Application of DNA methyl donors in the preparation of drugs for the treatment of severe acute pancreatitis and / or its complications, and methods for screening candidate drugs.

Abstract

This invention discloses the application of DNA methyl donors in the preparation of drugs for treating severe acute pancreatitis and / or its complications, as well as a method for screening candidate drugs. The application of DNA methyl donors provided by this invention in the preparation of drugs for treating severe acute pancreatitis and / or its complications, by supplementing the methyl donor, restores the methylation level of the Foxp1 enhancer, blocks the "FOXP1-CXCR4" axis upstream, significantly reduces pancreatic microthrombus formation and alleviates tissue necrosis, and avoids the systemic side effects of directly antagonizing CXCR4.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of a DNA methyl donor in the preparation of a drug for treating severe acute pancreatitis and / or its complications, and a method for screening candidate drugs. Background Technology

[0002] Severe acute pancreatitis (SAP) is a critical illness characterized by rapid onset, rapid progression, numerous complications, and high mortality. Its typical pathological features include pancreatic microcirculatory disturbances, inflammatory storms, and multiple organ dysfunction. Among these, microthrombus formation due to excessive platelet activation is a key pathological factor contributing to pancreatic microcirculatory disturbances and ischemic necrosis. Clinical studies show that SAP patients exhibit a decrease in platelet count and a significant increase in platelet activation markers (such as P-selectin and PF4) in the early stages, and the degree of platelet activation is positively correlated with the severity of pancreatic necrosis.

[0003] Chemokine receptor CXCR4 is one of the important functional receptors on the platelet surface. Its binding to its ligand CXCL12 significantly enhances platelet adhesion, aggregation, and spreading. Previous studies have reported significantly elevated platelet CXCR4 expression in SAP animal models and patients. The use of CXCR4 antagonists requires systemic blockade of this receptor, which can adversely affect normal physiological processes such as hematopoietic stem cell homing and immune cell migration, thus limiting its clinical application. Therefore, how to achieve precise upstream regulation of CXCR4 function without directly blocking its function has become an urgent technical problem to be solved.

[0004] In recent years, studies have found that abnormal gene expression in disease states is not only regulated by transcription factors but also closely related to epigenetic modifications such as DNA methylation. Abnormal DNA demethylation can lead to abnormally high expression of specific genes under inflammatory conditions. However, research on the relationship between abnormal DNA methylation and CXCR4 expression regulation in platelets and their precursor megakaryocytes is still relatively lacking, and there are no mature epigenetic intervention protocols for the prevention and treatment of SAP-related abnormal platelet activation.

[0005] Therefore, there is an urgent need to establish a new regulatory strategy that can regulate CXCR4 expression at the epigenetic level, thereby inhibiting abnormal platelet activation and for the effective treatment of severe acute pancreatitis. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides the application of DNA methyl donors in the preparation of drugs for treating severe acute pancreatitis and / or its complications, as well as a method for screening candidate drugs.

[0007] Preferably, the mechanism of action of the drug is as follows: under inflammatory stimulation, by providing DNA methyl donors, it inhibits DNA demethylation of the Foxp1 gene regulatory element in megakaryocytes, thereby downregulating the transcriptional expression of Foxp1, and thus inhibiting the transcription and expression of Cxcr4.

[0008] Preferably, the Foxp1 gene regulatory element is an enhancer region.

[0009] Preferably, the enhancer region is located downstream of the Foxp1 gene transcription start site.

[0010] Preferably, the DNA methyl donor is S-adenosylmethionine or a pharmaceutically acceptable salt thereof.

[0011] Preferably, the drug is used for: (1) Reduce pancreatic microthrombus formation; (2) Reduce pancreatic tissue necrosis; (3) Inhibit abnormal platelet activation, including increased platelet aggregation rate, increased spreading area, or increased P-selectin exposure; and / or (4) Improve microcirculatory disturbances caused by acute pancreatitis.

[0012] A second aspect of the present invention provides a method for screening candidate drugs for the prevention or treatment of severe acute pancreatitis and / or its complications, the method being performed in vitro and comprising the following steps: (1) Provide megakaryocyte or platelet systems expressing FOXP1 and CXCR4; (2) Add inflammatory stimuli to the system to establish an inflammation model; (3) Bring the candidate drug into contact with the inflammation model; (4) Detect the DNA methylation level of the Foxp1 gene enhancer region in the system, or detect the expression level of FOXP1 or CXCR4; (5) Compare the detection results of step (4) with the control group that has not been exposed to the candidate drug; If, compared with the control group, the candidate drug can inhibit DNA demethylation in the enhancer region of the Foxp1 gene, or reduce the expression levels of FOXP1 or CXCR4, then the candidate drug is deemed to have potential activity in preventing or treating severe acute pancreatitis and thrombotic diseases.

[0013] Preferably, the inflammatory stimulant in step (2) is TNF-α; and / or, the DNA demethylation of the Foxp1 gene enhancer region in step (4) is mediated by TET enzyme.

[0014] A third aspect of the present invention provides the application of a reagent for detecting the expression levels of CXCR4 and / or FOXP1 in the preparation of a diagnostic product for assisting in the assessment of the severity or prognosis of severe acute pancreatitis; wherein the diagnostic product is used to detect isolated platelet samples from subjects, and elevated expression levels of CXCR4 and / or FOXP1 in the platelet samples are positively correlated with high severity or poor prognosis of severe acute pancreatitis.

[0015] Preferably, the diagnostic product further includes a reagent for detecting the DNA methylation level in the enhancer region of the Foxp1 gene; wherein, the hypomethylation state of the enhancer region is positively correlated with increased Foxp1 transcriptional activity.

[0016] By adopting the above technical solution, the present invention has the following beneficial effects: The DNA methyl donor provided by this invention, in the preparation of drugs for treating severe acute pancreatitis and / or its complications, elucidates for the first time the epigenetic mechanism of platelet overactivation in severe acute pancreatitis (SAP), namely, the cascade reaction of "inflammation-TET enzyme-Foxp1 enhancer demethylation-FOXP1 upregulation-CXCR4 upregulation". Unlike the systemic side effects such as impaired hematopoietic stem cell mobilization that may result from the direct use of CXCR4 antagonists in existing technologies, this invention achieves specific inhibition of pathological CXCR4 overexpression by regulating the methylation state of the Foxp1 gene enhancer, preserving its physiological function and exhibiting higher safety.

[0017] Furthermore, this invention demonstrated using megakaryocyte-specific gene knockout mice (Foxp1 or Cxcr4 knockout) that blocking this pathway reduces platelet aggregation by approximately 28-38%, microthrombus formation by approximately 64%, and pancreatic necrosis by approximately 45%. Further, drug intervention using a DNA methyl donor (S-adenosylmethionine, SAMe) showed that SAMe significantly reversed the upregulation of FOXP1 and CXCR4, reducing pancreatic microthrombi by approximately 76%, necrosis by approximately 75%, and the overall injury score by approximately 66%, demonstrating significantly better efficacy than single anti-inflammatory therapy.

[0018] 3. In the application of the DNA methyl donor provided by this invention in the preparation of medicaments for treating severe acute pancreatitis and / or its complications, the preferred DNA methyl donor is S-adenosylmethionine (SAMe). S-adenosylmethionine is a clinically marketed drug with detailed human safety data. At an effective therapeutic dose (25 mg / kg), no bleeding tendency or other organ toxicity was observed. This "drug repurposing" strategy greatly reduces drug development risks, shortens the clinical translation cycle, and provides a new treatment option for severe acute pancreatitis and its complications (such as microcirculatory disturbances and multiple organ failure).

