Primary antibodies for Western blotting.

ATOX1 knockdown inhibits ES-/Cu-induced cuproptosis in AML cells. A, Cell Counting Kit-8 assay for detecting the viability of AML cells transfected with oe-NC or oe-ATOX1 treated with different concentrations of ES/Cu (0, 2, 10, 50, and 200 nmol/L and ES and CuCl2 combined in a 1:1 ratio). AML cells transfected with sh-NC or sh-ATOX1 were treated with 200 nmol/L ES/Cu for 72 hours. B, Cell Counting Kit-8 assay for detecting the viability of AML cells. C, EDU staining assay for detecting the cell proliferation of AML cells. Scale bar, 25 μm. D, Flow cytometry for detecting the cell death of AML cells. E, Western blot analysis of lipoylated DLAT expression in AML cells. F, Western blot analysis of Fe–S cluster protein (FDX1, LIAS, and ACO2) expression in AML cells. Data are shown as mean ± SD. n = 3. SSC, side scatter.

Animal experiments validate the therapeutic potential of the ALKBH5–ATOX1 axis for AML. Female BALB/c nude mice were subcutaneously injected with 1 × 107 NOMO-1 cells in the right axilla. A, Western blot analysis of ATOX1 and ALKBH5 expression in tumor tissues of AML model mice. B, The growth curve of tumor volume in AML model mice. C, Representative images and weight of tumors in AML. D and E, IHC assay for detecting Ki67 expression. Scale bar, 25 and 100 μm. F, Western blot analysis of lipoylated DLAT expression in tumor tissues of AML model mice. G, Western blot analysis of Fe–S cluster protein (FDX1 and LIAS) expression in tumor tissues of AML model mice. Data are shown as mean ± SD. n = 5.

Figure S2. The rescue experiment verified the phenotypic specificity mediated by ATOX1 deletion. AML cells were transfected with sh-ATOX1 and/or oe-ALKBH5 with silent mutations. A. Western blot analysis of ATOX1 expression in AML cells. B. CCK-8 assay for detecting the viability of AML cells. C-D. EDU staining assay for detecting the cell proliferation of AML cells. Scale bar: 25 μm. E. Flow cytometry for detecting the cell death of AML cells. Data are shown as the mean ± SD. n=3.

ATOX1 overexpression alleviates AML progression. A, qRT-PCR and Western blot analysis of ATOX1 expression in AML cells transfected with sh-NC or sh-ATOX1. AML cells were transfected with sh-ATOX1 or oe-ATOX1. B, Western blot analysis of ATOX1 expression in AML cells. C, Cell Counting Kit-8 assay for detecting the viability of AML cells. D, EDU staining assay for detecting the cell proliferation of AML cells. Scale bar, 25 μm. E, Flow cytometry for detecting the cell cycle of AML cells. F, Flow cytometry for detecting the cell death of AML cells. Mortality rate (%) represents the percentage of PI-positive (membrane-compromised necrotic/late apoptotic) cells. Data are shown as mean ± SD. n = 3. SSC, side scatter.

Figure S1. ATOX1 overexpression alleviates AML progression. AML cells were transfected with sh-ATOX1 or oe-ATOX1 for 7 days. A. CCK-8 assay for detecting the viability of AML cells. B. Flow cytometry for detecting the cell death of AML cells. Data are shown as the mean ± SD. n=3.

