 D and 2 H). To further verify its mechanism, we examined the markers associated with inflammation and oxidative stress. The results demonstrated that in the AAV8-shRNA-NUP85 group, the expression levels of inflammatory cytokines (IL-6, IL-1β, and TNF-α) were significantly downregulated in both the serum and liver tissues of mice (Fig. 2 I-J). Additionally, the MDA content in liver tissues was markedly reduced, whereas the activities of SOD and the levels of GSH were significantly enhanced (Fig. 2 K). The above results suggest that silencing of NUP85 can significantly improve AILI by inhibiting inflammatory responses and reducing oxidative stress, indicating that NUP85 may be a potential intervention target for the prevention and treatment of AILI. 3.3. NUP85 is elevated in AILI AML-12 cells To investigate the effects of APAP treatment on NUP85 expression in AML-12 cells, we examined changes in NUP85 expression following exposure to various concentrations of APAP for 6 h. The results indicated dose-dependent upregulation of NUP85 expression, with a significant increase at a dose of 10 mM (Fig. 3 A). The maximal NUP85 protein expression was observed 6 h following treatment with 10 mM APAP (Fig. 3 B). Based on these findings, all in vitro studies were performed with 10 mM APAP for a duration of 6 h. Western blotting and RT-qPCR assays revealed that the protein and mRNA levels of NUP85 were significantly reduced in the NUP85-siRNA group relative to the NC-siRNA group (Fig. 3 C and D). Conversely, NUP85 transfection with pcDNA3.1-3×Flag-c-NUP85 resulted in elevated NUP85 expression levels (Fig. 3 E and F). These results indicated that NUP85 was successfully knocked down and overexpressed in AML-12 cells after transfection with NUP85-siRNA and pcDNA3.1-3×Flag-c-NUP85, respectively. 3.4. NUP85 knockdown attenuates AILI by inhibiting NF-кB signaling pathway Following the effective knockdown and overexpression of NUP85, we investigated its impact on the expression of APAP-induced inflammatory cytokines (IL-6, IL-1β, and TNF-α) in AML-12 cells. The results indicated that the protein and mRNA levels of these inflammatory cytokines were significantly upregulated in APAP-treated AML-12 cells. After transfection of NUP85-siRNA, the expression of these cytokines were actually reduced (Fig. 4 A and B). On the contrary, the results showed that inflammatory cytokine expression levels were significantly upregulated in AML-12 cells overexpressing NUP85 (Fig. 4 C and D). These findings indicated that disruption of NUP85 attenuates inflammatory responses in AML-12 cells treated with APAP. The NF-κB signaling pathway was shown to be responsible for playing a crucial role in the inflammatory response of AILI [ 23 , 24 ][ 24 – 25 ]. To elucidate the mechanism by which NUP85 influences inflammation, we investigated the expression levels of key proteins involved in this signaling pathway. Western blotting result revealed that NUP85 knockdown inhibited the phosphorylation of P65 and IκBα. Compared to the NC-siRNA group, P65 was downregulated in the nucleus and upregulated in the cytoplasm in the NUP85-siRNA group, indicating that NUP85 knockdown inhibited P65 nuclear translocation (Fig. 4 E and 4 G-H). Conversely, overexpression of NUP85 resulted in enhanced phosphorylation of P65 and IκBα, thereby promoting the nuclear translocation of P65 (Fig. 4 F and 4 I-J). Collectively, these findings indicated that NUP85 plays a critical role in modulating NF-κB signaling pathway in AML-12 cells treated with APAP. 3.5. NUP85 knockdown alleviates oxidative stress in the liver of AILI by activating Nrf2-Keap1 signaling pathway To investigate the effect of NUP85 on oxidative stress in AML-12 cells, and the contents of MDA, SOD and GSH within the cells were detected. The results showed that, compared with the NC-siRNA group, NUP85 knockdown reduced the MDA content, while elevating the SOD activity and GSH content (Fig. 5 A). Conversely, NUP85 overexpression increased the MDA content and decreased both SOD activity and GSH content (Fig. 5 B). To further reveal its molecular mechanism, western blotting was used to detect the expression of proteins related to the Nrf2-Keap1 signaling pathway. The results indicated that in APAP-treated AML-12 cells, NUP85 knockdown significantly upregulated the Nrf2 protein expression and downregulated the Keap1 protein expression (Fig. 5 C and 5 E). In addition, compared with the NC-siRNA group, NUP85 knockdown promoted the nuclear translocation of Nrf2, as indicated by its increased expression in the nucleus and the decreased expression in the cytoplasm (Fig. 5 C and 5 F). Conversely, NUP85 overexpression significantly suppressed the activation of the Nrf2-Keap1 signaling pathway (Fig. 5 D, 5 G and 5 H). In summary, these results suggested that knockdown of NUP85 at the cellular level can effectively alleviate APAP-induced oxidative stress injury by activating the Nrf2-Keap1 signaling pathway. 3.6. Crocin-I targets NUP85 and inhibits its expression Further, we utilized virtual screening to identify potential inhibitors targeting NUP85 from a natural product database. Crocin-I was identified as a compound exhibiting specific binding affinity for NUP85. Molecular docking studies revealed that the binding pocket of NUP85 contains multiple hydrophobic amino acid residues, enabling crocin-I to interact with NUP85 via hydrophobic interactions and hydrogen bonding. Notably, crocin-I formed 14 hydrogen bonds with NUP85, suggesting that hydrogen bonding plays a critical role in stabilizing the interaction (Fig. 6 A). To assess the pharmacological effects of crocin-I, we performed a CCK-8 assay, which showed that crocin-I at 20 µM exhibited the most significant protective effect on AML-12 cells treated with APAP (Fig. 6 B). In vitro , we conducted a detailed investigation into the binding interaction between crocin-I and NUP85 at a concentration of 20 µM. The CETSA result showed that the NUP85 protein expression level gradually decreased with increasing temperature. However, in the crocin-I group, NUP85 protein expression was higher than in the control group at the same temperature, suggesting that the binding of crocin-I enhanced the stability of the NUP85 protein (Fig. 6 C). Additionally, IHC staining and RT-qPCR demonstrated that crocin-I treatment significantly reduced NUP85 expression levels (Fig. 6 D-E). These results confirmed that crocin-I may act as a targeted inhibitor of NUP85. 3.7. Crocin-I alleviates liver injury in AILI mice To evaluate the therapeutic potential of crocin-I in AILI, we orally administered crocin-I to C57BL/6J mice for 7 days prior to APAP treatment. Histological analysis indicated that compared with the APAP group, crocin-I (10 and 40 mg/kg) and NAC (60 mg/kg) significantly reduced the inflammatory cell infiltration and necrosis in liver tissues (Fig. 7 A and B). Moreover, crocin-I treatment significantly lowered serum ALT and AST levels in a dose-dependent manner (Fig. 7 C). These findings suggested that crocin-I offers protective effects against AILI, with efficacy comparable to NAC. 3.8. Crocin-I alleviates inflammation and oxidative stress in AILI mice To investigate the potential inhibitory effect of crocin-I on macrophage infiltration in damaged liver tissue, this study utilized F4/80 as a specific marker for immunohistochemical analysis. The results showed that the number of F4/80-positive macrophages was significantly reduced in the livers of mice administered crocin-I or NAC compared with the APAP-treated group (Fig. 8 A and B). The results of the RT-qPCR and western blotting showed that crocin-I inhibited the increase in IL-6, TNF-α, and IL-1β levels in the liver that was caused by APAP (Fig. 8 C and D). Compared to the group that received only APAP, the treated groups demonstrated significantly lower levels of IL-6, TNF-α, and IL-1β in serum, indicating that crocin-I significantly decreased systemic inflammation (Fig. 8 E). In addition, crocin-I reduced the MDA content in the liver induced by APAP, while increasing the activities of SOD and the content of GSH (Fig. 8 F). Therefore, crocin-I has an antioxidant stress effect on AILI. 4. Discussion DILI, particularly that induced by APAP, has emerged as a major challenge in global public health owing to its high incidence and potential fatality[ 2 ]. After excessive APAP, the oxidative stress and inflammatory response mediated by N-acetyl-p-benzoquinone imine (NAPQI) interweave with each other, forming a vicious cycle, which is the core pathological link leading to massive necrosis of the liver [ 25 ]. Currently, the NAC relied upon in clinical practice has problems such as limited efficacy and adverse reactions [ 3 ], while the pathological mechanism of AILI is complex, involving multiple aspects such as immune cell infiltration, inflammatory cytokine release, and imbalance of the antioxidant system[ 26 , 27 ]. Therefore, it is currently urgent to develop new therapeutic drugs targeting the key pathogenic factors. This study focuses on the potential target NUP85 and delves deeply into its role in AILI, providing new ideas for resolving the clinical treatment predicament. NUPs as a core component of the nuclear pore complex, in addition to their classic nuclear cytoplasmic transport function, have been proven to be widely involved in pathological processes such as inflammatory regulation and metabolic disorders in recent years [ 8 , 12 ]. Current studies have shown that NUP85 can alleviate abnormal lipid metabolism in the liver by regulating inflammatory responses [ 15 ], and it is highly expressed in the liver [ 25 ], suggested that NUP85 may play an important regulatory role in liver diseases. Notably, this study is the first to report that NUP85 mRNA and protein expression are significantly upregulated in mouse liver and AML-12 cells in the AILI model, with its expression positively correlated with the severity of liver injury. Moreover, both in vivo and in vitro experiments demonstrated that NUP85 silencing, achieved via AAV8-mediated knockdown in mice, markedly reduces serum ALT/AST levels, alleviates liver inflammatory infiltration and liver cells necrosis, and effectively inhibits APAP-induced inflammatory cytokine release and oxidative stress damage, collectively indicating a critical role of NUP85 in AILI progression. The above results indicated that NUP85 is highly expressed in AILI. Meanwhile, NUP85 high expression aggravates liver injury by amplifying inflammation and oxidative stress responses, clarifying the potential value of NUP85 as a therapeutic target for AILI. NF-κB signaling pathway is the core signaling pathway regulating inflammatory responses [ 23 ]. Notably, the activation of the NF-κB signaling pathway can promote the transcription of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, and aggravate liver injury by amplifying the inflammatory cascade reaction [ 28 ]. In addition, in the resting state of the NF-κB signaling pathway, the P65 subunit binds to the inhibitory protein IκBα to form a heterodimer, which is stably located in the cytoplasm. When the NF-κB signaling pathway is activated, IκBα is phosphorylated and degraded by the proteasome. Meanwhile, the released P65 subunit undergoes nuclear translocation and binds to the promoter region of the target gene, thereby initiating the transcriptional process of downstream pro-inflammatory cytokines and related genes [ 29 ]. Current studies have shown that the down-regulation of NUP85 can exert anti-tumor and anti-proliferative effects by inhibiting the activation of the NF-κB signaling pathway [ 30 ]. This study found that NUP85 silencing could significantly inhibit APAP-induced phosphorylation of IκBα and P65, reduce nuclear translocation of P65, while overexpression of NUP85 promoted the activation of the NF-κB signaling pathway. Therefore, NUP85 can promote the release of pro-inflammatory