[0019] 4. This invention provides a method for screening candidate drugs for the prevention or treatment of severe acute pancreatitis and / or its complications. It simulates the pathological state of megakaryocytes / platelets under the inflammatory environment of SAP. By detecting the recovery of DNA methylation level in the enhancer region of the Foxp1 gene, or the downregulation of FOXP1 / CXCR4 expression, it can rapidly and efficiently screen for potential active compounds that treat SAP and thrombotic diseases by regulating epigenetic mechanisms (such as inhibiting TET enzyme-mediated demethylation). This provides a reliable tool and evaluation system for the development of new antithrombotic drugs.

[0020] 5. Application of reagents for detecting CXCR4 and / or FOXP1 expression levels in the preparation of diagnostic products for assisting in the assessment of the severity or prognosis of severe acute pancreatitis (SAP). This involves using the expression levels of CXCR4 and / or FOXP1 in platelets, as well as the DNA methylation level in the enhancer region of the Foxp1 gene, as novel biomarkers. By detecting isolated platelet samples from subjects, physicians can be assisted in assessing the severity and prognostic risk of SAP patients (elevated expression levels or decreased methylation levels indicate severe disease or poor prognosis). This provides a convenient, sensitive, and specific detection method for early warning, stratified treatment, and efficacy monitoring of SAP in clinical practice. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 The relationship between microthrombus formation and pancreatic necrosis in severe acute pancreatitis; Figure 1 In the middle section, A represents coagulation indicators in patients with AP of varying severity. Figure 1 In the middle section B: Immunohistochemistry of CD42c-positive platelet thrombi in pancreatic tissue of SAP patients; Figure 1 C represents the gross morphology of the pancreas and H&E staining at different time points in the SAP mouse model. Figure 1 D in the middle represents the H&E staining pathology score; Figure 1 E represents the temporal changes in plasma enzyme levels. Figure 1 F represents the temporal changes of inflammatory factors. Figure 1 G represents the temporal changes in platelet-derived mediators. Figure 1 The middle H represents the microthrombus density map. Figure 1 In section I, correlation analysis of necrosis scores is performed. Figure 2 Results related to SAP-induced platelet overactivation; Figure 2 In the middle, A represents the spread of platelets on the fibrinogen-coated surface in SAP patients and healthy volunteers; Figure 2 2B represents the platelet aggregation response of SAP mice to collagen, U46619, and thrombin at different time points. Figure 2 C represents the temporal changes in platelet spreading function; Figure 3 The upregulation of CXCR4 in SAP mediates microthrombosis and necrosis-related outcomes. Figure 3 In section A: Flow cytometry detection of CXCR4 and CXCR7 expression on platelet surface in SAP patients and healthy controls; Figure 3 B represents the expression of Cxcr4 mRNA in platelets of SAP mice. Figure 3 C represents: Immunohistochemistry of CXCL12 in pancreatic tissue; Figure 3 D represents the effect of AMD3100 treatment on microthrombi. Figure 3 The Chinese E refers to the impact of AMD3100 treatment on pathology. Figure 3 The middle F represents the impact of AMD3100 treatment on pathological scores. Figure 3 The central term is: the effect of AMD3100 treatment on plasma enzymes (plasma lipase + plasma amylase). Figure 3 H represents the effect of AMD3100 treatment on plasma TXB2. Figure 3 The middle part (I) refers to the effect of AMD3100 treatment on inflammatory factors. Figure 4 The results suggest that platelet-specific CXCR4 deficiency inhibits platelet activation and is associated with pancreatic injury. Figure 4 A in the table refers to the Cxcr4MK-KO mouse construction strategy and Western blot validation. Figure 4 B stands for: platelet aggregation function. Figure 4 C represents: P-selectin exposure (P-selectin expression) graph. Figure 4 D represents the spreading function (mean platelet area) graph. Figure 4 E represents: Microthrombus density map. Figure 4 The image in Figure F shows the results of plasma TXB2 levels. Figure 4 G in the image represents the H&E staining results. Figure 4 H in the middle represents: pathological scoring chart; Figure 4 Figure I shows the results of plasma enzyme analysis. Figure 4 The image in the middle (J) shows the results of inflammatory factor analysis. Figure 5 Figure showing the results related to FOXP1 transcriptional activation of Cxcr4; Figure 5 A in the middle is a heatmap for screening candidate transcription factors; Figure 5B in the middle section refers to the in vitro differentiation and inflammatory stimulation process of megakaryocytes. Figure 5 C represents the dose-dependent expression of FOXP1 and CXCR4 proteins in TNF-α. Figure 5 D in the image shows the binding peak of FOXP1 at the Cxcr4 locus, as revealed by CUT & Tag sequencing. Figure 5 In the middle, E represents: ac: quantitative analysis of binding site distribution and signal intensity; d: motif analysis to identify co-transcriptional factors. Figure 5 F represents the change in bonding strength as shown by sliding window analysis. Figure 5 Heavy G is: dual-luciferase reporter gene verification; Figure 6 Megakaryocyte-specific FOXP1 loss reduces pancreatic injury-related outcomes by inhibiting CXCR4 expression; Figure 6 A in the table represents: a: the strategy for constructing Foxp1MK-KO mice; b and c: Western blot and immunofluorescence verification of decreased FOXP1 and CXCR4 expression; Figure 6 B represents the platelet aggregation result. Figure 6 The image in center C shows the platelet spreading results (average platelet area). Figure 6 D represents the result of P-selectin cell membrane level. Figure 6 E in the middle refers to plasma TXB2 levels; Figure 6 F represents the microthrombus density result; Figure 6 G in the middle represents the H&E pathology score; Figure 6 H: Plasma enzyme results Figure 6 I represents the results of inflammatory factors; Figure 7 The study investigated findings related to inflammation-induced DNA demethylation upregulating FOXP1 and methyl supplementation reducing pancreatic damage. Figure 7 A represents the TET enzyme activity in bone marrow megakaryocytes of SAP mice or TNF-α-treated mice. Figure 7 B represents the whole genome and promoter methylation level of the Foxp1 locus; Figure 7 Heavy C represents: methylation changes of different types of regulatory elements; Figure 7 D represents a lollipop diagram of demethylation of a single regulatory element; Figure 7 Figure E shows the effect of SAMe on TNF-α-induced upregulation of FOXP1 and CXCR4; Figure 7F shows the effect of SAMe treatment on pancreatic histopathology. Figure 7 G in the text refers to the pathological score of pancreatic tissue after SAMe treatment. Figure 7 H represents the effect of SAMe treatment on plasma enzymes (plasma amylase + plasma lipase). Figure 7 The first part, I, is about the effect of SAMe treatment on plasma TKB2. Figure 7 The central question is: What is the effect of SAMe treatment on inflammatory factors? Figure 7 The middle K is: the effect of SAMe treatment on microthrombi; Figure 8 For methylation analysis and SAMe treatment efficacy; Figure 8 In the middle, A represents the inter-sample correlation in WGBS. Figure 8 In the middle B section, the correlation between enhancer methylation and Foxp1 expression is indicated. Figure 8 C represents the individual correlation between four significant demethylation elements and FOXP1 expression; Figure 8 D in the middle represents: SAMe dose gradient analysis; Figure 8 E in the middle represents: H&E staining images of different doses of SAMe; Figure 8 F represents the pathological scores for different doses of SAMe. Figure 8 The value of G is: the effect of SAMe (25 mg / kg) treatment on plasma IL-1β and IL-1α. Detailed Implementation

[0023] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] The relevant English descriptions in the accompanying drawings of this invention are as follows: CD42c: A platelet-specific surface marker that identifies platelets and microthrombi. CD41: Megakaryocyte / Platelet Marker FOXP1: Forkhead Box Transcription Factor P1 CXCR4: CXC chemokine receptor type 4 CXCL12: CXC chemokine ligand 12 P-selectin (CD62P): a marker of platelet activation H&E: Hematoxylin-eosin staining TSS: Transcription start site RPKM: Number of valid reads per kilobase length per million segments RE: Relative expression level FC: Multiple Change MFI: Mean fluorescence intensity AOD: Average optical density.

