Application of inhibitor Acevaltrate targeting PCBP2 in preparation of medicine for treating allergic asthma

Abstract

The invention discloses application of an inhibitor Acevaltrate targeting PCBP2 in preparation of a medicine for treating allergic asthma, and belongs to the technical field of biological medicine. The invention discloses application of PCBP2 as a molecular target of a drug for treating allergic asthma. The core pathological mechanism of allergic asthma is closely related to M2 polarization of macrophages, and PCBP2 positively drives the polarization process by regulating an STAT6 signal channel. Acevaltrate can specifically inhibit the biological function of PCBP2 and block M2 polarization of macrophages induced by IL-4 / IL-13, so that airway inflammation is relieved, over-generation of IgE (immunoglobulin E) is inhibited, and airway remodeling is improved. The invention further provides a pharmaceutical composition containing the Acevaltrate, a drug combination scheme of the Acevaltrate and an existing asthma treatment drug, and a drug screening system based on a PCBP2-M2 polarization axis. The technology breaks through the limitation of an existing asthma treatment target spot, realizes the trans-boundary application of Acevaltrate from an anti-tumor drug to an anti-asthma drug, provides an accurate and efficient novel treatment strategy for allergic asthma, especially refractory asthma, and has remarkable clinical transformation value and wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically the application of Acevaltrate, an inhibitor targeting PCBP2, in the preparation of drugs for treating allergic asthma. Background Technology

[0002] Allergic asthma is a chronic inflammatory airway disease triggered by allergens, affecting over 350 million people worldwide, and the prevalence continues to rise, making it a significant public health issue. In my country, the prevalence of asthma in people aged 20 and above is 4.2%, and the prevalence in children has nearly doubled in the past thirty years, with significant regional differences. Its clinical manifestations include recurrent episodes of wheezing, shortness of breath, chest tightness, and cough. Characteristic pathological changes include airway hyperresponsiveness, reversible airflow limitation, mucus hypersecretion, and progressive airway remodeling. Immunological features are primarily marked by elevated serum IgE levels, a shift in the type 2 helper T cell (Th2) immune response, and eosinophilic infiltration. A small number of patients develop refractory asthma, responding poorly to traditional optimized treatment regimens based on inhaled corticosteroids, necessitating novel treatment strategies.

[0003] In the airway inflammatory microenvironment, the polarization state of macrophages has a decisive influence on the pathological process of asthma. Macrophages are highly plastic and can polarize into classical activated (M1) and alternative activated (M2) types. In the Th2-type inflammatory environment of asthma (mainly driven by IL-4 and IL-13), macrophages polarize into the M2 phenotype, highly expressing markers such as mannose receptor (CD206, gene name: Mrc1), arginase 1 (Arg1), and chitinase-like protein 3 (Ym1). M2 macrophages drive the persistence of airway inflammation and the progression of airway remodeling by promoting eosinophil recruitment and activation, enhancing Th2 immune responses, and stimulating airway fibroblast proliferation and collagen deposition. Therefore, targeted regulation of macrophage M2 polarization has become an important intervention direction in the field of asthma treatment.

[0004] Polycytosine-binding protein 2 (PCBP2) is a highly conserved RNA-binding protein that recognizes cytosine-rich RNA sequences through its KH domain. It plays a central role in post-transcriptional gene regulation and is also involved in innate immune regulation and ferroptosis suppression. Based on the stabilizing effect of PCBP2 on the mRNAs of cytokines such as IL-10 and TGF-β, it is theoretically possible that PCBP2 could promote the expression of M2-related factors through post-transcriptional regulation. However, this hypothesis has not yet been experimentally verified in the pathological context of asthma, and its function in allergic asthma remains a research gap.

[0005] Acevaltrate (acetylvaleric triester, ACE) is a natural iridoid compound. Current technology has only reported its use as a ferroptosis inducer in the treatment of colorectal cancer, with a mechanism of action targeting PCBP1 / 2 and GPX4 to interfere with the ferroptosis defense system. This study focuses entirely on the application of acevaltrate in cancer treatment, without involving any respiratory disease models, and without exploring its potential value in immune-inflammatory diseases. Notably, this study shows that acevaltrate has an inhibitory effect on PCBP2, which offers the possibility of its application in non-tumor diseases, but existing literature has not explored this further.