cytokines and the infiltration of inflammatory cells by activating the NF-κB signaling pathway, thereby exacerbating the inflammatory damage of AILI. Nrf2-Keap1 signaling pathway is a key defense system for cells against oxidative stress [ 31 ]. Under normal physiological conditions, Nrf2 binds to Keap1 and is ubiquitinized and degraded. Additionally, under oxidative stress conditions, Nrf2 dissociates from the Keap1 nucleus and translocates, initiating the expression of antioxidant genes such as SOD, GSH, and MDA [ 1 , 32 ]. Previous studies have demonstrated that promoting the activation of the Nrf2 signaling pathway can effectively alleviate liver injury induced by APAP [ 33 ]. In this study, NUP85 silencing could significantly up-regulate the expression level of Nrf2, promoting its nuclear translocation, and simultaneously increase intracellular SOD activity and GSH content, while reducing the accumulation of lipid peroxidation product MDA. Conversely, NUP85 overexpression inhibited the activation of the Nrf2-Keap1 signaling pathway, which is consistent with the research conclusion that NUPs regulated the nuclear transport of Nrf2 [ 34 ]. Therefore, it is suggested that NUP85 weakens the antioxidant capacity of liver cells by inhibiting the Nrf2-Keap1 signaling pathway, thereby aggravating AILI-induced oxidative stress injury. In summary, NUP85 promotes the occurrence and development of AILI through a dual mechanism of activating the NF-κB inflammatory signaling pathway and inhibiting the Nrf2-Keap1 antioxidant signaling pathway. Natural products have become an important resource for the research and development of AILI therapeutic drugs due to their advantages of multiple targets and low toxicity [ 35 , 36 ]. Crocin-I, as the main active component of saffron, has been confirmed to have various pharmacological activities such as anti-inflammation, anti-oxidation and neuroprotection [ 37 – 39 ], but its therapeutic potential in AILI remains unclear. This study identified via virtual screening that crocin-I specifically binds to NUP85, with molecular docking analysis revealing that the two molecules form a stable complex through hydrophobic interactions and hydrogen bonds. Furthermore, the CETSA experiment confirmed that crocin-I could enhance the stability of NUP85 protein, and in vivo experiments indicated that it could significantly down-regulate the NUP85 expression. Further studies have shown that pretreatment with crocin-I can dose-dependently reduce the serum transaminase level in mice induced by APAP, alleviate liver inflammatory infiltration and oxidative stress injury, and its therapeutic effect is comparable to that of NAC. The above results indicated that crocin-I exerts a protective effect on AILI by targeting and inhibiting the NUP85 expression and regulating the NF-κB and Nrf2-Keap1 signaling pathways, providing a new candidate drug for the clinical treatment of AILI. In conclusion, this study has for the first time confirmed the high expression of NUP85 in AILI. Meanwhile, NUP85 exacerbates liver inflammatory responses and oxidative stress damage by activating the NF-κB inflammatory signaling pathway and inhibiting the Nrf2-Keap1 antioxidant signaling pathway. Additionally, the natural active ingredient crocin-I can target NUP85 and inhibit its expression, effectively alleviating the pathological damage of AILI, and its therapeutic effect is comparable to that of the clinical drug NAC. This study not only revealed the pathological function and regulatory network of NUP85 in AILI but also clarified the feasibility of NUP85 as a therapeutic target for AILI and the potential application value of crocin-I, providing new theoretical basis and therapeutic strategies for the clinical prevention and treatment of AILI. Although this study has clarified the pathogenic effect and regulatory mechanism of NUP85 in AILI, it is still necessary to further explore the specific binding sites and selective action mechanisms of crocin-I and NUP85 to provide a molecular basis for drug optimization. Abbreviations APAP Acetaminophen AILI APAP-induced liver injury NUP85 Nucleoporin 85 DILI drug-induced liver injury NAC N-acetylcysteine NPC Nuclear pore complexes NUPs nuclear pore proteins IL-1β interleukin-1beta TNF-α tumor necrosis factor-alpha IL-6 interleukin-6 ALT Alanine aminotransferase AST aspartate aminotransferase ELISA enzyme-linked immunosorbent assay CETSA Cellular thermal shift assay H&E Hematoxylin and eosin IHC Immunohistochemistry MCD methionine-choline-deficient NAPQI N-acetyl-p-benzoquinone imine HCC hepatocellular carcinoma ROS reactive oxygen species Declarations Funding This study is supported by the National Natural Science Foundation of China (82373932); Outstanding Youth Talents Fund Project of Anhui Province (2023AH030114); Key Research and Development Project of Anhui Provincial (202204295107020023); Department of Education of Anhui Province Outstanding Young Teacher Training Project (YQZD2023023); Natural Science Project of Anhui Province (2208085MH252); Anhui University Outstanding Youth Program (2024AH020006); Natural Science Research Project of Anhui Educational Committee (2022AH050754); 2025 School-Level Tutoring & Ideological and Political Studio(0601067124); School-Level College Students' Innovative Training Program (0601067120,0601067122); 2024 Provincial Quality Project – School-Enterprise Cooperative Practical Education Base (0601067123) . CRediT authorship contribution statement Mengqi Han : Writing - original draft, Methodology, Investigation, Formal analysis. Shanshan Ge : Methodology, Investigation, Formal analysis. Guitao Zhao : Methodology, Investigation, Data curation. Pan-pan Yang and Zhipan Luo : Validation, Software, Investigation. Cheng Qian : Investigation, Formal analysis. Linxin Pan : Methodology, Data curation. Peng Fu : Conceptualization, Writing - review & editing. Tao Xu : Project administration, Funding acquisition. Zhaolin Chen : Writing - review & editing, Funding acquisition, Conceptualization. Declaration of competing interest The authors declare no competing interests. Data A v ailability Data will be made available on request. Clinical trial number Not applicable. References Zhang, J., M. Li, T. Zhao, J. Cao, Y. Liu, and Y. Wang et al. 2022. E Se tea alleviates acetaminophen-induced liver injury by activating the Nrf2 signaling pathway. Food & function 13:7240–7250. Wang, Y., L. Tian, Y. Wang, T. Zhao, A. Khan, and Y. Wang et al. 2021. Protective effect of Que Zui tea hot-water and aqueous ethanol extract against acetaminophen-induced liver injury in mice via inhibition of oxidative stress, inflammation, and apoptosis. Food & function 12:2468–2480. Tang, C., L. Cen, H. Zeng, X. Zhang, P. Liu, and Y. Chen et al. 2024. Inhibiting Hepatocyte Uric Acid Synthesis and Reabsorption Ameliorates Acetaminophen-Induced Acute Liver Injury in Mice. Cellular and molecular gastroenterology and hepatology 17:251–265. Chowdhury, A., J. Nabila, I. Adelusi Temitope, and S. Wang. 2020. Current etiological comprehension and therapeutic targets of acetaminophen-induced hepatotoxicity. Pharmacological research 161:105102. Li, H., Q. Weng, S. Gong, W. Zhang, J. Wang, and Y. Huang et al. 2023. Kaempferol prevents acetaminophen-induced liver injury by suppressing hepatocyte ferroptosis via Nrf2 pathway activation. Food & function 14:1884–1896. Li, Q., W. Zhang, N. Cheng, Y. Zhu, H. Li, and S. Zhang et al. 2023. Pectolinarigenin ameliorates acetaminophen-induced acute liver injury via attenuating oxidative stress and inflammatory response in Nrf2 and PPARa dependent manners. Phytomedicine: international journal of phytotherapy and phytopharmacology 113:154726. Yu, Q., J. Zhang, J. Li, Y. Song, J. Pan, and C. Mei et al. 2024. Sirtuin 5-Mediated Desuccinylation of ALDH2 Alleviates Mitochondrial Oxidative Stress Following Acetaminophen-Induced Acute Liver Injury. Advanced science (Weinheim, Baden-Wurttemberg, Germany). ; 11:e2402710. Beck, M., and E. Hurt. 2017. The nuclear pore complex: understanding its function through structural insight. Nature reviews Molecular cell biology 18:73–89. Nakano, H., W. Wang, C. Hashizume, T. Funasaka, H. Sato, and R. W. Wong. 2011. Unexpected role of nucleoporins in coordination of cell cycle progression. Cell cycle (Georgetown Tex) 10:425–433. Coyne, A. N., and J. D. Rothstein. 2022. Nuclear pore complexes - a doorway to neural injury in neurodegeneration. Nature reviews Neurology 18:348–362. Nord, M. S., C. Bernis, S. Carmona, D. C. Garland, A. Travesa, and D. J. Forbes. 2020. Exportins can inhibit major mitotic assembly events in vitro: membrane fusion, nuclear pore formation, and spindle assembly. Nucleus (Austin Tex) 11:178–193. Holzer, K., A. Ori, A. Cooke, D. Dauch, E. Drucker, and P. Riemenschneider et al. 2019. Nucleoporin Nup155 is part of the p53 network in liver cancer. Nature communications 10:2147. Lin, C. S., Y. Liang, S. G. Su, Y. L. Zheng, X. Yang, and N. Jiang et al. 2022. Nucleoporin 93 mediates β-catenin nuclear import to promote hepatocellular carcinoma progression and metastasis. Cancer letters 526:236–247. Wu, Y. C., Q. Yan, S. Q. Yue, L. X. Pan, D. S. Yang, and L. S. Tao et al. 2024. NUP85 alleviates lipid metabolism and inflammation by regulating PI3K/AKT signaling pathway in nonalcoholic fatty liver disease. International journal of biological sciences 20:2219–2235. Zhang, Z., S. Li, Y. Zhang, R. Xu, J. Zhou, and Y. Wang et al. 2025. NUP85 siRNA loaded red blood cell-derived extracellular vesicles alleviate hepatic steatosis in MASLD. Journal of nanobiotechnology 24:63. Taniuchi, K., T. Yawata, M. Tsuboi, T. Ueba, and T. Saibara. 2019. Efficient delivery of small interfering RNAs targeting particular mRNAs into pancreatic cancer cells inhibits invasiveness and metastasis of pancreatic tumors. Oncotarget 10:2869–2886. Terashima, Y., N. Onai, M. Murai, M. Enomoto, V. Poonpiriya, and T. Hamada et al. 2005. Pivotal function for cytoplasmic protein FROUNT in CCR2-mediated monocyte chemotaxis. Nature immunology 6:827–835. Yuan, K., X. Zhang, L. Lv, J. Zhang, W. Liang, and P. Wang. 2016. Fine-tuning the expression of microRNA-155 controls acetaminophen-induced liver inflammation. International immunopharmacology 40:339–346. Zhu, Y., L. Lei, X. Wang, L. Chen, W. Li, and J. Li et al. 2023. The E3 ubiquitin ligase NEDD4-1 protects against acetaminophen-induced liver injury by targeting VDAC1 for degradation. Acta pharmaceutica Sinica B 13:1616–1630. Zeng, Y., R. Wu, F. Wang, S. Li, L. Li, and Y. Li et al. 2023. Liberation of daidzein by gut microbial β-galactosidase suppresses acetaminophen-induced hepatotoxicity in mice. Cell host & microbe 31:766–780e7. Elshal, M., and M. E. Abdelmageed. 2022. Diacerein counteracts acetaminophen-induced hepatotoxicity in mice via targeting NLRP3/caspase-1/IL-1β and IL-4/MCP-1 signaling pathways. Archives of pharmacal research 45:142–158. Martinez Molina, D., R. Jafari, M. Ignatushchenko, T. Seki, E. A. Larsson, and C. Dan et al. 2013. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay . vol. 341 84–87. New York, NY: Science. Barnabei, L., E. Laplantine, W. Mbongo, F. Rieux-Laucat, and R. Weil. 2021. NF-κB: At the Borders of Autoimmunity and Inflammation. Frontiers in immunology 12:716469. Yang, R., C. Song, J. Chen, L. Zhou, X. Jiang, and X. Cao et al. 2020. Limonin ameliorates acetaminophen-induced hepatotoxicity by activating Nrf2 antioxidative pathway and inhibiting NF-κB inflammatory response via upregulating Sirt1. Phytomedicine: international journal of phytotherapy and phytopharmacology 69:153211. Cai, X., H. Cai, J. Wang, Q. Yang, J. Guan, and J. Deng et al. 2022. Molecular pathogenesis of acetaminophen-induced liver injury and its treatment options. Journal of Zhejiang University Science B 23:265–285. Zhao, R., Q. Zhang, W. Liu, Y. Lin, Y. He, and D. Chang et al. 2023. Pien Tze Huang attenuated acetaminophen-induced liver injury by autophagy mediated-NLRP3 inflammasome inhibition. Journal of ethnopharmacology 311:116285. Wang, Q., X. Peng, Y. Chen, X. Tang, Y. Qin, and M. He et al. 2023. Piezo1 alleviates acetaminophen-induced acute liver injury by activating Nrf2 and reducing mitochondrial reactive oxygen species. Biochemical and biophysical research communications 652:88–94. Jiang, W. P., J. S. Deng, S. S. Huang, S. H. Wu, C. C. Chen, and J. C. Liao et al. 2021. Sanghuangporus sanghuang Mycelium Prevents Paracetamol-Induced Hepatotoxicity through Regulating the MAPK/NF-κB, Keap1/Nrf2/HO-1, TLR4/PI3K/Akt, and CaMKKβ/LKB1/AMPK Pathways and Suppressing Oxidative Stress and Inflammation. Antioxidants (Basel, Switzerland). ; 10. Lawrence, T. 2009. The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harbor perspectives in biology 1:a001651. Satoh, M., T. Akatsu, Y. Ishkawa, Y. Minami, and M. Nakamura. 