[0025] This invention reveals a complete "inflammation-epigenetics-transcription-function" regulatory chain: inflammatory stimulation can cause DNA demethylation in the regulatory region of the Foxp1 gene in megakaryocytes, and the demethylation further upregulates the transcriptional expression level of Foxp1; after the expression of FOXP1 increases, it drives the enhanced transcription of the Cxcr4 gene, which significantly increases the expression of CXCR4 on the platelet surface, thereby amplifying the platelet response to chemokine signals, inducing platelet overactivation and promoting microthrombus formation.

[0026] Specific mechanism: Under inflammatory stimulation, inflammatory factors (such as TNF-α) can significantly activate the activity of TET enzymes in megakaryocytes. These TET enzymes catalyze the demethylation of 5-methylcytosine in the regulatory elements of the Foxp1 gene, particularly resulting in significant DNA demethylation changes in the enhancer region of the Foxp1 gene. After demethylation of the enhancer region, its transcriptional activation capacity is significantly enhanced, thereby promoting Foxp1 gene transcription and further upregulating FOXP1 protein expression. Upregulated FOXP1 expression enhances the transcriptional activity of the Cxcr4 gene by forming a chromatin loop between the enhancer and the Cxcr4 gene promoter, leading to a significant increase in CXCR4 protein expression on the platelet surface. Increased CXCR4 expression significantly enhances the chemotactic response of platelets to its ligand CXCL12, amplifying platelet activation signal transduction and ultimately leading to platelet overactivation.

[0027] Genomic localization analysis revealed that the mouse Cxcr4 gene is located on chromosome 1, Chr1: 128,515,936-128,520,036. Based on CUT & Tag sequencing results, a systematic analysis of the binding of FOXP1 at the entire Cxcr4 locus was performed. The results showed that the FOXP1 binding signal was not concentrated in the traditional promoter region, but was mainly enriched in the gene body region downstream of the transcription start site (TSS), which had high binding confidence. Under inflammatory stimulation, the enrichment intensity of FOXP1 in this gene body region was significantly increased by approximately 2.17 times compared to the control group, suggesting that FOXP1's regulation of CXCR4 in the inflammatory microenvironment exhibits a stimulation-dependent enhancement characteristic.

[0028] Further motif analysis of the FOXP1 binding peaks within the chr1 range (128, 518, 127-128, 519, 439 base pairs) of the Cxcr4 gene body region identified three core binding motifs, corresponding to typical DNA binding sites of three transcription factors: ZNF263, SP1, and KLF5. These three motifs showed almost no high-confidence enrichment peaks at rest, only exhibiting significant FOXP1 enrichment signals after inflammatory stimulation, indicating that FOXP1 recruitment depends on inflammatory signal-driven transcriptional complex recombination.

[0029] In addition to the identified downstream region of TSS (chr1: 128518127-128519439), other FOXP1 binding sites may exist at the Cxcr4 locus: Alternative site 1: TSS upstream promoter region (within -2000 bp) Although CUT&Tag showed low FOXP1 enrichment in this region (only 0.68-fold), it may play a role in certain cell types or under certain stimulating conditions, which can be further verified by chromatin immunoprecipitation (ChIP).

[0030] Substitution site 2: Distal enhancer (distance from TSS > 5kb) Genomic studies suggest that Cxcr4 may have long-range regulatory elements, and 3D chromatin conformation capture (Hi-C or 4C) can identify long-range interactions. These remote elements may be selectively activated in different tissues or disease states.

[0031] Alternation site 3: Intron region of the gene The Cxcr4 gene contains one intron, which may contain regulatory elements. The intron enhancer plays a role in the transcriptional elongation stage, and ATAC-seq can identify open chromatin regions within the intron.

[0032] Comprehensive analysis of localization and motif characteristics reveals that FOXP1's transcriptional regulation of Cxcr4 does not follow the classic "direct promoter binding" model. Instead, it primarily functions by binding to regulatory elements with enhancer properties within the gene body region. Further protein-protein interaction and motif analysis indicates that FOXP1 itself does not act as a direct DNA-binding factor, but rather indirectly mediates the regulation of Cxcr4 transcription by forming a co-transcriptional complex with DNA-binding proteins such as ZNF263, SP1, and KLF5. Furthermore, the enhancer-like region bound by FOXP1 can form a spatial looping structure between the enhancer and promoter through chromatin three-dimensional conformational changes, thereby achieving remote regulation of transcription initiation region activation.

[0033] Cooperative transcriptional regulation mechanism Based on motif enrichment analysis of FOXP1 binding peak sites, a variety of transcription factors synergistically interact with FOXP1 were systematically identified, with ZNF263, SP1, and KLF5 being the most significant. ZNF263, a member of the zinc finger protein family, primarily provides sequence specificity for DNA binding; SP1, a broad-spectrum transcription factor, participates in the stability of the transcription initiation complex; and KLF5, a Krüppel-like factor, significantly enhances gene transcription activity. These three constitute the core synergistic network for FOXP1-dependent regulation of Cxcr4 expression. A dual-luciferase reporter system was constructed for the Cxcr4 promoter region, and site-directed mutagenesis was performed on the FOXP1 promoter binding sites. The results showed that mutating only the FOXP1 binding sites in the promoter region reduced Cxcr4 transcriptional activity by only about 19%, suggesting that direct promoter regulation has a limited contribution to overall transcriptional activation. This result further confirms that the main transcriptional activation of Cxcr4 originates from an indirect mechanism of "enhancer-co-factor-chromatin spatial circular regulation." Localization analysis of the functional enhancer within the CXCR4 gene revealed that this enhancer is approximately 597 bp from the transcription start site. Under inflammatory stimulation, CTCF-mediated chromatin spatial structure was significantly enhanced, and the physical proximity between the enhancer and the promoter was significantly increased, thereby promoting a significant upregulation of Cxcr4 transcription levels.

[0034] Preferably, in addition to ZNF263, SP1, and KLF5, other transcription factors may be involved in FOXP1-mediated Cxcr4 regulation: NF-κB family: classic downstream transcription factors stimulated by inflammation, which may synergistically activate Cxcr4 with FOXP1. Inhibiting the NF-κB pathway may be a combination therapy strategy.

[0035] The AP-1 complex, composed of Fos and Jun proteins, is widely involved in the transcription of inflammatory genes and may interact with the Cxcr4 promoter through TRE (TPA response element).

[0036] STAT transcription factors: key effectors in cytokine signaling pathways. STAT3 or STAT5 may be involved in Cxcr4 regulation, and JAK inhibitors may have synergistic therapeutic effects.

[0037] DNA methylation regulatory mechanism In both in vivo SAP-induced and TNF-α-stimulated models, TET enzyme activity was significantly increased in bone marrow-derived megakaryocytes. TET enzyme can catalyze the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), thereby initiating an active DNA demethylation process, suggesting that inflammatory stimulation can reshape the epigenetic state of the Foxp1 gene through a TET-mediated demethylation pathway.

[0038] Whole-genome bisulfite sequencing (WGBS) results showed that, at the whole-genome level, TNF-α stimulation only caused a slight decrease in overall methylation (83.4% ± 1.3% in the PBS group and 77.7% ± 5.8% in the TNF-α group). The Foxp1 promoter region, however, showed a slight trend of hypermethylation (from 54.4 ± 10.9% to 69.6 ± 7.3%). In contrast, the overall demethylation level of Foxp1 enhancer elements reached 5.7%, with six enhancers showing demethylation exceeding 10%, and one site reaching statistical significance. Furthermore, overall demethylation in open chromatin regions reached 11.9%, and two CTCF binding sites also showed demethylation exceeding 15%.

[0039] Correlation analysis showed that the methylation level of Foxp1 enhancers was significantly negatively correlated with FOXP1 protein expression, and the site with the highest degree of demethylation was most significantly associated with the upregulation of FOXP1 protein, clearly indicating that enhancer demethylation is a key functional epigenetic mechanism driving the increase in FOXP1 expression.