Cuproptosis, a regulated cell death caused by copper-dependent enzyme overactivation in the tricarboxylic acid cycle, leads to proteotoxic stress. Although the copper chaperone human antioxidant protein 1 (ATOX1) plays a key role in cuproptosis, its link to acute myeloid leukemia (AML) progression remains unclear. In this study, elesclomol (ES) or disulfiram (DSF)/Cu was used to induce cuproptosis, and bathocuproine disulfonic acid (BCS) was used to inhibit it. An AML xenograft mouse model was also established to validate their effects in tumor tissue. Our study demonstrated that ATOX1 is downregulated in AML. Knockdown of ATOX1 promoted cell viability and proliferation, reduced the proportion of cells in the G2-M phase, and decreased cell death. In contrast, overexpression of ATOX1 produced the opposite outcomes. Moreover, ATOX1 knockdown attenuated ES-/Cu-induced cuproptosis in AML cells, whereas ATOX1 overexpression enhanced it. This promoting effect of ATOX1 overexpression was effectively counteracted by the copper chelator BCS. Delving deeper, we discovered that ATOX1 is subject to N6-methyladenosine (m6A) modification mediated by AlkB homolog 5 (ALKBH5). Consequently, ALKBH5 can influence cuproptosis in AML cells by regulating ATOX1 expression. In vivo, the role of the ALKBH5-ATOX1 axis in AML progression has also been confirmed. In conclusion, the demethylase ALKBH5 downregulates ATOX1 by reducing its m6A levels, thereby modulating cuproptosis in AML-a mechanism that offers potential novel insights and therapeutic targets for AML treatment.Significance: This study reveals that in AML, the demethylase ALKBH5 downregulates ATOX1 expression by reducing its m6A modification, thereby inhibiting cuproptosis and promoting AML progression. This mechanism provides a novel potential therapeutic target.

Diosmetin is a bioactive flavonoid that exhibits well-documented antioxidant, anti-inflammatory, and anti-tumor properties. However, its potential to attenuate acute pancreatitis (AP) progression through gut microbiota modulation has not yet been elucidated. In this study, mice were pretreated with varying oral doses of diosmetin for 1 week before AP induction via intraperitoneal (i.p.) caerulein injections. The therapeutic efficacy and optimal dosage were determined through histopathological analysis of pancreatic tissue and serological biomarker assessment. Additionally, transcriptomic profiling and western blot were employed to elucidate the underlying signaling pathways. Furthermore, based on integrated metagenomic and metabolomic analyses, a core gut microbiota-metabolite-gene interaction network modulated by diosmetin was constructed. Finally, fecal microbiota transplantation (FMT) experiments validated the critical role of gut microbiota in the effects of diosmetin against AP. The results showed that medium-dose diosmetin treatment significantly attenuated pancreatic histopathological damage and acinar cell apoptosis in AP mice, while suppressing the activation of the MAPK inflammatory signaling pathway. Notably, diosmetin treatment was associated with restored microbial diversity, altered bacterial community structure, and changes in key metabolic pathways, reversing gut microbiota dysbiosis. Specifically, a diosmetin-responsive interaction network was constructed, highlighting associations between core bacterial taxa (Butyricimonas faecalis, Enterocloster bolteae, Roseburia intestinalis), key metabolites (3-indoleacrylic acid, 2-methoxy-4-vinylphenol, nitrite), and MAPK pathway-related genes. Finally, the protective effect of diosmetin was further substantiated by FMT, suggesting a potential role of the gut microbiota in this process. In conclusion, diosmetin ameliorated pancreatic injury in a murine model of caerulein-induced AP by modulating gut microbiota composition and associated metabolic profiles. These findings suggested that diosmetin represented a promising therapeutic option for AP, offering a scientific foundation for its clinical application and the underlying mechanisms involved.

ALKBH5-mediated m6A modification regulating ATOX1 expression affects cuproptosis in AML cells. AML cells transfected with oe-ATOX1 and/or oe-ALKBH5 were treated with 200 nmol/L ES/Cu for 72 hours. A, Western blot analysis of ATOX1 and ALKBH5 expression in AML cells. B, CCK-8 assay for detecting the viability of AML cells. C, EDU staining assay for detecting the cell proliferation of AML cells. Scale bar, 25 μm. D, Flow cytometry for detecting the cell death of AML cells. E, Western blot analysis of lipoylated DLAT expression in AML cells. F, Western blot analysis of Fe–S cluster protein (FDX1, LIAS, and ACO2) expression in AML cells. Data are shown as mean ± SD. n = 3. SSC, side scatter.