2007. A Novel Activator of C-C Chemokine, FROUNT, is Expressed With C-C Chemokine Receptor 2 and its Ligand in Failing Human Heart. Journal of Cardiac Failure 13:114–119. Kobayashi, M., and M. Yamamoto. 2005. Molecular mechanisms activating the Nrf2-Keap1 pathway of antioxidant gene regulation. Antioxidants & redox signaling 7:385–394. Shen, Z., Y. Wang, Z. Su, R. Kou, K. Xie, and F. Song. 2018. Activation of p62-keap1-Nrf2 antioxidant pathway in the early stage of acetaminophen-induced acute liver injury in mice. Chemico-biological interactions 282:22–28. Lv, H., L. Hong, Y. Tian, C. Yin, C. Zhu, and H. Feng. 2019. Corilagin alleviates acetaminophen-induced hepatotoxicity via enhancing the AMPK/GSK3β-Nrf2 signaling pathway. Cell communication and signaling: CCS 17:2. Yoshimura, S. H., S. Otsuka, M. Kumeta, M. Taga, and K. Takeyasu. 2013. Intermolecular disulfide bonds between nucleoporins regulate karyopherin-dependent nuclear transport. Journal of cell science 126:3141–3150. Gouda, N. A., S. O. Alshammari, M. A. S. Abourehab, Q. A. Alshammari, and A. Elkamhawy. 2023. Therapeutic potential of natural products in inflammation: underlying molecular mechanisms, clinical outcomes, technological advances, and future perspectives. Inflammopharmacology 31:2857–2883. Yan, M., L. Ye, S. Yin, X. Lu, X. Liu, and S. Lu et al. 2018. Glycycoumarin protects mice against acetaminophen-induced liver injury predominantly via activating sustained autophagy. British journal of pharmacology 175:3747–3757. Zhang, L., M. Jing, and Q. Liu. 2021. Crocin alleviates the inflammation and oxidative stress responses associated with diabetic nephropathy in rats via NLRP3 inflammasomes. Life sciences 278:119542. Zhu, A., C. Lao, Z. Wang, Y. Chen, and C. Bai. 2019. Characterization of Crocetin-Monoglucuronide as a Neuron-Protective Metabolite of Crocin-1. Molecular nutrition & food research 63:e1900024. Xiao, Q., Z. Xiong, C. Yu, J. Zhou, Q. Shen, and L. Wang et al. 2019. Antidepressant activity of crocin-I is associated with amelioration of neuroinflammation and attenuates oxidative damage induced by corticosterone in mice. Physiology & behavior 212:112699. 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-9224000","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":620094256,"identity":"2bc29e24-a5b8-41dd-aec2-01b1b17822ab","order_by":0,"name":"Mengqi Han","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mengqi","middleName":"","lastName":"Han","suffix":""},{"id":620094257,"identity":"82d0026f-4b20-4cfa-9805-05bbbc398f57","order_by":1,"name":"shanshan Ge","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"shanshan","middleName":"","lastName":"Ge","suffix":""},{"id":620094258,"identity":"d2c34d8d-0da3-42e7-97f0-58973c79fda2","order_by":2,"name":"Guitao Zhao","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guitao","middleName":"","lastName":"Zhao","suffix":""},{"id":620094259,"identity":"edb261fb-0384-4dd3-8b16-768dcdbc22a8","order_by":3,"name":"Panpan Yang","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Panpan","middleName":"","lastName":"Yang","suffix":""},{"id":620094260,"identity":"751be709-66df-47d1-b4c1-a4d2e947b47f","order_by":4,"name":"Zhipan Luo","email":"","orcid":"","institution":"First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhipan","middleName":"","lastName":"Luo","suffix":""},{"id":620094261,"identity":"2f7d4e6b-c625-48e2-9127-0696c0c5e0ec","order_by":5,"name":"Cheng Qian","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Qian","suffix":""},{"id":620094262,"identity":"efc52b93-4017-4df9-8bcc-c265014c4d39","order_by":6,"name":"Linxin Pan","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Linxin","middleName":"","lastName":"Pan","suffix":""},{"id":620094263,"identity":"db49b830-97ed-46c6-a5c2-16b6e67f3175","order_by":7,"name":"Peng Fu","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Fu","suffix":""},{"id":620094264,"identity":"476826d0-e39f-4354-833d-7966d133cb9b","order_by":8,"name":"Tao Xu","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Xu","suffix":""},{"id":620094265,"identity":"cc341c37-6865-4800-84ac-567f44ef24bd","order_by":9,"name":"Zhaolin Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYFAC5oYDHwxs5GA8YrQwNj6cUZFmDFVNnJZmY54zhxMbiNbC33+wTXJmW1r6hvPnD35gqLBObGA/ewCvFokDB9skPrbZ5G64kcwswXAmPbGBJy8BrxYDxkawLUAtzGwMjG1AF0rwGODXwszYJs3bdjjd4PxhoJZ/xGhhg3g/weBAMlBLAxFaJM5AAtlw5o1kY4mEY+nGbTw5+LXw9x8+AIpKeb7zBx9++FBjLdvPfga/FlSQAMRsJKgfBaNgFIyCUYADAADlw0dIS5tqmwAAAABJRU5ErkJggg==","orcid":"","institution":"University of Science and Technology of China","correspondingAuthor":true,"prefix":"","firstName":"Zhaolin","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2026-03-25 13:54:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9224000/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9224000/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106760777,"identity":"452dd8ca-e3e8-4748-b2fd-2d487fa47349","added_by":"auto","created_at":"2026-04-13 08:50:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":853468,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression level of NUP85 is elevated in AILI mice (\u003cstrong\u003eA\u003c/strong\u003e) Representative images of liver tissues. (\u003cstrong\u003eB\u003c/strong\u003e) Serum ALT and AST levels in control and model mice. (\u003cstrong\u003eC\u003c/strong\u003e) H\u0026amp;E staining revealing liver histopathology (defective areas indicated by white circles) in both control and model mice. Scale bar = 50 µm. (\u003cstrong\u003eD\u003c/strong\u003e) Representative immunohistochemical staining of NUP85 in liver tissue of mice in the control group and the model group. Scale bar = 50 µm. (\u003cstrong\u003eE\u003c/strong\u003e) Histopathological scores of liver tissues based on H\u0026amp;E staining in different groups. (\u003cstrong\u003eF\u003c/strong\u003e) Quantitative analysis of NUP85 positive expression in livers of different groups. (\u003cstrong\u003eG\u003c/strong\u003e) Western blotting analysis of NUP85 expression in liver tissues. (\u003cstrong\u003eH\u003c/strong\u003e)RT-qPCR analysis of NUP85 expression in liver tissues. All experimental results in this study were repeated at least three times. \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 versus the control group.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9224000/v1/3ce7db78f64ea25f9bdacad9.png"},{"id":107480416,"identity":"cca8d80d-e076-476d-b655-5ba6bd24e0a9","added_by":"auto","created_at":"2026-04-22 02:10:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":978500,"visible":true,"origin":"","legend":"\u003cp\u003eNUP85 knockdown alleviates AILI (\u003cstrong\u003eA\u003c/strong\u003e) Analysis of small animals imaging. (\u003cstrong\u003eB-C\u003c/strong\u003e) Western blotting and RT-qPCR analysis of NUP85 expression in liver tissues. (\u003cstrong\u003eD\u003c/strong\u003e) Representative immunohistochemical staining of NUP85, H\u0026amp;E staining, and immunohistochemical staining of F4/80 in liver tissues in the APAP+AAV8-shRNA-NUP85 group and the APAP+AAV8-empty group. Scale bar = 50 µm. (\u003cstrong\u003eE\u003c/strong\u003e) Quantitative analysis of NUP85 positive expression in liver tissues from different groups. (\u003cstrong\u003eF\u003c/strong\u003e) Serum ALT and AST levels in different groups. (\u003cstrong\u003eG\u003c/strong\u003e)Histopathological scores of liver tissues based on H\u0026amp;E staining in different groups. (\u003cstrong\u003eH\u003c/strong\u003e)Quantitative analysis of F4/80 positive expression in liver tissues from different groups. (\u003cstrong\u003eI\u003c/strong\u003e)ELISA measurement of serum IL-6, IL-1β, and TNF-α levels. (\u003cstrong\u003eJ\u003c/strong\u003e)RT-qPCR analysis of IL-6, TNF-α, and IL-1β expression in liver tissues. \u0026nbsp;\u0026nbsp;(\u003cstrong\u003eK\u003c/strong\u003e)Levels of liver SOD, MDA and GSH. All experimental results in this study were repeated at least three times. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 , \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 versus the control group.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9224000/v1/015dcfd4c9e0441b696a802e.png"},{"id":106960371,"identity":"b2ed8d28-7ce7-4be9-922e-528889f04d59","added_by":"auto","created_at":"2026-04-15 09:20:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":551638,"visible":true,"origin":"","legend":"\u003cp\u003eNUP85 expression is elevated in APAP-treated AML-12 cells. (\u003cstrong\u003eA\u003c/strong\u003e) Western blotting analysis of NUP85 protein expression at different APAP concentrations. (\u003cstrong\u003eB\u003c/strong\u003e) The expression levels of NUP85 protein at different time points after APAP treatment. (\u003cstrong\u003eC-F\u003c/strong\u003e) Western blotting and RT-qPCR analysis for expression levels of NUP85 after transfection of NUP85-siRNA or pcDNA3.1-3xFlag-c-NUP85 in APAP-treated AML-12 cells. All experimental results of this study were replicated at least three times. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 versus the control group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 versus the NC-siRNA group or pcDNA3.1-3xFlag-c group.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9224000/v1/92f71a0058a47a448e8423d9.png"},{"id":106960392,"identity":"e740e16b-d4ae-4782-87a0-428ad4eb13fb","added_by":"auto","created_at":"2026-04-15 09:20:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":954728,"visible":true,"origin":"","legend":"\u003cp\u003eNUP85 modulates NF-κB signaling pathway in APAP­treated AML-12 cells. (\u003cstrong\u003eA-D\u003c/strong\u003e) Western blotting and RT-qPCR analysis for expression levels of IL-1β, IL-6 and TNF-α after transfection of NUP85-siRNA or pcDNA3.1-3xFlag-c-NUP85 in APAP-treated AML-12 cells. (\u003cstrong\u003eE-J\u003c/strong\u003e) Western blotting analysis for expression levels of P-P65, P65, P-IκBα and IκBα. All experimental results of this study were replicated at least three times. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 versus the control group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 versus the NC-siRNA group or pcDNA3.1-3xFlag-c group.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9224000/v1/30b199e4aca38224cf5e777d.png"},{"id":106760780,"identity":"c7a099ae-2cde-4202-b2ca-825012dddd2c","added_by":"auto","created_at":"2026-04-13 08:50:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":827362,"visible":true,"origin":"","legend":"\u003cp\u003eNUP85 regulates APAP-induced oxidative stress through the Nrf 2-Keap 1 signaling pathway (\u003cstrong\u003eA-B\u003c/strong\u003e) Levels of AML-12 cells SOD, MDA and GSH. (\u003cstrong\u003eC-H\u003c/strong\u003e) Western blotting analysis for expression levels of Nrf2 and Keap-1. All experimental results of this study were replicated at least three times. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 versus the control group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 versus the NC-siRNA group or pcDNA3.1-3xFlag-c group.