[0040] The Foxp1 locus exhibits a complex regulatory pattern of "enhancer demethylation + promoter hypermethylation." Enhancer demethylation establishes the basis for transcriptional activation (competence), while promoter hypermethylation maintains precise quantitative control over transcriptional levels. This dual epigenetic checkpoint mechanism prevents abnormally low-threshold activation while simultaneously enabling highly efficient and strongly responsive transcriptional activation under inflammatory stimulation.

[0041] In addition to changes in DNA methylation, this invention suggests that other epigenetic regulatory mechanisms may also be involved in the regulation of Foxp1 gene expression and may serve as alternatives to or supplements to the method of this invention.

[0042] First, regarding histone modifications, the enhancer or promoter regions of the Foxp1 gene may exhibit synergistic changes with multiple modification types. For example, H3K27ac, a classic marker of active enhancer, may show an enrichment trend in the Foxp1 enhancer region, thereby promoting its transcriptional activation; H3K4me3, a common modification of active promoters, may increase with upregulation of Foxp1 expression; conversely, a reduction in the repressive modification H3K27me3 may relieve transcriptional repression of Foxp1, further promoting its expression. Therefore, regulating histone acetylation or methylation levels (e.g., by applying histone deacetylase inhibitors, HDAC inhibitors) can serve as an alternative intervention strategy for regulating Foxp1 transcription and expression in this invention.

[0043] Secondly, regarding chromatin remodeling, the SWI / SNF complex may play a crucial role in the chromatin opening process at the Foxp1 gene locus. The deletion or alteration of the activity of its core subunits BRG1 or BRM may directly affect the accessibility of Foxp1-related enhancers or promoters, thereby influencing transcriptional levels. Therefore, regulation of the chromatin remodeling process (including compounds that inhibit the activity of the SWI / SNF complex) theoretically has potential therapeutic value and can serve as a supplementary intervention method in this invention.

[0044] Furthermore, non-coding RNAs may also participate in the regulation of Foxp1 expression. For example, specific microRNAs can affect the stability or translation efficiency of Foxp1 mRNA; long non-coding RNAs (lncRNAs) may participate in conformational changes in the Foxp1 regulatory region by binding to chromatin; and circular RNAs (circRNAs) can act as competitive binding molecules for microRNAs, thereby indirectly regulating the expression level of FOXP1. These various RNA regulatory mechanisms can serve as alternatives to or parallel pathways to the Foxp1 regulatory system described in this invention, providing expanded possibilities for the development of further therapeutic strategies.

[0045] In vitro cell model validation: A megakaryocyte differentiation model was established using fetal hepatocytes induced by TPO, and the simulated inflammatory microenvironment was treated with TNF-α (1 ng / mL, 12 h). Western blot results showed that both FOXP1 and CXCR4 proteins were significantly and synergistically upregulated after inflammatory stimulation, validating the in vitro reproducibility of the molecular regulatory axis. Megakaryocyte / platelet-specific Cxcr4MK-KO mice (Cxcr4fl / fl; Pf4-Cre+ / -) were constructed, exhibiting a 28–38% reduction in platelet aggregation, a 64% reduction in microthrombus formation, and a 45% reduction in pancreatic necrosis. Simultaneously, Foxp1MK-KO mice (Foxp1fl / fl; Pf4-Cre+ / -) were constructed, showing a significant decrease in CXCR4 expression and exhibiting high consistency with Cxcr4MK-KO mice in platelet aggregation, microthrombus formation, and pancreatic necrosis phenotypes, confirming that FOXP1 exerts its biological function by regulating CXCR4.

[0046] Intervention was performed using the methyl donor S-adenosylmethionine (SAMe). In in vitro experiments, 240 μM SAMe significantly reversed TNF-α-induced upregulation of FOXP1 and CXCR4 expression. In in vivo experiments, intraperitoneal injection of 25 mg / kg SAMe inhibited microthrombus formation by approximately 76%, reduced pancreatic necrosis by approximately 75%, decreased the overall injury score by approximately 66%, and significantly improved plasma enzyme indices and inflammatory factor levels.

[0047] Alternative technologies for pharmacological intervention In addition to using S-adenosylmethionine (SAMe) as a methyl donor for intervention, this invention also proposes a variety of alternative or complementary pharmacological regulatory strategies for intervening in the FOXP1-CXCR4 signaling axis and its mediated abnormal platelet activation process.

[0048] One approach involves using TET enzyme inhibitors as demethylation blockers. Directly inhibiting TET enzyme activity with small molecule compounds such as Bobcat339 can block DNA demethylation, inhibiting demethylation activation in the Foxp1 enhancer region at its source. This type of intervention is more specific to its target than SAMe, but its long-term safety, biotoxicity, and tissue specificity still require further systematic evaluation.

[0049] Secondly, DNMT agonists are used to enhance DNA methylation maintenance. These drugs enhance the catalytic activity of DNA methyltransferases (DNMTs), thereby strengthening the maintenance of methylation of Foxp1 gene enhancers or key regulatory elements and inhibiting their abnormal activation. This strategy is complementary to the methyl donor mechanism of SAMe, but the number of clinically available compounds is limited, and novel agonists need to be developed specifically for this purpose.

[0050] Third, epigenetic synergistic regulation can be achieved using histone methyltransferase regulators. For example, EZH2 inhibitors (such as Tazemetostat) have been used in clinical cancer treatment. They affect gene transcriptional repression by regulating H3K27me3 levels, and are expected to indirectly regulate Foxp1 transcriptional activity. However, the efficacy and safety of these drugs in severe acute pancreatitis (SAP) and related platelet hyperresponsiveness still need further validation in animal models.

[0051] Fourth, direct targeted intervention using FOXP1 protein degraders. PROTAC (proteolytic-targeting chimera) technology can be used to construct protein-degrading molecules targeting FOXP1, achieving rapid and complete clearance of FOXP1 protein through the ubiquitin-proteasome pathway. This strategy can achieve more direct and efficient functional inhibition at the post-transcriptional level, but its in vivo stability, off-target effects, and immune safety require systematic evaluation.

[0052] In addition to conventional in vivo drug delivery methods, this invention also proposes a variety of delivery schemes that can improve targeting and treatment efficiency.

[0053] Firstly, a nanoparticle-targeted delivery system is employed. Lipid nanoparticles (LNPs) are used to encapsulate siRNA or regulatory molecules targeting Foxp1, and further, platelet membrane coating technology is utilized to construct a homologous targeted nanodelivery system. This significantly improves the drug accumulation efficiency in megakaryocytes and platelets, reducing systemic exposure and adverse reactions.

[0054] Secondly, long-term regulation can be achieved using cell therapy strategies. This involves gene editing or Foxp1 knockdown of autologous hematopoietic stem cells in vitro, followed by reinfusion into the body. This allows the cells to differentiate into a platelet population with low CXCR4 expression and controlled function, thus enabling long-term stable management of high-risk thrombosis. This approach is particularly suitable for chronic high-risk patients requiring long-term intervention, but its genetic stability, tumor risk, and immune safety still require rigorous evaluation.

[0055] Based on this, the present invention forms a complete and reproducible technical route, including: screening transcription factors by RT-qPCR and determining the strongest positive correlation between FOXP1 and CXCR4; accurately locating the binding site of FOXP1 at the Cxcr4 locus using CUT&Tag sequencing; establishing the enhancer-mediated indirect regulatory mode through motif analysis and dual-luciferase assays; identifying functional demethylation elements by combining TET enzyme activity detection and WGBS; conducting multi-level causal verification through tissue-specific gene knockout mice and SAP models; and finally ensuring the transformation from mechanism to treatment through SAMe pharmacological intervention.

[0056] In addition to platelet hyperactivation and microthrombus formation associated with severe acute pancreatitis (SAP), the FOXP1-CXCR4 regulatory axis disclosed in this invention can also be extended to a variety of disease areas closely related to platelet hyperresponsiveness, inflammatory response and thrombosis.