Figure S3. ALKBH5-mediated m6A modification regulating ATOX1 expression affects cuproptosis in AML cells. A. CCK-8 assay for detecting the viability of AML cells transfected with oe-NC or oe-ATOX1 treated with different concentrations of DSF/Cu (0 nM, 2 nM, 10 nM, 50 nM, and 200 nM) for 72 h. AML cells transfected with oe-ATOX1 and/or oe-ALKBH5 were treated with 200 nM DSF/Cu for 72 h. B. CCK-8 assay for detecting the viability of AML cells. C. EDU staining assay for detecting the cell proliferation of AML cells. Scale bar: 25 μm. D. Flow cytometry for detecting the cell cycle of AML cells. E. Flow cytometry for detecting the cell death of AML cells. F. Western blot analysis of lipoylated DLAT expression in AML cells. G. Western blot analysis of Fe-S cluster proteins (FDX1 and LIAS) expression in AML cells. Data are shown as the mean ± SD. n=3.

Sepsis-associated coagulopathy contributes to organ dysfunction and mortality. Macrophage-mediated inflammation disrupts anticoagulant homeostasis, but the role of glutathione S-transferase omega 1 (GSTO1) in this process remains unclear. Clinical samples from sepsis patients were analyzed for correlations among GSTO1, ANXA5, and coagulation parameters were evaluated using Spearman rank correlation analysis. In vitro, loss- and gain-of-function approaches were employed in macrophages to examine the regulatory axis among GSTO1, ANXA5, and HUWE1. Protein interactions, post-translational modifications (S-glutathionylation and ubiquitination), and protein stability were assessed using co-immunoprecipitation, Western blotting, and cycloheximide chase assays. Macrophage polarization and tissue factor activity were detected with a commercial assay kit and flow cytometry. Coagulation function was assessed by incubating macrophage-conditioned media with human plasma and measuring APTT, PT, and TT. In vivo, myeloid-specific GSTO1 knockout mice and pharmacological GSTO1 inhibition were utilized to dissect the function of GSTO1 in a cecal ligation and puncture (CLP)-induced septic mouse model. The results indicated that upregulation of GSTO1 was associated with coagulation abnormalities in sepsis, where it promoted macrophage-mediated coagulopathy. GSTO1 reduced ANXA5 protein stability by facilitating its S-glutathionylation at Cys314 in a catalytic activity-dependent manner. Additionally, GSTO1 promoted HUWE1-dependent ubiquitination and subsequent degradation of ANXA5. ANXA5 overexpression rescued the loss of anticoagulant function caused by GSTO1-induced inflammation in macrophages. In vivo, knockout or inhibition of GSTO1 reduced the incidence of coagulopathy in a CLP-induced septic mouse model. GSTO1 facilitated ANXA5 degradation via S-glutathionylation and HUWE1-mediated ubiquitination, promoting macrophage inflammation and anticoagulation impairment, identifying GSTO1 as a candidate for translational exploration in sepsis-associated coagulopathy.

ATOX1 overexpression promotes ES-/Cu-induced cuproptosis in AML cells. AML cells transfected with oe-NC or oe-ATOX1 were treated with 200 nmol/L ES/Cu for 72 hours. A, Cell Counting Kit-8 assay for detecting the viability of AML cells. B, EDU staining assay for detecting the cell proliferation of AML cells. Scale bar, 25 μm. C, Flow cytometry for detecting the cell death of AML cells. D, Western blot analysis of lipoylated DLAT expression in AML cells. E, Western blot analysis of Fe–S cluster protein (FDX1, LIAS, and ACO2) expression in AML cells. F, Cell Counting Kit-8 assay for detecting the viability of AML cells transfected with oe-NC or oe-ATOX1 and sh-NC or sh-FDX1 treated with different concentrations of ES/Cu (0, 2, 10, 50, and 200 nmol/L) for 72 hours. Data are shown as mean ± SD. n = 3. SSC, side scatter.