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9224000/v1/b02e5318f6bce44606a0e430.png"},{"id":106960004,"identity":"1222ee4d-dc6d-4454-bb55-df7384815df6","added_by":"auto","created_at":"2026-04-15 09:17:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":702034,"visible":true,"origin":"","legend":"\u003cp\u003eCrocin-I targets NUP85 and inhibits its expression. (\u003cstrong\u003eA\u003c/strong\u003e) The binding site of crocin-I to NUP85. (\u003cstrong\u003eB\u003c/strong\u003e) Cell viability was determined by CCK-8 assay. (\u003cstrong\u003eC\u003c/strong\u003e) CETSA analysis of NUP85 expression levels in AML-12 cells cultured with or without crocin-I. (\u003cstrong\u003eD\u003c/strong\u003e) Representative immunohistochemical staining of NUP85 in liver tissue of mice in the control group and the model group. Scale bar = 50 µm. (\u003cstrong\u003eE\u003c/strong\u003e) RT-qPCR analysis of NUP85 expression in liver tissues from mice. All experimental results of this study were replicated at least three times. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 versus the APAP group.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9224000/v1/20d4b9469c2553e89c14a364.png"},{"id":106960375,"identity":"3687d453-d45b-4bf0-b92c-cbad18b53583","added_by":"auto","created_at":"2026-04-15 09:20:40","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1037396,"visible":true,"origin":"","legend":"\u003cp\u003eCrocin-I alleviates liver injury in APAP-treated mice. (\u003cstrong\u003eA\u003c/strong\u003e) Representative images and H\u0026amp;E staining (defective areas marked by white circles) of liver tissue from different groups. (\u003cstrong\u003eB\u003c/strong\u003e) Histopathological scores of liver tissues based on H\u0026amp;E staining in different groups. (\u003cstrong\u003eC\u003c/strong\u003e) Serum ALT and AST levels in different groups. All experimental results of this study were replicated at least three times. \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 versus the control group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 versus the APAP group.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-9224000/v1/e9ff4a01d226f99e4b6fe89b.png"},{"id":106993984,"identity":"d46d3f91-549a-4ffe-97d7-1ba967e360c6","added_by":"auto","created_at":"2026-04-15 15:01:34","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1082525,"visible":true,"origin":"","legend":"\u003cp\u003eCrocin-I alleviates inflammation in APAP-treated mice. (\u003cstrong\u003eA\u003c/strong\u003e) Representative immunohistochemical staining of F4/80 in liver tissue of mice in the control group and the model group. Scale bar = 50 µm. (\u003cstrong\u003eB\u003c/strong\u003e) Quantitative analysis of F4/80 positive expression in liver tissues from different groups. (\u003cstrong\u003eC-D\u003c/strong\u003e) RT-qPCR and Western blotting analysis of IL-6, TNF-α, and IL-1β expression levels in liver tissues from different groups. (\u003cstrong\u003eE\u003c/strong\u003e) Levels of serum IL-6, TNF-α and IL-1β. （\u003cstrong\u003eF\u003c/strong\u003e）Levels of liver SOD, MDA and GSH. All experimental results of this study were replicated at least three times. \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 versus the control group. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001 versus the APAP group.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-9224000/v1/b1e527f0936e0e487a85a900.png"},{"id":108146940,"identity":"b6262833-32c5-4952-b18e-e3ae95d2e9c2","added_by":"auto","created_at":"2026-04-29 21:40:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7351299,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9224000/v1/d6260659-19fb-4fd7-834b-8ef37bfe647d.pdf"},{"id":106959745,"identity":"b51bdfeb-9537-47e9-89aa-8d248b5a2537","added_by":"auto","created_at":"2026-04-15 09:14:20","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":914167,"visible":true,"origin":"","legend":"","description":"","filename":"abstractfigure.png","url":"https://assets-eu.researchsquare.com/files/rs-9224000/v1/d9ae758b53fb121824004c6e.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeting nucleoporin NUP85 ameliorates acetaminophen-induced liver injury by inhibiting inflammation and oxidative stress in hepatocytes","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAcetaminophen (APAP), a commonly used over-the-counter antipyretic and analgesic drug in clinical practice, has good safety at therapeutic doses[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, excessive intake APAP can lead to acute liver injury, which may progress to liver failure or even death in severe cases [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Notably, excessive intake APAP is one of the main causes of drug-induced liver injury (DILI) worldwide. Although N-acetylcysteine (NAC) is currently the first-line drug for the clinical treatment of APAP-induced liver injury (AILI), its therapeutic window is relatively narrow and its efficacy for severe liver injury is limited [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, it is of great clinical significance to deeply clarify the pathogenesis of AILI, explore new therapeutic targets and develop highly effective and low-toxicity targeted drugs.\u003c/p\u003e \u003cp\u003eThe pathophysiological process of AILI is complex, among which inflammatory response and oxidative stress are the core driving factors [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It is worth nothing that the reactive oxygen species (ROS) produced by excessive metabolism of APAP can directly damage liver cells, simultaneously activate innate immune cells such as kupffer cells, release a large number of inflammatory cytokines, recruit neutrophils and macrophages to infiltrate the liver, form a vicious cycle of \"oxidative stress - inflammation\", and aggravate liver injury [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, screening and identifying key targets that can regulate inflammation and oxidative stress responses is expected to provide potential intervention strategies and new ideas for the clinical treatment of AILI.\u003c/p\u003e \u003cp\u003eNuclear pore complexes (NPCs) are large molecular complexes of the nuclear envelope that play a role in DNA repair, the structure of chromatin, and nucleocytoplasmic transport [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. NPCs are composed of various nuclear pore proteins (NUPs), including NUP93, NUP155, and NUP85[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. More and more evidence indicates that NUPs are associated with oxidative stress and inflammation in liver diseases[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. For instance, NUP93 is highly expressed in hepatocellular carcinoma (HCC) tissues and correlates with poor clinical prognosis. Downregulation of NUP93 inhibits tumor cell metastasis and proliferation, whereas its upregulation promotes malignant progression [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In recent years, it has been documented that the knockdown of NUP85 alleviates the lipid metabolism disorder, inflammatory response and oxidative stress of liver induced by the methionine-choline-deficient (MCD) diet, exerting a protective effect on the liver [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In the Human Protein Atlas, NUP85 is widely expressed in various organs, especially the liver [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The amino acid sequence of NUP85 is highly conserved between mice and humans, making it a promising therapeutic target [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In addition, NUP85 is presumed to play a significant role in oxidative stress and inflammatory responses related to AILI. However, NUP85 expression characteristics, biological functions, and regulatory mechanisms in AILI remain unclear at present. Therefore, this study systematically explored the role and molecular mechanism of NUP85 in AILI through \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments, and screened natural active drugs targeting NUP85, providing new targets and strategies for the clinical treatment of AILI.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Reagent\u003c/h2\u003e \u003cp\u003eAnti-NUP85 (15027-1-AP), anti-P65 (66535-1-Ig), anti-IкBα (66418-1-Ig), anti-IL-1β (66737-1-Ig), anti-β-actin (66009-1-Ig), anti-TNF-α (60291-1-Ig), anti-Keap1 (10503-2-AP), anti-LaminB1 (66095-1-Ig), anti-phospho-IкBα (P-IкBα, 68999-1-Ig), and anti-IL-6 (66146-1-Ig) were purchased from Proteintech (Wuhan, China). Anti-F4/80 (sc-52664) was provided by Santa Cruz Biotechnology (CA, USA). Anti-phospho-p65 (P-P65, AF2006) and anti-Nrf2 (AF0639) were purchased from Affinity (USA). The SOD (A001-2-2), MDA (A003-1-2) and GSH (A006-2-1) kits were all provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). DMEM/F12 was purchased from HyClone (Beijing, China). Fetal bovine serum and Opti-MEM were purchased from Gibco (USA).IL-1β, IL-6, and TNF-α ELISA kits were procured from ELK Biotechnology Co., Ltd. (Wuhan, China). Reverse transcription kit and SYBR Green Premix Pro Tag HS qPCR kit were purchased from Accurate Biology (Hunan, China). Cell Counting Kit-8 (CCK-8) was obtained from Biosharp (Beijing, China). APAP, crocin-I, and NAC were purchased from Med Chem Express (Shanghai, China). Nucleus and cytoplasmic protein extraction kits were bought from Beyotime (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Animal experiments\u003c/h2\u003e \u003cp\u003e Male C57BL/6J mice (6\u0026ndash;8 weeks old, 20\u0026ndash;22 g body weight) were obtained from Gempharmatech Co., Ltd. Mice were maintained under controlled laboratory conditions on a standard 12 h/12 h light cycle, and all animal procedures followed the ethical guidelines outlined for the Care and Use of Laboratory Animals. Animal protocols were conducted in accordance with guidelines approved by the Experimental Animal Ethics Committee of the First Affiliated Hospital of USTC (2023-N(A)-99). After a 7-day adaptation period, the mice were randomly assigned to seven experimental groups (n\u0026thinsp;=\u0026thinsp;10/group): (1) control group, (2) APAP group, (3) APAP+AAV8-empty group, (4) APAP+AAV8-shRNA-NUP85 group, (5) APAP+C10 group, (6) APAP+C40 group, (7) APAP\u0026thinsp;+\u0026thinsp;NAC group. The APAP\u0026thinsp;+\u0026thinsp;AAV8-empty group and the APAP\u0026thinsp;+\u0026thinsp;AAV8-shRNA-NUP85 group were respectively injected with AAV8-empty plasmid or AAV8-shRNA-NUP85 plasmid via the tail vein. The APAP+C10 and APAP+C40 groups received crocin-I for one week at doses of 10 mg/kg and 40 mg/kg, respectively. APAP\u0026thinsp;+\u0026thinsp;NAC group gavaged daily administration of NAC (60 mg/kg) for 7 days. After the last administration of the mice, they were fasted overnight and received a single intraperitoneal injection of acetaminophen (300 mg/kg), except for the control group mice, which injected the same volume of PBS instead. Liver tissues and serum samples were collected for additional investigation after 24 h[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Cell culture\u003c/h2\u003e \u003cp\u003eThe AML-12 cells were obtained from the School of Pharmacy at Anhui Medical University. AML-12 cells were cultured in DMEM/F12 containing 10% fetal bovine serum in a 37℃ incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. AML-12 cells were stimulated with 10 mM APAP for 6 h to establish the AILI model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. SiRNA and plasmid transfection\u003c/h2\u003e \u003cp\u003eWhen the AML-12 cells reached 70% confluence, they were transfected with NC-siRNA, NUP85-siRNA, pcDNA3.1-3\u0026times;Flag-c, and pcDNA3.1-3\u0026times;Flag-c-NUP85 by Lipofectamine 2000. After culturing AML-12 cells in Opti-MEM for 6 h, the medium was replaced with DMEM/F12 supplemented with 10% FBS, and the cells were subsequently treated with 10 mM APAP for an additional 6 h. The sequences of NUP85-siRNA and the negative control siRNA are provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Cell proliferation assay\u003c/h2\u003e \u003cp\u003eApproximately 5 \u0026times; 10\u0026sup3; AML-12 cells were plated into each well of 96-well plates. Following the standard procedure, the plates were placed in a cell culture incubator for incubation. Following a 3 h cell adherence period, the cells were incubated with crocin-I at a range of concentrations (0, 2.5, 5, 10, 20, 40, and 80 \u0026micro;M) for 24 h. Subsequently, they were exposed to APAP for an additional 6 h. Following this treatment, the culture medium in each well was replaced with 90 \u0026micro;L of fresh media supplemented with 10 \u0026micro;L of CCK-8 reagent. Following a 1 h incubation period, absorbance readings were taken at 450 nm using a microplate reader (SpectraMax iD3, Molecular Devices, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Biochemical analysis\u003c/h2\u003e \u003cp\u003eSerum samples were obtained by centrifuging whole blood at 2000 \u0026times; g for 15 min at 4\u0026deg;C. Serum ALT and AST activities were quantified using commercially available enzymatic assay kits following the manufacturer\u0026rsquo;s protocols.