[0057] In the field of thrombotic diseases, it can be used for the prevention and treatment of diseases such as deep vein thrombosis, pulmonary embolism, and ischemic stroke.

[0058] In the field of inflammatory diseases, it can be used for systemic inflammatory diseases such as sepsis, acute respiratory distress syndrome (ARDS), and inflammatory bowel disease, which are accompanied by abnormal platelet activation.

[0059] In the field of cardiovascular diseases, it can be used to intervene in thrombosis-related complications in diseases such as acute myocardial infarction, atherosclerosis, and unstable angina.

[0060] In the area of ​​tumor-associated thrombosis, the FOXP1-CXCR4 axis may be involved in abnormal platelet activation in the tumor microenvironment and the formation of thrombi associated with tumor metastasis. Treatment for this type of disease requires parameter balancing and dosage optimization between antithrombotic therapy and tumor progression control to achieve the best comprehensive therapeutic effect.

[0061] The following detailed description is based on specific embodiments.

[0062] In the implementation of this invention, the key experimental conditions and detection parameters are set as follows.

[0063] Regarding cell experimental conditions, megakaryocyte differentiation was induced using thrombopoietin (TPO), with a final TPO concentration controlled at 15 IU / mL and a culture time of 4–5 days to ensure that fetal liver cells fully differentiated into megakaryocytes. In the inflammatory stimulation model, tumor necrosis factor-α (TNF-α) was used as the stimulating factor, with an effective concentration set at 1 ng / mL and a stimulation duration of 12 hours, to simulate the effect of the inflammatory microenvironment on megakaryocytes. In the methylation regulation intervention experiment, S-adenosylmethionine (SAMe) was used as the methyl donor in in vitro and in vivo experiments, with an in vitro treatment concentration of 240 μM and an in vivo administration dose of 25 mg / kg. The animal disease model was induced using severe acute pancreatitis (SAP) via retrograde injection of 4% sodium taurocholate into the pancreatic duct at a rate controlled at 0.4 mL / h for 10 min to ensure model stability and reproducibility.

[0064] Regarding detection indicators and judgment criteria, transcriptional levels were detected using real-time quantitative PCR (RT-qPCR), with the control group as a baseline. A significant upregulation was defined as a relative upregulation of the target gene exceeding 1.5-fold. Protein expression levels were detected using Western blot, with a significant upregulation defined as a relative upregulation exceeding 1.3-fold. Platelet function evaluation included platelet aggregation rate, spreading area, and P-selectin exposure levels to comprehensively reflect platelet activation status. Histopathological evaluation used hematoxylin and eosin (H&E) staining, and the degree of pancreatic tissue damage was quantitatively assessed according to the Xuanfu Xu scoring system. Microthrombus formation was detected using immunohistochemical staining targeting CD42c, with the proportion of positively stained area used as a quantitative indicator of microthrombus burden.

[0065] Example 1: Transcription factor screening and identification of FOXP1 This embodiment provides a screening method for upstream transcriptional regulatory factors of CXCR4, used to identify key transcription factors that are significantly associated with CXCR4 expression.

[0066] Whole blood was collected from severely acute pancreatitis (SAP) mice (12 h post-modeling) and sham-operated control mice. After centrifugation to remove erythrocytes and leukocytes, platelets were isolated and purified. Total RNA was extracted from platelets using standard kits, and the mRNA expression levels of target genes were detected using RT-qPCR. Based on the Cistrome database and relevant literature search results, candidate transcription factors with potential Cxcr4 regulatory functions were selected for screening, including: Foxp1 (forkhead box transcription factor), Bcl6 (B-cell lymphoma 6 protein), Myc (proto-oncogene transcription factor), Notch1 (Notch signaling pathway core transcription factor), Ezh2 (histone methyltransferase), and Foxo1 (forkhead box transcription factor O1).

[0067] RT-qPCR test results (see) Figure 5 China A and Figure 3 Figure B shows that compared with the control group, the mRNA expression level of Foxp1 in SAP mouse platelets was upregulated by approximately 4.87-fold (P<0.05), and the mRNA expression level of Cxcr4 was upregulated by approximately 1.63-fold (P<0.05); no significant changes were observed in the expression of other candidate transcription factors between the SAP group and the control group. These results indicate that Foxp1 is significantly upregulated in SAP platelets, and its upregulation is consistent with that of CXCR4. Therefore, FOXP1 is identified as the most likely key transcription factor regulating CXCR4 expression.

[0068] Example 2: Establishment of an in vitro megakaryocyte inflammation model This embodiment constructs an in vitro megakaryocyte inflammation model to verify the regulatory role of inflammatory factors on the expression of FOXP1 and CXCR4.

[0069] Fetal liver tissue was isolated from C57BL / 6 pregnant mice at gestational age of 13.5–14.5 days and dispersed mechanically to obtain suspension cells. These cells were seeded into a culture system containing DMEM basal medium, 15% fetal bovine serum (FBS), 15 IU / mL recombinant mouse thrombopoietin (TPO), and conventional penicillin / streptomycin, and cultured at 37 ℃ and 5% CO2 for 5–7 days. After culture, the cells showed a significant increase in volume, polyploid nuclei, and morphological characteristics consistent with megakaryocytes.

[0070] Subsequently, different concentrations of recombinant mouse TNF-α (0, 0.5, 1, 2 ng / mL) were added to the differentiated megakaryocytes. After 12 h of stimulation, cell lysates were collected, and the expression levels of FOXP1 and CXCR4 proteins were detected by Western blot. The results are as follows: Figure 5As shown in B-5C: Treatment with TNF-α 0.5 ng / mL upregulated FOXP1 protein expression by approximately 1.73-fold and CXCR4 by approximately 1.25-fold; treatment with 1 ng / mL upregulated FOXP1 by approximately 3.04-fold and CXCR4 by approximately 1.41-fold, representing the optimal stimulation conditions; treatment with 2 ng / mL upregulated FOXP1 and CXCR4 by approximately 1.93-fold and 1.33-fold, respectively. Therefore, 1 ng / mL and 12 h were determined to be the preferred inflammatory stimulation conditions for inducing FOXP1 / CXCR4 axis activation in this model.

[0071] Example 3: CUT & Tag sequencing to identify the binding site of FOXP1 at the Cxcr4 locus. This embodiment uses CUT&Tag sequencing technology to identify the binding mode and key regulatory regions of FOXP1 at the Cxcr4 locus.

[0072] The megakaryocyte model established in Example 2 was used, divided into a control group (untreated megakaryocytes) and a TNF-α treatment group (1 ng / mL, 12 h). 1 × 10⁵ cells were collected from each group, and the standard CUT&Tag experimental procedure was followed: cells were first fixed by incubating with concanavalin A-coated magnetic beads, then permeabilized, followed by incubation overnight at 4 °C with primary antibody against FOXP1 (catalog number 61309, Proteintech). After washing, the secondary antibody-pA-Tn5 transposase complex was added and incubated. The transposase was then activated by Mg²⁺, inserting a sequencing adapter at the antibody binding site. After the reaction, DNA was extracted, a library was constructed, and high-throughput sequencing was performed on the Illumina platform.

[0073] Sequencing data analysis results show that ( Figure 5 DE: Single-sample sequencing depth exceeded 20 million reads, with an alignment rate greater than 90% to the GRCm39 / mm39 mouse reference genome. Peak calling was performed using SEACR software at an FDR < 0.01 threshold, and motif analysis was performed using MEME Suite. Within the Cxcr4 locus Chr1: 128515936–128521036 range, the control group showed only weak binding signals, failing to reach the peak threshold, with an average RPKM of 25.7. The TNF-α group showed a significant enrichment peak, mainly located in the gene body region downstream of the transcription start site (TSS), with an overall average RPKM of 40.27 (approximately a 1.57-fold increase). Among them, the RPKM in the gene body region was 44.30 (approximately a 2.17-fold increase), while the RPKM in the promoter region was 23.71 (approximately a 0.68-fold decrease).