ATOX1 expression is affected by ALKBH5-mediated modification of m6A methylation. A, RNA pull-down assay for identifying the interaction of ATOX1 RNA with METTL3, ALKBH5, ELAVL1, and HNRNPC. AML cells were transfected with oe-NC or oe-ALKBH5. B, RIP-qPCR assay for detecting the m6A methylation level of ATOX1 in AML cells. C, qRT-PCR of ATOX1 transcripts in ActD-treated AML cells. D, Western blot analysis of ATOX1 expression in AML cells. AML cells were transfected with sh-NC or sh-ALKBH5. E, Western blot analysis of ATOX1 expression in AML cells. Data are shown as mean ± SD. n = 3.

Acute pancreatitis (AP) has caused great concern worldwide due to its serious threat to human health. Astragalin is a bioactive natural flavonoid compound with several pharmacological activities, but it remains unclear about its effect on AP. The objective of this experiment was to explore the mitigating efficacy of astragalin on caerulein-induced AP model and examine the underlying mechanisms. Following the assessment of astragalin’s direct effects on pancreatic acinar cells using an in vitro AP model, an in vivo mouse model was established to further validate its efficacy and elucidate the underlying mechanisms. Pancreatic histopathology, amylase, and lipase levels of mice were observed to determine the optimal therapeutic dose of astragalin. The network pharmacology and RNA sequencing technology were used to reveal the possible targets and pathways. Subsequent molecular docking and western blot were conducted to validate the association between astragalin and key target molecules, as well as the NLRP3 signaling pathway. Combined with metagenomics and metabolomics analysis, the astragalin effective gut microbiota-metabolite-gene network was constructed. Moreover, fecal microbiota transplantation experiments were performed to clarify the importance of gut microbiota in astragalin-mediated alleviation of AP. The results showed that astragalin attenuated caerulein-induced injury in AR42J cells in vitro. Consistent with these findings, in vivo experiments revealed that astragalin treatment significantly improved pancreatic pathological injury, cell apoptosis, and systemic inflammatory response in AP mice, particularly at high doses. The integrated analysis of network pharmacology and transcriptomics revealed that the NLRP3 signaling pathway was a key molecular pathway, which was further validated using western blot. Docking analysis showed that 12 target genes had good docking activity with astragalin. More intriguingly, it was found that astragalin could reverse gut microbiota dysbiosis by restoring microbial diversity, altering bacterial community composition, and modulating key metabolic pathways. Specifically, astragalin-effective correlation networks were constructed with Lachnoclostridium sp. YL32, Roseburia intestinalis, Ruminococcus gnavus, Lachnospiraceae bacterium Choco86, Anaerobutyricum hallii, etc. as the core strains, 22 metabolites, including 5-Methoxytryptophan, D-Serine, L-Tryptophan, L-Methionine, etc. as core metabolites, and NLRP3 pathway-related genes as the main regulatory targets. Furthermore, fecal microbiota transplantation experiments confirmed the involvement of gut microbiota in AP remission. Collectively, these findings identify astragalin as a promising therapeutic agent for AP, targeting both the NLRP3 signaling cascade and gut microbial homeostasis.

Acute pancreatitis (AP) is a self-limiting inflammatory disorder, but severe cases can lead to persistent organ failure with high mortality. Metabolic dysregulation and inflammatory activation play critical roles in AP pathogenesis, highlighting the metabolic-inflammation crosstalk as a potential therapeutic target. Although riboflavin, an essential water-soluble vitamin, has been implicated in modulating disease processes, its role in AP remains unclear. In this study, untargeted metabolomics identified significant riboflavin downregulation in an AP mouse model. Subsequent in vivo experiments demonstrated that riboflavin intervention (25, 50, and 100 mg/kg) ameliorated pancreatic injury and systemic inflammation, with 50 mg/kg exhibiting optimal efficacy. Targeted metabolomics revealed elevated acetate levels following riboflavin supplementation. At the same time, transcriptomic and molecular biology assays showed riboflavin-mediated downregulation of HDAC3, a key acetate downstream target, and suppression of NF-κB pathway activation. In vitro, riboflavin and acetate mitigated pancreatic acinar cell damage, including apoptosis and necrosis, and inhibited NF-κB signaling. Rescue experiments using the HDAC3 inhibitor RGFP966 further provided pharmacological evidence for a mechanistic link between the acetate-HDAC3 axis and riboflavin's protective effects. Collectively, these findings reveal that riboflavin alleviates AP, and its effect is associated with the modulation of the acetate-HDAC3 axis, offering a novel therapeutic strategy for this condition.