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. ELISA assay\u003c/h2\u003e \u003cp\u003eELISA assay was used to detect the expression levels of three inflammatory cytokines: IL-1β, TNF-α, and IL-6. The experimental procedures were conducted in strict accordance with the detailed instructions provided in the kit's manual. Absorbance values were subsequently measured at 450 nm using a microplate reader. The concentrations of cytokines in the test samples were quantified based on a standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Antioxidant Enzymes and Lipid Peroxidation Assay\u003c/h2\u003e \u003cp\u003e The levels of molecules related to oxidative stress in liver tissues and AML-12 cells were detected by using SOD, GSH, and MDA kits, and the experimental steps were strictly carried out in accordance with the operation guidelines of the kit instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Hematoxylin and eosin (H\u0026amp;E) staining\u003c/h2\u003e \u003cp\u003eMice liver tissue sections (5 \u0026micro;m thick) were deparaffinized in xylene and rehydrated through a graded ethanol series (100%, 95%, and 70%). Nucleus were stained with hematoxylin for 5 min, followed by rinsing in running tap water. Sections were counterstained with eosin for 2 min and dehydrated through an ascending ethanol series (70%, 95%, and 100%) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Following coverslipping, tissue sections were scanned using a slide scanner (Panoramic MIDI II, 3DHISTECH, Hungary) and subsequently analyzed with CaseViewer software (version 2.4, 3DHISTECH, Hungary) for histopathological assessment. Based on previous studies, the degree of liver injury in mice is quantified and analyzed by assessing the degree of inflammatory cell infiltration and the extent of tissue necrosis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Immunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eLiver tissue sections underwent standard dewaxing and rehydration processes. Antigen retrieval was then performed by heat-mediated epitope restoration in citrate buffer (0.01 M, pH 6.0) maintained at 95\u0026deg;C for 20 min. Following this, endogenous peroxidase activity was effectively blocked through 15 min incubation in 3% hydrogen peroxide at ambient temperature. To minimize nonspecific antibody binding, tissue sections were blocked with 3% bovine serum albumin for 1 h at room temperature. Primary antibodies targeting NUP85 (1:300) and F4/80 (1:100) were applied for overnight incubation at 4\u0026deg;C, followed by a 1 h incubation with species-matched horseradish peroxidase (HRP)-conjugated secondary antibodies at 37\u0026deg;C. Chromogenic visualization was performed using diaminobenzidine (DAB), and nuclei were counterstained with hematoxylin. Finally, slices were dehydrated through an ethanol series, cleared in xylene, and permanently mounted with a neutral resinous medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Real-time fluorescence quantitative PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cells and tissue specimens using TRIzol according to the manufacturer's instructions. The RNA was reverse-transcribed into cDNA using the reverse transcription kit, and then real-time fluorescence quantitative PCR (RT-qPCR) was performed using the SYBR Green Premix Pro Tag HS qPCR kit. GAPDH served as the endogenous reference gene, and relative gene expression levels were calculated using the comparative 2\u003csup\u003e\u0026minus;∆∆ct\u003c/sup\u003e method. Primer sequences are listed in Table S2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Western blotting\u003c/h2\u003e \u003cp\u003eLiver tissues or cells were homogenized in RIPA buffer containing a mixture of protease and phosphatase inhibitors. Protein extracts were loaded onto 10% acrylamide gels, electrophoresed and transferred to polyvinylidenedifluoride (PVDF) membranes. Membranes were incubated with specific antibodies, followed by secondary antibodies conjugated to horseradish peroxidase. The primary antibody included NUP85 (1:1000), p-P65 (1:1000), P65 (1:1000), TNF-α (1:1000), IкBα (1:6000), P-IкBα (1:3000), IL-6 (1:1000), IL-1β (1:1000), Keap1 (1:1000), Nrf2 (1:10000), LaminB1 (1:6000), and β-actin (1:6000). Finally, the grayscale values of the protein bands were analyzed using ImageJ software (version 1.8.0; National Institutes of Health) to determine the relative expression levels of the target proteins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Molecular docking\u003c/h2\u003e \u003cp\u003eMolecular docking of crocin-I (Pub Chem CID: 5281233) to protein NUP85 (Uniprot ID: Q8R480) using Auto Dock Vina 1.1.2 software. Protein pre-processing was accomplished using PyMol 2.4. Auto Dock Tools 1.5.6 was used to generate PDBQT files for the docking simulation. The docking conformation with the lowest binding energy and highest clustering frequency is considered to be the most potent binding mode between ligand and protein. Finally, the docking results were visualized using PLIP and PyMOL 2.4 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14. Cellular thermal shift assay (CETSA)\u003c/h2\u003e \u003cp\u003eAML-12 cells were divided into two groups for the experiment: one group received 20 \u0026micro;M crocin-I treatment for 6 h, and the other acted as a control with no crocin-I administration. Following this, the cells were collected and subjected to three freeze-thaw cycles using liquid nitrogen. The cell lysates were then centrifuged at 20 000 \u0026times; g for 30 min to obtain the supernatants. Each set of supernatants was divided into six aliquots and heated at different temperatures (37, 45, 50, 55, 60, and 65\u0026deg;C) for 3 min. Finally, the expression levels of NUP85 in the samples were analyzed by western blotting[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.15. Statistical analysis\u003c/h2\u003e \u003cp\u003eAt least three replicate experiments verified all findings in this study. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). One-way ANOVA was employed to assess inter-group differences. Statistical significance was analyzed using GraphPad Prism version 9.0, with significance levels defined as \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1. NUP85 is upregulated in AILI mice\u003c/h2\u003e \u003cp\u003eTo assess the effects of APAP overdose in mice, a dose of 300 mg/kg was administered intraperitoneally for 24 h. Macroscopic examination revealed that livers in the APAP group exhibited increased volume and a paler colouration compared to those in the control group, indicating significant morphological changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Additionally, serum levels of ALT and AST were significantly elevated in the APAP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Histological examination via H\u0026amp;E staining revealed that the liver tissues in the control group exhibited intact lobular architecture with no evidence of pathological alterations. In contrast, the liver of APAP-treated mice demonstrated marked inflammatory cell infiltration and hepatocellular necrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The above results indicated that the AILI model has been successfully established. To explore the correlation between NUP85 and AILI, NUP85 expression in liver tissues from APAP-treated mice was examined. NUP85 was highly upregulated in the APAP group, as shown by IHC staining, western blotting, and RT-qPCR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF-H). All of these results suggested that NUP85 expression level was significantly elevated in mice administered with APAP, which may exacerbate liver injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2. NUP85 knockdown alleviates liver injury in AILI mice\u003c/h2\u003e \u003cp\u003eTo investigate the biological role of NUP85 in AILI, we established a mouse model of NUP85 gene silencing via tail vein injection of AAV8-shRNA-NUP85. The results of western blotting, RT-qPCR, and immunohistochemical analysis revealed that this intervention significantly downregulated the protein and mRNA expression levels of NUP85 in the mouse liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-E). Specifically, the expression levels of serum ALT and AST were significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), and the degree of liver injury was significantly alleviated as shown by H\u0026amp;E staining of liver tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Moreover, immunohistochemical analysis revealed a significant reduction in the number of F4/80-positive macrophage infiltrations in the liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). To further verify its mechanism, we examined the markers associated with inflammation and oxidative stress. The results demonstrated that in the AAV8-shRNA-NUP85 group, the expression levels of inflammatory cytokines (IL-6, IL-1β, and TNF-α) were significantly downregulated in both the serum and liver tissues of mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-J). Additionally, the MDA content in liver tissues was markedly reduced, whereas the activities of SOD and the levels of GSH were significantly enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). The above results suggest that silencing of NUP85 can significantly improve AILI by inhibiting inflammatory responses and reducing oxidative stress, indicating that NUP85 may be a potential intervention target for the prevention and treatment of AILI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3. NUP85 is elevated in AILI AML-12 cells\u003c/h2\u003e \u003cp\u003eTo investigate the effects of APAP treatment on NUP85 expression in AML-12 cells, we examined changes in NUP85 expression following exposure to various concentrations of APAP for 6 h. The results indicated dose-dependent upregulation of NUP85 expression, with a significant increase at a dose of 10 mM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The maximal NUP85 protein expression was observed 6 h following treatment with 10 mM APAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Based on these findings, all \u003cem\u003ein vitro\u003c/em\u003e studies were performed with 10 mM APAP for a duration of 6 h. Western blotting and RT-qPCR assays revealed that the protein and mRNA levels of NUP85 were significantly reduced in the NUP85-siRNA group relative to the NC-siRNA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D). Conversely, NUP85 transfection with pcDNA3.1-3\u0026times;Flag-c-NUP85 resulted in