[0074] Further analysis revealed that the high-confidence FOXP1 binding region was concentrated in the chr1:128518127–128519439 segment, and three core motifs were identified in this region, which highly matched the classic binding sequences of ZNF263, SP1, and KLF5, respectively. These results indicate that under inflammatory conditions, FOXP1 primarily binds to enhancer-like elements within the Cxcr4 gene, exerting its transcriptional regulatory effect on Cxcr4 through this region, rather than simply relying on direct promoter binding.

[0075] Example 4: Dual-luciferase reporter gene validation of the effect of FOXP1 on Cxcr4 promoter activity This embodiment utilizes a dual-luciferase reporter system to verify the regulatory role of FOXP1 on Cxcr4 motor activity and to evaluate the contribution of direct promoter binding to overall regulation.

[0076] Based on the CUT&Tag sequencing results in Example 3, the Cxcr4 promoter region (-547 to +90 bp relative to TSS) was amplified and cloned into the pGL3-basic firefly luciferase vector to construct the wild-type promoter reporter plasmid pCxcr4-wt-luc. Simultaneously, four site-directed mutagenesis were performed on the predicted FOXP1 binding motif to construct the FOXP1 binding site mutant plasmid pCxcr4-mut-luc. Additionally, the CMV promoter-driven full-length Foxp1 cDNA expression plasmid pFoxp1 and the corresponding empty vector pΔFoxp1 were constructed.

[0077] HEK293T cells were seeded in 24-well plates and transfected with the following plasmid combinations as experimental and control groups: (1)p Cxcr4-wt-luc+pΔFoxp1; (2) pCxcr4-wt-luc+pFoxp1; (3) p Cxcr4-mut-luc+pΔFoxp1; (4) pCxcr4-mut-luc+pFoxp1; (5) pGL3-basic+pΔFoxp1; (6) pGL3-basic+pFoxp1.

[0078] Simultaneously, pRL-TK Renilla luciferase plasmid was co-transfected as an internal control. Forty-eight hours after transfection, the ratio of firefly to Renilla luciferase activity was determined using a dual-luciferase assay kit and then standardized.

[0079] The results show that ( Figure 5 (Middle FG): The normalized fluorescence ratio of group (1) pCxcr4-wt-luc+pΔFoxp1 was recorded as 1.0 (baseline); The ratio of pCxcr4-wt-luc+pFoxp1 in group (2) was 1.15, suggesting that FOXP1 overexpression could increase the activity of the Cxcr4 promoter by about 1.15 times. Group (3) is similar to group (1), with a value of 0.93; The ratio of pCxcr4-mut-luc+pFoxp1 in group (4) was still about 0.93, which was about 19% lower than the pre-mutation FOXP1 activation.

[0080] The above results indicate that although FOXP1 can directly activate the Cxcr4 promoter, mutations at the promoter binding site only lead to a limited decrease in transcriptional activity. This suggests that the main regulation of Cxcr4 by FOXP1 originates from indirect regulation mediated by enhancer-co-factor-chromatin loop structure, rather than direct promoter binding.

[0081] Example 5: Construction of megakaryocyte / platelet-specific Cxcr4 and Foxp1 gene knockout mice In this embodiment, megakaryocyte / platelet-specific CXCR4 and Foxp1 gene knockout mice were constructed to verify in vivo the role of the FOXP1-CXCR4 axis in platelet function and pancreatic injury.

[0082] 5.1 Construction of Cxcr4MK-KO mice Cxcr4f / f mice (constructed by the Shanghai Southern Model Organisms Center using CRISPR / Cas9 technology) were used to cross with Pf4-Cre mice (Jackson Laboratory, serial number 008535). The first generation was crossbred with Cxcr4f / f × Pf4-Cre to obtain Cxcr4fl / -; Pf4-Cre+ / - and Cxcr4fl / -; Pf4-Cre- / - mice. Cxcr4fl / -; Pf4-Cre+ / - were then crossed with Cxcr4fl / -; Pf4-Cre- / - to obtain Cxcr4fl / fl; Pf4-Cre+ / - (denoted as Cxcr4MK-KO, experimental group) and Cxcr4fl / fl; Pf4-Cre- / - (denoted as Cxcr4fl / fl, control group).

[0083] Genomic DNA was extracted from mouse tail tissue, and genotyping was performed using PCR. Further Western blot analysis of CXCR4 protein levels in platelet lysates revealed significantly reduced CXCR4 protein expression in Cxcr4MK-KO mouse platelets, confirming specific knockout of Cxcr4 in the megakaryocyte / platelet lineage.

[0084] 5.2 Construction of Foxp1MK-KO mice Using the same strategy, Foxp1f / f mice (constructed using CRISPR / Cas9 technology at the Shanghai Southern Model Organisms Center) and Pf4-Cre mice were bred. First, Foxp1f / f × Pf4-Cre mice were crossed to obtain Foxp1fl / -; Pf4-Cre+ / - and Foxp1fl / -; Pf4-Cre- / - mice. These were then mated to obtain Foxp1fl / fl; Pf4-Cre+ / - (denoted as Foxp1MK-KO, experimental group) and Foxp1fl / fl; Pf4-Cre- / - (denoted as Foxp1fl / fl, control group). Genotyping was performed using tail DNA PCR, and FOXP1 protein expression in platelet lysates was detected by Western blot, while FOXP1 signaling in bone marrow megakaryocytes was detected by immunofluorescence. The results showed that FOXP1 expression was significantly decreased in platelets and megakaryocytes of Foxp1MK-KO mice, indicating that Foxp1 knockout of the megakaryocyte / platelet lineage was successful.

[0085] 5.3 Basic phenotypic analysis Basic phenotypic assessments were performed on the two gene knockout mice, including body weight, general appearance, complete blood count (white blood cell count, red blood cell count, platelet count, and mean platelet volume), and reproductive capacity. The results showed that the above indicators in both Cxcr4MK-KO and Foxp1MK-KO mice were within the normal range and showed no significant differences compared to the corresponding control group, indicating that gene knockout does not cause significant developmental defects or hematopoietic system disorders (see [link to relevant documentation]). Figure 4 China A Figure 6 (A)

[0086] Example 6: Establishment of SAP mouse model and functional verification of gene knockout mice In this embodiment, the gene knockout mice constructed in Example 5 were applied to the SAP model to verify the function of the FOXP1 / CXCR4 axis in platelet activation and pancreatic injury.

[0087] 6.1 Methods for Inducing SAP Models Mice aged 8–10 weeks were selected, fasted overnight but with free access to water, and anesthetized with an intraperitoneal injection of sodium pentobarbital at a dose of 50 mg / kg. Under aseptic conditions, a midline abdominal incision was made to expose the duodenum. A 30G needle was used to puncture the bile and pancreatic ducts through the duodenal wall, and the ducts were temporarily clamped proximal to the common hepatic duct. Subsequently, 4% sodium taurocholate solution was injected retrogradely into the bile and pancreatic ducts at a rate of 0.4 mL / h for 10 minutes, after which the injection was stopped, the clamp was released, and the abdominal cavity was sutured until the mice regained consciousness. The sham-operated group underwent only laparotomy and suturing, without the injection of sodium taurocholate.

[0088] 6.2 Functional phenotype of Cxcr4MK-KO mice in SAP Cxcr4fl / fl and Cxcr4MK-KO mice were randomly divided into four groups (n=8-9 in each group): (1) Cxcr4fl / fl+Sham; (2) Cxcr4fl / fl+SAP; (3) Cxcr4MK-KO+Sham; (4) Cxcr4MK-KO+SAP.