Background Acute pancreatitis (AP) initiates as primarily sterile local inflammation that triggers pro-inflammatory response, which is subsequently counterbalanced by an anti-inflammatory response. Immune checkpoints, such as PD-1/PD-L1, play a pivotal role in modulating these responses to prevent excessive immune activation and associated inflammatory damage. This study aimed to investigate the underlying mechanisms of these processes in both murine and human AP. Methods We conducted a comprehensive integration of data from cerulein-induced AP mouse models (CER-AP), utilizing single-cell RNA sequencing and digital spatial profiling for pancreatic samples, as well as single-cell Cytometry by Time Of Flight (CyTOF) for blood samples. Additionally, bulk-RNA sequencing performed on blood samples from AP patients was employed to investigate innate and adaptive immune changes at early stage of the disease. Results Across the four analytical approaches, we observed consistent immune cell type distributions. Our integrative analysis revealed a significant imbalance between increased innate immune cells, including neutrophils, macrophages, and monocytes, and decreased adaptive immune cells, including CD4+ and CD8+ T cells, in early-stage AP. Notably, the PD-1/PD-L1 related pathway exhibited substantial alterations, especially in the acinar cells, T cells, B cells, macrophages, and neutrophils at the early stage of disease. Moreover, we observed a significant reduction in PD-L1 expression in the blood and regulatory T cells of CyTOF mice at the CyTOF level. Conclusion This multi-omics analysis deciphers a distinct imbalance between increased innate immunity and decreased adaptive immunity during the early phase of AP. The PD-L1 checkpoint emerges as a key regulator of immune homeostasis and a critical factor in the pathogenesis of AP.

BACKGROUND: Acute pancreatitis (AP) is an acute abdominalgia with complicated pathogenesis and high mortality, which is lacking in specific means for clinical diagnosis and treatment. Currently, numerous traditional Chinese medicines have demonstrated remarkable efficacy in AP. Given their multi-target and multi-compound actions, we hypothesize that an underlying common mechanism may contribute to their therapeutic effects. This study aimed to identify key therapeutic targets and potential strategies for AP by investigating the shared pharmacological effects of medicinal plants through network pharmacology analysis and experimental validation. METHODS: We systematically searched the literature for medicinal herbs that have been reported in AP treatment. Next, we utilized the TCMSP database to identify active compounds that were present in at least two medicinal herbs. Key active compounds and targets were determined through Cytoscape analysis and a PPI network, followed by KEGG pathway enrichment analysis. Combined the core targets identified by Cytoscape and the targets enriched in the PI3K/AKT signaling pathway, molecular docking was performed to assess the binding affinity between the intersecting targets and active compounds. Finally, high-affinity compounds were screened, and linarin’s optimal binding profile led to its selection for further in vivo and in vitro experimental validation. RESULTS: A total of 37 medicinal herbs were retrieved from the literature search. We identified 62 common compounds and 968 targets from medicinal herbs, further taking intersection to 319 targets for anti-AP. Based on this, “compound-target” and “target” networks were constructed, and the top 12 key active compounds and 11 targets were selected. KEGG analysis indicated that the PI3K/AKT pathway might be closely related to pancreatic protection. Molecular docking results showed that linarin exhibited good binding affinity with all core intersecting targets, particularly with AKT1. Subsequently, both in vivo and in vitro experiments demonstrated that linarin could alleviate AP-induced pancreatic damage and systemic inflammation. To further validate the mechanistic involvement of PI3K/AKT signaling pathway, we employed the PI3K/AKT activator 740 Y-P, which was found to effectively reverse linarin-mediated downregulation of PI3K/AKT activation, thereby confirming the crucial role of this pathway in linarin’s protective effects. CONCLUSION: Exploring therapeutic strategies based on common mechanisms and targets may be an effective approach. This study revealed that linarin and AKT1 were potential therapeutic compounds and targets for AP in the preclinical stage, which could provide theoretical support and new insights for the drug discovery of AP.