elevated NUP85 expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and F). These results indicated that NUP85 was successfully knocked down and overexpressed in AML-12 cells after transfection with NUP85-siRNA and pcDNA3.1-3\u0026times;Flag-c-NUP85, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4. NUP85 knockdown attenuates AILI by inhibiting NF-кB signaling pathway\u003c/h2\u003e \u003cp\u003eFollowing the effective knockdown and overexpression of NUP85, we investigated its impact on the expression of APAP-induced inflammatory cytokines (IL-6, IL-1β, and TNF-α) in AML-12 cells. The results indicated that the protein and mRNA levels of these inflammatory cytokines were significantly upregulated in APAP-treated AML-12 cells. After transfection of NUP85-siRNA, the expression of these cytokines were actually reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and B). On the contrary, the results showed that inflammatory cytokine expression levels were significantly upregulated in AML-12 cells overexpressing NUP85 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). These findings indicated that disruption of NUP85 attenuates inflammatory responses in AML-12 cells treated with APAP. The NF-κB signaling pathway was shown to be responsible for playing a crucial role in the inflammatory response of AILI [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e][\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To elucidate the mechanism by which NUP85 influences inflammation, we investigated the expression levels of key proteins involved in this signaling pathway. Western blotting result revealed that NUP85 knockdown inhibited the phosphorylation of P65 and IκBα. Compared to the NC-siRNA group, P65 was downregulated in the nucleus and upregulated in the cytoplasm in the NUP85-siRNA group, indicating that NUP85 knockdown inhibited P65 nuclear translocation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-H). Conversely, overexpression of NUP85 resulted in enhanced phosphorylation of P65 and IκBα, thereby promoting the nuclear translocation of P65 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI-J). Collectively, these findings indicated that NUP85 plays a critical role in modulating NF-κB signaling pathway in AML-12 cells treated with APAP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5. NUP85 knockdown alleviates oxidative stress in the liver of AILI by activating Nrf2-Keap1 signaling pathway\u003c/h2\u003e \u003cp\u003eTo investigate the effect of NUP85 on oxidative stress in AML-12 cells, and the contents of MDA, SOD and GSH within the cells were detected. The results showed that, compared with the NC-siRNA group, NUP85 knockdown reduced the MDA content, while elevating the SOD activity and GSH content (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Conversely, NUP85 overexpression increased the MDA content and decreased both SOD activity and GSH content (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). To further reveal its molecular mechanism, western blotting was used to detect the expression of proteins related to the Nrf2-Keap1 signaling pathway. The results indicated that in APAP-treated AML-12 cells, NUP85 knockdown significantly upregulated the Nrf2 protein expression and downregulated the Keap1 protein expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). In addition, compared with the NC-siRNA group, NUP85 knockdown promoted the nuclear translocation of Nrf2, as indicated by its increased expression in the nucleus and the decreased expression in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Conversely, NUP85 overexpression significantly suppressed the activation of the Nrf2-Keap1 signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). In summary, these results suggested that knockdown of NUP85 at the cellular level can effectively alleviate APAP-induced oxidative stress injury by activating the Nrf2-Keap1 signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Crocin-I targets NUP85 and inhibits its expression\u003c/h2\u003e \u003cp\u003eFurther, we utilized virtual screening to identify potential inhibitors targeting NUP85 from a natural product database. Crocin-I was identified as a compound exhibiting specific binding affinity for NUP85. Molecular docking studies revealed that the binding pocket of NUP85 contains multiple hydrophobic amino acid residues, enabling crocin-I to interact with NUP85 via hydrophobic interactions and hydrogen bonding. Notably, crocin-I formed 14 hydrogen bonds with NUP85, suggesting that hydrogen bonding plays a critical role in stabilizing the interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). To assess the pharmacological effects of crocin-I, we performed a CCK-8 assay, which showed that crocin-I at 20 \u0026micro;M exhibited the most significant protective effect on AML-12 cells treated with APAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). \u003cem\u003eIn vitro\u003c/em\u003e, we conducted a detailed investigation into the binding interaction between crocin-I and NUP85 at a concentration of 20 \u0026micro;M. The CETSA result showed that the NUP85 protein expression level gradually decreased with increasing temperature. However, in the crocin-I group, NUP85 protein expression was higher than in the control group at the same temperature, suggesting that the binding of crocin-I enhanced the stability of the NUP85 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Additionally, IHC staining and RT-qPCR demonstrated that crocin-I treatment significantly reduced NUP85 expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-E). These results confirmed that crocin-I may act as a targeted inhibitor of NUP85.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Crocin-I alleviates liver injury in AILI mice\u003c/h2\u003e \u003cp\u003eTo evaluate the therapeutic potential of crocin-I in AILI, we orally administered crocin-I to C57BL/6J mice for 7 days prior to APAP treatment. Histological analysis indicated that compared with the APAP group, crocin-I (10 and 40 mg/kg) and NAC (60 mg/kg) significantly reduced the inflammatory cell infiltration and necrosis in liver tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and B). Moreover, crocin-I treatment significantly lowered serum ALT and AST levels in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). These findings suggested that crocin-I offers protective effects against AILI, with efficacy comparable to NAC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Crocin-I alleviates inflammation and oxidative stress in AILI mice\u003c/h2\u003e \u003cp\u003eTo investigate the potential inhibitory effect of crocin-I on macrophage infiltration in damaged liver tissue, this study utilized F4/80 as a specific marker for immunohistochemical analysis. The results showed that the number of F4/80-positive macrophages was significantly reduced in the livers of mice administered crocin-I or NAC compared with the APAP-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and B). The results of the RT-qPCR and western blotting showed that crocin-I inhibited the increase in IL-6, TNF-α, and IL-1β levels in the liver that was caused by APAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and D). Compared to the group that received only APAP, the treated groups demonstrated significantly lower levels of IL-6, TNF-α, and IL-1β in serum, indicating that crocin-I significantly decreased systemic inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). In addition, crocin-I reduced the MDA content in the liver induced by APAP, while increasing the activities of SOD and the content of GSH (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). Therefore, crocin-I has an antioxidant stress effect on AILI.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eDILI, particularly that induced by APAP, has emerged as a major challenge in global public health owing to its high incidence and potential fatality[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. After excessive APAP, the oxidative stress and inflammatory response mediated by N-acetyl-p-benzoquinone imine (NAPQI) interweave with each other, forming a vicious cycle, which is the core pathological link leading to massive necrosis of the liver [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Currently, the NAC relied upon in clinical practice has problems such as limited efficacy and adverse reactions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], while the pathological mechanism of AILI is complex, involving multiple aspects such as immune cell infiltration, inflammatory cytokine release, and imbalance of the antioxidant system[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, it is currently urgent to develop new therapeutic drugs targeting the key pathogenic factors. This study focuses on the potential target NUP85 and delves deeply into its role in AILI, providing new ideas for resolving the clinical treatment predicament.\u003c/p\u003e \u003cp\u003eNUPs as a core component of the nuclear pore complex, in addition to their classic nuclear cytoplasmic transport function, have been proven to be widely involved in pathological processes such as inflammatory regulation and metabolic disorders in recent years [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Current studies have shown that NUP85 can alleviate abnormal lipid metabolism in the liver by regulating inflammatory responses [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and it is highly expressed in the liver [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], suggested that NUP85 may play an important regulatory role in liver diseases. Notably, this study is the first to report that NUP85 mRNA and protein expression are significantly upregulated in mouse liver and AML-12 cells in the AILI model, with its expression positively correlated with the severity of liver injury. Moreover, both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments demonstrated that NUP85 silencing, achieved via AAV8-mediated knockdown in mice, markedly reduces serum ALT/AST levels, alleviates liver inflammatory infiltration and liver cells necrosis, and effectively inhibits APAP-induced inflammatory cytokine release and oxidative stress damage, collectively indicating a critical role of NUP85 in AILI progression. The above results indicated that NUP85 is highly expressed in AILI. Meanwhile, NUP85 high expression aggravates liver injury by amplifying inflammation and oxidative stress responses, clarifying the potential value of NUP85 as a therapeutic target for AILI.\u003c/p\u003e \u003cp\u003eNF-κB signaling pathway is the core signaling pathway regulating inflammatory responses [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Notably, the activation of the NF-κB signaling pathway can promote the transcription of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, and aggravate liver injury by amplifying the inflammatory cascade reaction [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In addition, in the resting state of the NF-κB signaling pathway, the P65 subunit binds to the inhibitory protein IκBα to form a heterodimer, which is stably located in the cytoplasm. When the NF-κB signaling pathway is activated, IκBα is phosphorylated and degraded by the proteasome. Meanwhile, the released P65 subunit undergoes nuclear translocation and binds to the promoter region of the target gene, thereby initiating the transcriptional process of downstream pro-inflammatory cytokines and related genes [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Current studies have shown that the down-regulation of NUP85 can exert anti-tumor and anti-proliferative effects by inhibiting the activation of the NF-κB signaling pathway [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This study found that NUP85 silencing could significantly inhibit APAP-induced phosphorylation of IκBα and P65, reduce nuclear translocation of P65, while overexpression of NUP85 promoted the activation of the NF-κB signaling pathway. Therefore, NUP85 can promote the release of pro-inflammatory cytokines and the infiltration of inflammatory cells by activating the NF-κB signaling pathway, thereby exacerbating the inflammatory damage of AILI.