[0089] Mice were sacrificed 12 hours after SAP induction, and blood and pancreatic tissue were collected. Platelet function tests showed that the platelet aggregation rate in the Cxcr4MK-KO+SAP group was approximately 28-38% lower than that in the Cxcr4fl / fl+SAP group (P<0.01), the platelet spreading area was reduced by approximately 42% (P<0.05), and the P-selectin exposure level was reduced by approximately 28% (P<0.01). CD42c immunohistochemical results indicated that the proportion of pancreatic microthrombus-positive area in the Cxcr4MK-KO+SAP group was approximately 64% lower than that in the Cxcr4fl / fl+SAP group (P<0.001). H&E pathological scoring showed that the pancreatic necrosis score was reduced by approximately 45% (P<0.001) and the overall damage score was reduced by approximately 54% (P<0.001) in the Cxcr4MK-KO+SAP group, without an increased risk of bleeding. Plasma amylase and lipase levels decreased by approximately 29% (P<0.05 and P<0.01), respectively, and TXB2 decreased by approximately 23% (P<0.01), indicating that Cxcr4 knockout significantly improved SAP-induced pancreatic injury and platelet hyperresponsiveness.

[0090] 6.3 Functional phenotype of Foxp1MK-KO mice in SAP Using the same experimental grouping and SAP induction method as in 6.2, functional evaluations were performed on Foxp1MK-KO and its control mice. Results showed that the Foxp1MK-KO + SAP group exhibited a decrease in platelet aggregation rate of approximately 33–37%, a reduction in microthrombus formation of approximately 57%, a decrease in pancreatic necrosis score of approximately 55%, and significant improvements in related plasma markers, highly consistent with the phenotype of Cxcr4MK-KO mice.

[0091] 6.4 Validation of CXCR4 Expression CXCR4 expression was detected in Foxp1MK-KO and control mice. Western blot results showed that in the sham group, the platelet CXCR4 protein level of Foxp1MK-KO mice was reduced by approximately 86.8% compared with the control group (P<0.01); in the SAP group, the platelet CXCR4 protein level of Foxp1MK-KO mice was reduced by approximately 86.3% (P<0.01). Immunofluorescence detection showed that the CXCR4 signal intensity in bone marrow megakaryocytes of Foxp1MK-KO mice was reduced by approximately 53.0% and 66.0% under sham and SAP conditions, respectively (P<0.01). These results indicate that FOXP1 drives platelet activation and pancreatic damage by regulating CXCR4 expression. The consistency of functional phenotypes in the two gene knockout models suggests that this pathway has a single and well-defined pathological pathway (see [link to relevant documentation]). Figure 4 A, Figure 6 A).

[0092] Example 7: Whole-genome bisulfite sequencing analysis of DNA methylation changes at the Foxp1 locus This embodiment analyzes the DNA methylation reprogramming characteristics of Foxp1 gene-related regions under inflammatory stimulation using whole-genome bisulfite sequencing (WGBS).

[0093] Using the megakaryocyte model described in Example 2, cells were divided into a PBS control group and a TNF-α (1 ng / mL) treatment group. Genomic DNA (≥500 ng) was extracted from each group, and sulfite conversion was performed using the EZ DNA Methylation-Gold Kit. Subsequently, methylated sequencing libraries were constructed using the Accel-NGS Methyl-Seq DNA Library Kit, and sequencing was performed on the Illumina NovaSeq platform in PE150 mode. The sequencing yield of each sample was greater than 120 Gb.

[0094] Sequencing reads were aligned to the GRCm39 / mm39 reference genome using Bismark software, achieving an alignment rate greater than 95%, an average genome coverage of approximately 26×, and a sulfite conversion rate greater than 99.5%. Differential methylation analysis was performed using MethylKit, with a minimum coverage depth of 10×, a differential threshold of |Δmethylation|>10%, and q<0.05.

[0095] The results show that ( Figure 7 Chinese AD, Figure 8In the AC group, the average whole-genome methylation level was 83.4% ± 1.3% in the control group and 77.7% ± 5.8% in the TNF-α group, with no statistically significant difference (P = 0.232). The methylation level in the Foxp1 promoter region (2 kb upstream of the TSS) increased from 54.4% ± 10.9% to 69.6% ± 7.3%, showing a slight hypermethylation trend (P = 0.125). Among the 21 identified Foxp1 enhancer elements, the average demethylation was approximately 6%, with 6 enhancers showing demethylation greater than 10% and 1 element showing approximately 18%, which was statistically significant (q = 0.032). In the two open chromatin regions, the average demethylation was approximately 12%, with one region showing demethylation greater than 15%. Among the five CTCF binding sites, the overall methylation change averaged approximately 1%, but two sites showed demethylation greater than 15%.

[0096] The five enhancer elements with the highest demethylation levels were further extracted, their average methylation levels were calculated, and Pearson correlation analysis was performed with FOXP1 protein expression levels measured by Western blot. The results showed a significant negative correlation between the two (r = -0.823, P = 0.044). Analysis of the top four significantly demethylated elements showed correlation coefficients of r = -0.89 (P = 0.018), r = -0.85 (P = 0.032), r = -0.78 (P = 0.067), and r = -0.82 (P = 0.045), respectively. These results indicate that inflammatory stimulation can induce targeted DNA demethylation of Foxp1 regulatory elements (especially enhancers), and there is a functional causal relationship between this demethylation change and the upregulation of FOXP1 protein expression.

[0097] Example 8: SAMe reverses DNA demethylation and alleviates pancreatic damage This embodiment verifies the intervention effect of DNA methyl donor SAMe on the FOXP1 / CXCR4 axis and SAP pathological process in vivo and in vitro.

[0098] 8.1 In vitro SAMe treatment Using the megakaryocyte inflammation model from Example 2, cells were divided into a PBS control group, a TNF-α group (1 ng / mL), and a TNF-α + SAMe combined treatment group. Different concentration gradients of SAMe were used in the combined treatment group: 0, 240, 480, and 960 μM. After 12 h of stimulation, Western blot was used to detect the protein levels of FOXP1 and CXCR4. The results showed that at a concentration of 240 μM, FOXP1 expression was reduced by approximately 94% compared to the TNF-α treatment group alone, and CXCR4 expression was reduced by approximately 76.4%, which was the optimal concentration; at 480 μM, the reductions were approximately 85% and 66%, respectively; at 960 μM, the inhibitory effect was similar, but cell viability was significantly decreased. Therefore, 240 μM was determined to be the preferred intervention concentration of SAMe in this in vitro experiment. Figure 7 E, etc.

[0099] 8.2 In vivo SAMe treatment and dosage exploration Simultaneously with the establishment of the SAP mouse model, mice were treated with different doses of SAMe via intraperitoneal injection, divided into a carrier group (physiological saline), a low-dose group (12.5 mg / kg), a medium-dose group (25 mg / kg), and a high-dose group (37.5 mg / kg). Mice were sacrificed 12 hours after model establishment, and blood and pancreatic tissue were collected for multi-parameter evaluation.

[0100] 8.3 Evaluation of the efficacy of SAMe 25 mg / kg treatment group At a dose of 25 mg / kg, platelet aggregation rate was reduced by approximately 46% (P<0.01), spreading area was reduced by approximately 38% (P<0.05), and P-selectin membrane expression level was reduced by approximately 52% (P<0.01) compared to the carrier SAP group. CD42c immunohistochemical results showed a reduction of approximately 76% in the proportion of pancreatic microthrombi (P<0.001); H&E pathological analysis indicated a reduction of approximately 75% in pancreatic necrosis score (P<0.001) and approximately 66% in overall injury score (P<0.001), with no increase in bleeding risk observed. Plasma amylase levels were reduced by approximately 42% (P<0.01), lipase by approximately 47% (P<0.01), and TXB2 by approximately 63% (P<0.001), indicating that 25 mg / kg SAMe significantly alleviated SAP-related pancreatic injury and excessive platelet activation. Figure 7 Middle FK, Figure 8 DG (China)

[0101] 8.4 Dose-response relationship analysis A moderate therapeutic effect was observed at a dose of 12.5 mg / kg; at a dose of 25 mg / kg, the improvement in various functional indicators reached 60-75%, which was the optimal dose for overall efficacy; although 37.5 mg / kg still had some therapeutic effect, it was not significantly better than 25 mg / kg. Therefore, 25 mg / kg was determined to be the preferred dose for in vivo SAMe treatment in this invention.