BACKGROUND:The mortality rate for severe cases of acute pancreatitis (AP), a common gastrointestinal emergency, is as high as 30%. Our previous study has shown that rutaecarpine (Rut) has a therapeutic effect on AP. AIM:To investigate the role of F-box and WD repeat domain containing 11 (FBXW11) in AP models and to assess whether Rut mitigates AP by regulating FBXW11. METHODS:AP rat model was established and treated with Rut, followed by biochemical analysis of serum amylase and lipase, hematoxylin and eosin staining of pancreatic tissue, and immunohistochemistry detection of pancreatic Ly6G, CD11b, and myeloperoxidase. Assay kits were used to detect oxidative stress-related indicators in pancreatic tissue and inflammatory factors in serum. AR42J cells were treated with cerulein to model AP and subjected to Cell Counting Kit-8 viability assay, flow cytometry apoptosis assay, and immunofluorescence detection of reactive oxygen species to elucidate the mechanistic involvement of the enhancer of zeste homolog 2 (EZH2)-FBXW11 axis in Rut-mediated protection against AP. The EZH2-histone H3 binding and H3 methylation were evaluated using co-immunoprecipitation. RESULTS:Rut treatment ameliorated AP severity, as evidenced by reduced serum levels of pancreatic enzymes (amylase and lipase) and attenuated histological damage. Rut also decreased inflammatory markers (interleukin-1 beta, interleukin-6, and tumor necrosis factor alpha), tissue oxidative stress (malondialdehyde), and neutrophil infiltration (Ly6G, CD11b, and myeloperoxidase) levels in rats with AP. Moreover, Rut restored pancreatic antioxidant capacity (glutathione and superoxide dismutase). In vitro, Rut pre-incubation enhanced cell viability and suppressed cerulein-induced apoptosis and oxidative stress. Rut increased EZH2 expression while decreasing FBXW11 expression. FBXW11 overexpression eliminated the protective effect of Rut against AP. Further analysis revealed that EZH2 binds to H3 and upregulates H3 methylation levels, thereby inhibiting FBXW11 expression. CONCLUSION:Collectively, our findings demonstrate that Rut ameliorates AP by upregulating EZH2, thereby enhancing H3 methylation and suppressing FBXW11 expression.

Acute pancreatitis (AP) is an inflammatory disease of the pancreas. Despite of a steadily increasing in morbidity and mortality, there is still no effective therapy. Gut microbial dysbiosis and its derived-metabolites disorder have been shown to play an important role in the development of AP, however, little is known regarding the crosstalk between gut microbiota and metabolites. In this study, we assessed the alterations in gut microbiota and metabolites by constructing three AP mouse models by means of metagenomic and metabolomic sequencing, and further clarified their relationship by correlation analysis. The results revealed that each model exhibited unique flora and metabolite profiles. KEGG analysis showed that the differential flora and metabolite-enriched pathway functions were correlated with lipid metabolism and amino acid metabolism. Moreover, two core differential bacterial species on Burkholderiales bacterium YL45 and Bifidobacterium pseudolongum along with eleven differential metabolites appeared to exert certain effects during the course of AP. In conclusion, further exploration of the crosstalk between microbiota and derived metabolites may provide novel insights and strategies into the diagnosis and treatment of AP.