\u003c/p\u003e \u003cp\u003eNrf2-Keap1 signaling pathway is a key defense system for cells against oxidative stress [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Under normal physiological conditions, Nrf2 binds to Keap1 and is ubiquitinized and degraded. Additionally, under oxidative stress conditions, Nrf2 dissociates from the Keap1 nucleus and translocates, initiating the expression of antioxidant genes such as SOD, GSH, and MDA [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Previous studies have demonstrated that promoting the activation of the Nrf2 signaling pathway can effectively alleviate liver injury induced by APAP [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this study, NUP85 silencing could significantly up-regulate the expression level of Nrf2, promoting its nuclear translocation, and simultaneously increase intracellular SOD activity and GSH content, while reducing the accumulation of lipid peroxidation product MDA. Conversely, NUP85 overexpression inhibited the activation of the Nrf2-Keap1 signaling pathway, which is consistent with the research conclusion that NUPs regulated the nuclear transport of Nrf2 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, it is suggested that NUP85 weakens the antioxidant capacity of liver cells by inhibiting the Nrf2-Keap1 signaling pathway, thereby aggravating AILI-induced oxidative stress injury. In summary, NUP85 promotes the occurrence and development of AILI through a dual mechanism of activating the NF-κB inflammatory signaling pathway and inhibiting the Nrf2-Keap1 antioxidant signaling pathway.\u003c/p\u003e \u003cp\u003eNatural products have become an important resource for the research and development of AILI therapeutic drugs due to their advantages of multiple targets and low toxicity [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Crocin-I, as the main active component of saffron, has been confirmed to have various pharmacological activities such as anti-inflammation, anti-oxidation and neuroprotection [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], but its therapeutic potential in AILI remains unclear. This study identified via virtual screening that crocin-I specifically binds to NUP85, with molecular docking analysis revealing that the two molecules form a stable complex through hydrophobic interactions and hydrogen bonds. Furthermore, the CETSA experiment confirmed that crocin-I could enhance the stability of NUP85 protein, and in vivo experiments indicated that it could significantly down-regulate the NUP85 expression. Further studies have shown that pretreatment with crocin-I can dose-dependently reduce the serum transaminase level in mice induced by APAP, alleviate liver inflammatory infiltration and oxidative stress injury, and its therapeutic effect is comparable to that of NAC. The above results indicated that crocin-I exerts a protective effect on AILI by targeting and inhibiting the NUP85 expression and regulating the NF-κB and Nrf2-Keap1 signaling pathways, providing a new candidate drug for the clinical treatment of AILI.\u003c/p\u003e \u003cp\u003eIn conclusion, this study has for the first time confirmed the high expression of NUP85 in AILI. Meanwhile, NUP85 exacerbates liver inflammatory responses and oxidative stress damage by activating the NF-κB inflammatory signaling pathway and inhibiting the Nrf2-Keap1 antioxidant signaling pathway. Additionally, the natural active ingredient crocin-I can target NUP85 and inhibit its expression, effectively alleviating the pathological damage of AILI, and its therapeutic effect is comparable to that of the clinical drug NAC. This study not only revealed the pathological function and regulatory network of NUP85 in AILI but also clarified the feasibility of NUP85 as a therapeutic target for AILI and the potential application value of crocin-I, providing new theoretical basis and therapeutic strategies for the clinical prevention and treatment of AILI.\u003c/p\u003e \u003cp\u003eAlthough this study has clarified the pathogenic effect and regulatory mechanism of NUP85 in AILI, it is still necessary to further explore the specific binding sites and selective action mechanisms of crocin-I and NUP85 to provide a molecular basis for drug optimization.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAPAP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAcetaminophen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAILI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAPAP-induced liver injury\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNUP85\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNucleoporin 85\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDILI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edrug-induced liver injury\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNAC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eN-acetylcysteine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNPC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNuclear pore complexes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNUPs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enuclear pore proteins\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIL-1β\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einterleukin-1beta\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNF-α\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etumor necrosis factor-alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIL-6\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einterleukin-6\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eALT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlanine aminotransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003easpartate aminotransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eenzyme-linked immunosorbent assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCETSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCellular thermal shift assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eH\u0026amp;E\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHematoxylin and eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunohistochemistry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMCD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emethionine-choline-deficient\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNAPQI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eN-acetyl-p-benzoquinone imine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehepatocellular carcinoma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is supported by the National Natural Science Foundation of China (82373932); Outstanding Youth Talents Fund Project of Anhui Province (2023AH030114); Key Research and Development Project of Anhui Provincial (202204295107020023); \u0026nbsp; Department of Education of Anhui Province Outstanding Young Teacher Training Project (YQZD2023023); Natural Science Project of Anhui Province (2208085MH252); Anhui University Outstanding Youth Program (2024AH020006); Natural Science Research Project of Anhui Educational Committee (2022AH050754); 2025 School-Level Tutoring \u0026amp; Ideological and Political Studio(0601067124); School-Level College Students\u0026apos; Innovative Training Program (0601067120,0601067122); 2024 Provincial Quality Project \u0026ndash; School-Enterprise Cooperative Practical Education Base (0601067123) .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMengqi Han\u003c/strong\u003e: Writing - original draft, Methodology, Investigation, Formal analysis. \u003cstrong\u003eShanshan Ge\u003c/strong\u003e: Methodology, Investigation, Formal analysis. \u003cstrong\u003eGuitao Zhao\u003c/strong\u003e: Methodology, Investigation, Data curation. \u003cstrong\u003ePan-pan Yang\u003c/strong\u003e and \u003cstrong\u003eZhipan Luo\u003c/strong\u003e: Validation, Software, Investigation. \u003cstrong\u003eCheng Qian\u003c/strong\u003e: Investigation, Formal analysis. \u003cstrong\u003eLinxin Pan\u003c/strong\u003e: Methodology, Data curation. \u003cstrong\u003ePeng Fu\u003c/strong\u003e: Conceptualization, Writing - review \u0026amp; editing. \u003cstrong\u003eTao Xu\u003c/strong\u003e: Project administration, Funding acquisition. \u003cstrong\u003eZhaolin Chen\u003c/strong\u003e: Writing - review \u0026amp; editing, Funding acquisition, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData A\u003c/strong\u003e\u003cstrong\u003ev\u003c/strong\u003e\u003cstrong\u003eailability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhang, J., M. Li, T. Zhao, J. Cao, Y. Liu, and Y. Wang et al. 2022. E Se tea alleviates acetaminophen-induced liver injury by activating the Nrf2 signaling pathway. \u003cem\u003eFood \u0026amp; function\u003c/em\u003e 13:7240\u0026ndash;7250.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Y., L. Tian, Y. Wang, T. Zhao, A. Khan, and Y. Wang et al. 2021. Protective effect of Que Zui tea hot-water and aqueous ethanol extract against acetaminophen-induced liver injury in mice via inhibition of oxidative stress, inflammation, and apoptosis. \u003cem\u003eFood \u0026amp; function\u003c/em\u003e 12:2468\u0026ndash;2480.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang, C., L. Cen, H. Zeng, X. Zhang, P. Liu, and Y. Chen et al. 2024. Inhibiting Hepatocyte Uric Acid Synthesis and Reabsorption Ameliorates Acetaminophen-Induced Acute Liver Injury in Mice. \u003cem\u003eCellular and molecular gastroenterology and hepatology\u003c/em\u003e 17:251\u0026ndash;265.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChowdhury, A., J. Nabila, I. Adelusi Temitope, and S. Wang. 2020. Current etiological comprehension and therapeutic targets of acetaminophen-induced hepatotoxicity. \u003cem\u003ePharmacological research\u003c/em\u003e 161:105102.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, H., Q. Weng, S. Gong, W. Zhang, J. Wang, and Y. Huang et al. 2023. Kaempferol prevents acetaminophen-induced liver injury by suppressing hepatocyte ferroptosis via Nrf2 pathway activation. \u003cem\u003eFood \u0026amp; function\u003c/em\u003e 14:1884\u0026ndash;1896.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, Q., W. Zhang, N. Cheng, Y. Zhu, H. Li, and S. Zhang et al. 2023. Pectolinarigenin ameliorates acetaminophen-induced acute liver injury via attenuating oxidative stress and inflammatory response in Nrf2 and PPARa dependent manners. \u003cem\u003ePhytomedicine: international journal of phytotherapy and phytopharmacology\u003c/em\u003e 113:154726.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu, Q., J. Zhang, J. Li, Y. Song, J. Pan, and C. Mei et al. 2024. Sirtuin 5-Mediated Desuccinylation of ALDH2 Alleviates Mitochondrial Oxidative Stress Following Acetaminophen-Induced Acute Liver Injury. Advanced science (Weinheim, Baden-Wurttemberg, Germany). ; 11:e2402710.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeck, M., and E. Hurt. 2017. The nuclear pore complex: understanding its function through structural insight. \u003cem\u003eNature reviews Molecular cell biology\u003c/em\u003e 18:73\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakano, H., W. Wang, C. Hashizume, T. Funasaka, H. Sato, and R. W. Wong. 2011. Unexpected role of nucleoporins in coordination of cell cycle progression. \u003cem\u003eCell cycle (Georgetown Tex)\u003c/em\u003e 10:425\u0026ndash;433.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoyne, A. N., and J. D. Rothstein. 2022. Nuclear pore complexes - a doorway to neural injury in neurodegeneration. \u003cem\u003eNature reviews Neurology\u003c/em\u003e 18:348\u0026ndash;362.