[0102] Example 9: Human Sample Validation and Clinical Translation Correlation Analysis This embodiment tests human SAP patient samples to verify the applicability of animal model results under clinical conditions.

[0103] 9.1 Source of Clinical Samples Seven patients with acute pancreatitis (SAP) were recruited. The inclusion criteria were onset time <72 hours and compliance with the 2012 revised Atlanta classification criteria; patients with chronic pancreatitis, malignant tumors, and hematological diseases were excluded. Seven age- and sex-matched healthy volunteers were recruited concurrently as controls, all of whom were confirmed to be free of acute and chronic diseases through routine physical examinations.

[0104] 9.2 Platelet CXCR4 Expression Detection Platelet CXCR4 protein expression was detected by flow cytometry using anti-CXCR4-PE antibody for labeling, with two stimulation conditions: thrombin-free (0.01 U / mL, 20 min) and thrombin-free. Results showed that at baseline, the mean fluorescence intensity (MFI) of platelet CXCR4 in SAP patients was 59.67±0.11, higher than that in the control group (53.37±3.06); under thrombin stimulation, the MFI in SAP patients was 68.91±11.85, while that in the control group was 56.60±5.72 (P<0.05). Calculations showed that CXCR4 expression in SAP patients was approximately 1.12-fold and 1.21-fold higher than that in the control group at baseline and under stimulation conditions, respectively. Figure 1 B, Figure 2 A, Figure 3 A).

[0105] 9.3 Platelet Function Testing Platelet spreading assays were performed using a fibrinogen-coated matrix. Results showed that the platelet spreading area in SAP patients was 40.32±3.22 μm², significantly larger than the 25.26±3.01 μm² in the healthy control group (P<0.01), indicating that platelets in SAP patients were in a state of hyperresponsiveness.

[0106] 9.4 Detection of microthrombi in necrotic tissue Immunohistochemical staining of CD42c in tissue specimens obtained from debridement of pancreatic necrosis in two SAP patients revealed numerous CD42c-positive microthrombus structures within the necrotic tissue. These results indicate that platelet CXCR4 upregulation and functional overactivation also exist in human SAP, consistent with animal experimental results, supporting the clinical relevance and translational potential of the mechanism described in this invention.

[0107] Example 10: Mechanism Integration and Summary of Action Paths Based on the results of Examples 1 to 9, this example systematically integrates the mechanism of action of the FOXP1-CXCR4 regulatory axis in SAP, forming a complete pathological-molecular-functional-therapeutic chain.

[0108] First, during the inflammation-triggered phase (see Example 6), SAP-induced inflammatory factors (such as TNF-α, IL-1β, IL-6, etc.) are produced by the body and act on bone marrow megakaryocytes. Second, during the epigenetic reprogramming phase (see Example 7), TNF-α stimulation activates TET enzymes, promoting targeted DNA demethylation in the Foxp1 gene enhancer and open chromatin regions, altering the local chromatin state. Subsequently, during the transcription factor activation phase (see Examples 1-2), the demethylated enhancer regions promote Foxp1 gene transcription, leading to the accumulation of FOXP1 protein, which then participates in target gene regulation as a regulatory core.

[0109] During the Cxcr4 transcriptional regulation phase (see Examples 3-4), FOXP1, along with co-transcription factors such as ZNF263, SP1, and KLF5, binds to the enhancer-like element within the Cxcr4 gene. This forms an enhancer-promoter chromatin loop, effectively activating the Cxcr4 transcription initiation region and leading to a significant upregulation of CXCR4 expression. During the platelet phenotypic alteration phase (see Example 6), platelets with high CXCR4 expression differentiated from the aforementioned megakaryocytes exhibit enhanced responses to CXCL12 signaling, manifested as significantly improved aggregation, spreading, and degranulation abilities.

[0110] In the pathological cascade stage, CXCR4 mediates platelet chemotaxis and aggregation towards damaged pancreatic tissue. Overactivated platelets form numerous microthrombi within microvessels, leading to pancreatic microcirculatory disturbances and ischemic necrosis (see Example 6). Regarding therapeutic intervention (see Examples 8-9), SAMe is used to reverse Foxp1 regulatory element demethylation, block FOXP1 upregulation, and inhibit CXCR4 expression, thereby reducing platelet activation, microthrombus formation, and pancreatic necrosis, achieving significant improvement in the course of SAP. This invention thus establishes a continuous pathway of action from inflammatory stimulation, epigenetic reprogramming, transcriptional regulation to platelet function alterations and tissue damage, and provides feasible pharmacological intervention strategies.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of DNA methyl donors in the preparation of medicaments for the treatment of severe acute pancreatitis and / or its complications.

2. The application according to claim 1, characterized in that, The mechanism of action of the drug is as follows: under inflammatory stimulation, by providing DNA methyl donors, it inhibits DNA demethylation of the Foxp1 gene regulatory element in megakaryocytes, thereby downregulating the transcriptional expression of Foxp1, and subsequently inhibiting the transcription and expression of Cxcr4.

3. The application according to claim 2, characterized in that, The Foxp1 gene regulatory element is an enhancer region.

4. The application according to claim 3, characterized in that, The enhancer region is located downstream of the transcription start site of the Foxp1 gene.

5. The application according to any one of claims 1-4, characterized in that, The DNA methyl donor is S-adenosylmethionine or a pharmaceutically acceptable salt thereof.

6. The application according to claim 5, characterized in that, The drug is used for: (1) Reduce pancreatic microthrombus formation; (2) Reduce pancreatic tissue necrosis; (3) Inhibit abnormal platelet activation, including increased platelet aggregation rate, increased spreading area, or increased P-selectin exposure; and / or (4) Improve microcirculatory disturbances caused by acute pancreatitis.

7. A method for screening candidate drugs for the prevention or treatment of severe acute pancreatitis and / or its complications, characterized in that, The method is performed in vitro and includes the following steps: (1) Provide megakaryocyte or platelet systems expressing FOXP1 and CXCR4; (2) Add inflammatory stimuli to the system to establish an inflammation model; (3) Bring the candidate drug into contact with the inflammation model; (4) Detect the DNA methylation level of the Foxp1 gene enhancer region in the system, or detect the expression level of FOXP1 or CXCR4; (5) Compare the detection results of step (4) with the control group that has not been exposed to the candidate drug; If, compared with the control group, the candidate drug can inhibit DNA demethylation in the enhancer region of the Foxp1 gene, or reduce the expression levels of FOXP1 or CXCR4, then the candidate drug is deemed to have potential activity in preventing or treating severe acute pancreatitis and thrombotic diseases.

8. The method according to claim 7, characterized in that, The inflammatory stimulant mentioned in step (2) is TNF-α; and / or, the DNA demethylation of the Foxp1 gene enhancer region mentioned in step (4) is mediated by TET enzyme.

9. The application of reagents for detecting CXCR4 and / or FOXP1 expression levels in the preparation of diagnostic products for the auxiliary assessment of the severity or prognosis of severe acute pancreatitis; characterized in that, The diagnostic product is used to detect isolated platelet samples from subjects, and elevated expression levels of CXCR4 and / or FOXP1 in the platelet samples are positively correlated with high severity or poor prognosis of severe acute pancreatitis.

10. The application according to claim 9, characterized in that, The diagnostic product also includes reagents for detecting DNA methylation levels in the enhancer region of the Foxp1 gene; wherein, the hypomethylation state of the enhancer region is positively correlated with increased Foxp1 transcriptional activity.

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