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNord, M. S., C. Bernis, S. Carmona, D. C. Garland, A. Travesa, and D. J. Forbes. 2020. Exportins can inhibit major mitotic assembly events in vitro: membrane fusion, nuclear pore formation, and spindle assembly. \u003cem\u003eNucleus (Austin Tex)\u003c/em\u003e 11:178\u0026ndash;193.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHolzer, K., A. Ori, A. Cooke, D. Dauch, E. Drucker, and P. Riemenschneider et al. 2019. Nucleoporin Nup155 is part of the p53 network in liver cancer. \u003cem\u003eNature communications\u003c/em\u003e 10:2147.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin, C. S., Y. Liang, S. G. Su, Y. L. Zheng, X. Yang, and N. Jiang et al. 2022. Nucleoporin 93 mediates β-catenin nuclear import to promote hepatocellular carcinoma progression and metastasis. \u003cem\u003eCancer letters\u003c/em\u003e 526:236\u0026ndash;247.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu, Y. C., Q. Yan, S. Q. Yue, L. X. Pan, D. S. Yang, and L. S. Tao et al. 2024. NUP85 alleviates lipid metabolism and inflammation by regulating PI3K/AKT signaling pathway in nonalcoholic fatty liver disease. \u003cem\u003eInternational journal of biological sciences\u003c/em\u003e 20:2219\u0026ndash;2235.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, Z., S. Li, Y. Zhang, R. Xu, J. Zhou, and Y. Wang et al. 2025. NUP85 siRNA loaded red blood cell-derived extracellular vesicles alleviate hepatic steatosis in MASLD. \u003cem\u003eJournal of nanobiotechnology\u003c/em\u003e 24:63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaniuchi, K., T. Yawata, M. Tsuboi, T. Ueba, and T. Saibara. 2019. Efficient delivery of small interfering RNAs targeting particular mRNAs into pancreatic cancer cells inhibits invasiveness and metastasis of pancreatic tumors. \u003cem\u003eOncotarget\u003c/em\u003e 10:2869\u0026ndash;2886.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTerashima, Y., N. Onai, M. Murai, M. Enomoto, V. Poonpiriya, and T. Hamada et al. 2005. Pivotal function for cytoplasmic protein FROUNT in CCR2-mediated monocyte chemotaxis. \u003cem\u003eNature immunology\u003c/em\u003e 6:827\u0026ndash;835.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan, K., X. Zhang, L. Lv, J. Zhang, W. Liang, and P. Wang. 2016. Fine-tuning the expression of microRNA-155 controls acetaminophen-induced liver inflammation. \u003cem\u003eInternational immunopharmacology\u003c/em\u003e 40:339\u0026ndash;346.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu, Y., L. Lei, X. Wang, L. Chen, W. Li, and J. Li et al. 2023. The E3 ubiquitin ligase NEDD4-1 protects against acetaminophen-induced liver injury by targeting VDAC1 for degradation. \u003cem\u003eActa pharmaceutica Sinica B\u003c/em\u003e 13:1616\u0026ndash;1630.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng, Y., R. Wu, F. Wang, S. Li, L. Li, and Y. Li et al. 2023. Liberation of daidzein by gut microbial β-galactosidase suppresses acetaminophen-induced hepatotoxicity in mice. \u003cem\u003eCell host \u0026amp; microbe\u003c/em\u003e 31:766\u0026ndash;780e7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElshal, M., and M. E. Abdelmageed. 2022. Diacerein counteracts acetaminophen-induced hepatotoxicity in mice via targeting NLRP3/caspase-1/IL-1β and IL-4/MCP-1 signaling pathways. \u003cem\u003eArchives of pharmacal research\u003c/em\u003e 45:142\u0026ndash;158.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartinez Molina, D., R. Jafari, M. Ignatushchenko, T. Seki, E. A. Larsson, and C. Dan et al. 2013. \u003cem\u003eMonitoring drug target engagement in cells and tissues using the cellular thermal shift assay\u003c/em\u003e. vol. 341 84\u0026ndash;87. New York, NY: Science.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarnabei, L., E. Laplantine, W. Mbongo, F. Rieux-Laucat, and R. Weil. 2021. NF-κB: At the Borders of Autoimmunity and Inflammation. \u003cem\u003eFrontiers in immunology\u003c/em\u003e 12:716469.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, R., C. Song, J. Chen, L. Zhou, X. Jiang, and X. Cao et al. 2020. Limonin ameliorates acetaminophen-induced hepatotoxicity by activating Nrf2 antioxidative pathway and inhibiting NF-κB inflammatory response via upregulating Sirt1. \u003cem\u003ePhytomedicine: international journal of phytotherapy and phytopharmacology\u003c/em\u003e 69:153211.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai, X., H. Cai, J. Wang, Q. Yang, J. Guan, and J. Deng et al. 2022. Molecular pathogenesis of acetaminophen-induced liver injury and its treatment options. \u003cem\u003eJournal of Zhejiang University Science B\u003c/em\u003e 23:265\u0026ndash;285.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao, R., Q. Zhang, W. Liu, Y. Lin, Y. He, and D. Chang et al. 2023. Pien Tze Huang attenuated acetaminophen-induced liver injury by autophagy mediated-NLRP3 inflammasome inhibition. \u003cem\u003eJournal of ethnopharmacology\u003c/em\u003e 311:116285.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Q., X. Peng, Y. Chen, X. Tang, Y. Qin, and M. He et al. 2023. Piezo1 alleviates acetaminophen-induced acute liver injury by activating Nrf2 and reducing mitochondrial reactive oxygen species. \u003cem\u003eBiochemical and biophysical research communications\u003c/em\u003e 652:88\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang, W. P., J. S. Deng, S. S. Huang, S. H. Wu, C. C. Chen, and J. C. Liao et al. 2021. Sanghuangporus sanghuang Mycelium Prevents Paracetamol-Induced Hepatotoxicity through Regulating the MAPK/NF-κB, Keap1/Nrf2/HO-1, TLR4/PI3K/Akt, and CaMKKβ/LKB1/AMPK Pathways and Suppressing Oxidative Stress and Inflammation. Antioxidants (Basel, Switzerland). ; 10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLawrence, T. 2009. The nuclear factor NF-kappaB pathway in inflammation. \u003cem\u003eCold Spring Harbor perspectives in biology\u003c/em\u003e 1:a001651.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSatoh, M., T. Akatsu, Y. Ishkawa, Y. Minami, and M. Nakamura. 2007. A Novel Activator of C-C Chemokine, FROUNT, is Expressed With C-C Chemokine Receptor 2 and its Ligand in Failing Human Heart. \u003cem\u003eJournal of Cardiac Failure\u003c/em\u003e 13:114\u0026ndash;119.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKobayashi, M., and M. Yamamoto. 2005. Molecular mechanisms activating the Nrf2-Keap1 pathway of antioxidant gene regulation. \u003cem\u003eAntioxidants \u0026amp; redox signaling\u003c/em\u003e 7:385\u0026ndash;394.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen, Z., Y. Wang, Z. Su, R. Kou, K. Xie, and F. Song. 2018. Activation of p62-keap1-Nrf2 antioxidant pathway in the early stage of acetaminophen-induced acute liver injury in mice. \u003cem\u003eChemico-biological interactions\u003c/em\u003e 282:22\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLv, H., L. Hong, Y. Tian, C. Yin, C. Zhu, and H. Feng. 2019. Corilagin alleviates acetaminophen-induced hepatotoxicity via enhancing the AMPK/GSK3β-Nrf2 signaling pathway. \u003cem\u003eCell communication and signaling: CCS\u003c/em\u003e 17:2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshimura, S. H., S. Otsuka, M. Kumeta, M. Taga, and K. Takeyasu. 2013. Intermolecular disulfide bonds between nucleoporins regulate karyopherin-dependent nuclear transport. \u003cem\u003eJournal of cell science\u003c/em\u003e 126:3141\u0026ndash;3150.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGouda, N. A., S. O. Alshammari, M. A. S. Abourehab, Q. A. Alshammari, and A. Elkamhawy. 2023. Therapeutic potential of natural products in inflammation: underlying molecular mechanisms, clinical outcomes, technological advances, and future perspectives. \u003cem\u003eInflammopharmacology\u003c/em\u003e 31:2857\u0026ndash;2883.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan, M., L. Ye, S. Yin, X. Lu, X. Liu, and S. Lu et al. 2018. Glycycoumarin protects mice against acetaminophen-induced liver injury predominantly via activating sustained autophagy. \u003cem\u003eBritish journal of pharmacology\u003c/em\u003e 175:3747\u0026ndash;3757.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, L., M. Jing, and Q. Liu. 2021. Crocin alleviates the inflammation and oxidative stress responses associated with diabetic nephropathy in rats via NLRP3 inflammasomes. \u003cem\u003eLife sciences\u003c/em\u003e 278:119542.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu, A., C. Lao, Z. Wang, Y. Chen, and C. Bai. 2019. Characterization of Crocetin-Monoglucuronide as a Neuron-Protective Metabolite of Crocin-1. \u003cem\u003eMolecular nutrition \u0026amp; food research\u003c/em\u003e 63:e1900024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiao, Q., Z. Xiong, C. Yu, J. Zhou, Q. Shen, and L. Wang et al. 2019. Antidepressant activity of crocin-I is associated with amelioration of neuroinflammation and attenuates oxidative damage induced by corticosterone in mice. \u003cem\u003ePhysiology \u0026amp; behavior\u003c/em\u003e 212:112699.\u003c/span\u003e\u003c/li\u003e\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":"NUP85, AILI, Inflammation, Oxidative stress, Crocin-I","lastPublishedDoi":"10.21203/rs.3.rs-9224000/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9224000/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLiver injury induced by excessive acetaminophen (APAP) is the main cause of drug-induced liver failure, and its pathological process is closely related to oxidative stress and inflammatory response. Nucleoporin 85 (NUP85, also known as FROUNT) plays a regulatory role in liver diseases, but its function and mechanism in APAP-induced liver injury (AILI) remain unclear. This study explored the role of NUP85 and targeted intervention strategies through \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments. The results showed that NUP85 expression was significantly upregulated in mouse and AML-12 cells after APAP treatment. Furthermore, targeted knockdown of NUP85 \u003cem\u003ein vivo\u003c/em\u003e reduced serum transaminase expression level, alleviated liver pathological damage, decreased macrophage infiltration and inflammatory cytokines secretion, and improved oxidative stress. \u003cem\u003eIn vitro\u003c/em\u003e experiments demonstrated that silencing NUP85 attenuates inflammation via inhibition of NF‑κB signaling pathway and mitigates oxidative stress via activation of the Nrf2\u0026ndash;Keap1 signaling pathway, while overexpression of NUP85 showed the opposite effect. It was found through virtual screening that crocin-I can specifically bind to NUP85 and inhibit its expression. \u003cem\u003eIn vivo\u003c/em\u003e experiments confirmed that crocin-I alleviates AILI in a dose-dependent manner and exerts comparable anti-inflammatory and antioxidant effects to the clinical drug N-acetylcysteine (NAC). In conclusion, NUP85 aggravates AILI by regulating the NF-κB and Nrf2-Keap1 signaling pathways. Crocin-I can target NUP85 to exert liver-protective effects, providing new targets and candidate drugs for the clinical treatment of AILI.\u003c/p\u003e","manuscriptTitle":"Targeting nucleoporin NUP85 ameliorates acetaminophen-induced liver injury by inhibiting inflammation and oxidative stress in hepatocytes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-13 08:50:24","doi":"10.21203/rs.3.rs-9224000/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":"1f510d77-c19b-4a77-83f2-435ff137798e","owner":[],"postedDate":"April 13th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-04-29T21:26:23+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-29T21:39:35+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-13 08:50:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9224000","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9224000","identity":"rs-9224000","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}