[0006] Currently, there is still a lack of understanding regarding the treatment methods and immune regulation mechanisms of allergic asthma, especially in the areas of macrophage function regulation, RNA-binding protein-mediated post-transcriptional regulation, and novel small molecule intervention strategies. Specifically, this manifests in the following ways: (1) First-line clinical drugs such as inhaled corticosteroids and long-acting β2 receptor agonists have limited efficacy in refractory asthma, and long-term use of corticosteroids easily leads to systemic side effects; (2) Existing drugs mostly focus on symptom control and broad-spectrum anti-inflammatory effects, lacking specific intervention methods targeting M2 macrophage polarization; (3) The function of PCBP2 in asthma immune regulation is not yet clear, and related technical solutions have not been established; (4) The known therapeutic uses of acevaltrate are mainly limited to the field of oncology, and there is a lack of technical inspiration for its application in inflammatory immune diseases; (5) There are currently no asthma immunomodulators targeting RNA-binding proteins. Therefore, developing asthma treatment drugs based on new targets and mechanisms has significant clinical needs and scientific value. Summary of the Invention

[0007] This invention addresses the limitations of existing asthma treatments in terms of target selection and clinical needs. For the first time, it reveals the crucial role of the "PCBP2-macrophage M2 polarization" regulatory axis in the pathogenesis of allergic asthma, establishes a novel use for Acevaltrate as a therapeutic agent targeting this axis, and provides a complete technical system covering drug use, drug composition, molecular target, and combination therapy.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: the application of Acevaltrate, an inhibitor targeting PCBP2, in the preparation of drugs for treating allergic asthma.

[0009] The proposed use is based on the innovative discovery that Acevaltrate effectively blocks IL-4 and IL-13-induced macrophage M2 polarization by specifically inhibiting the biological function of the RNA-binding protein PCBP2, thereby improving the pathological state of asthma at multiple levels. Specifically, Acevaltrate, through this mechanism, can significantly reduce airway inflammation dominated by the Th2 immune response, inhibit excessive IgE production, and effectively improve airway remodeling processes, including goblet cell proliferation, excessive mucus secretion, and abnormal collagen deposition.

[0010] To validate this use, the present invention provides ample in vivo pharmacodynamic evidence. In a classic mouse ovalbumin (OVA)-induced allergic asthma model, daily administration of Acevaltrate (at doses of 20 mg / kg and 50 mg / kg) via gavage produced the following clear and quantifiable therapeutic effects: (1) Flow cytometry analysis confirmed that Acevaltrate treatment significantly reduced the expression of CD206, a key marker of macrophage M2 polarization, in bronchoalveolar lavage fluid; (2) Effectively reduces serum total IgE levels in a dose-dependent manner; (3) Significant improvement in lung lesions at the histopathological level, specifically manifested as a significant decrease in the score of peri-airway inflammatory cell infiltration shown by HE staining, a significant reduction in bronchial epithelial goblet cell proliferation and mucus secretion revealed by PAS staining, and a significant reduction in the area of ​​collagen fiber deposition around the bronchial wall and blood vessels confirmed by Masson trichrome staining. (4) At the molecular level, Acevaltrate intervention significantly downregulated the mRNA and protein expression levels of key M2 polarization markers in alveolar macrophages. These systematic experimental results collectively confirm the clear efficacy of Acevaltrate in treating allergic asthma.

[0011] In a preferred embodiment, the drug uses PCBP2 as a molecular target for treating allergic asthma.

[0012] First, at the level of functional necessity, knocking down or eliminating PCBP2 in macrophages using small interfering RNA (SRNA) and CRISPR / Cas9 technologies significantly inhibited IL-4 / IL-13-induced M2 polarization, specifically manifested in the marked suppression of mRNA transcription and protein synthesis levels of key markers. This demonstrates from a genetic manipulation perspective that PCBP2 is a key regulatory node for macrophage differentiation into the M2 phenotype. Second, at the level of compound intervention, acevaltrate can inhibit PCBP2 protein levels in vitro in a concentration-dependent manner, simultaneously suppressing the expression of the aforementioned M2 polarization markers, thus establishing a direct pharmacological link of "Acevaltrate inhibiting PCBP2 function → blocking M2 polarization." Finally, at the mechanistic level, this invention systematically elucidates the network of action of PCBP2 in regulating M2 polarization. In vitro cell experiments showed that during IL-4 / IL-13-induced macrophage M2 polarization, the upregulation of PCBP2 expression was positively correlated with the phosphorylation level of the key signaling protein STAT6, and that PCBP2 knockdown or acevaltrate inhibition effectively suppressed STAT6 phosphorylation, indicating that the functional state of PCBP2 directly affects the activation of the classical signaling pathway of M2 polarization. This evidence collectively reveals the core role of PCBP2 in positively regulating the macrophage M2 polarization program by influencing key signal transduction and downstream gene expression, firmly establishing its scientific basis as an interventionable drug target.

[0013] In a preferred embodiment, the drug uses Acevaltrate or its pharmaceutically acceptable salts, solvates, prodrugs, or isomers with the same therapeutic activity as the active ingredient; wherein, a solvate refers to a complex of Acevaltrate and a solvent molecule (such as water); and a prodrug refers to a derivative that can be converted into Acevaltrate in vivo.

[0014] In a preferred embodiment, the drug further comprises a pharmaceutically acceptable carrier.

[0015] In a further preferred embodiment, the carrier includes excipients, diluents, disintegrants, adhesives, lubricants, or stabilizers.

[0016] In the preferred embodiment, the drug is administered orally.

[0017] In a preferred embodiment, the dosage form of the drug is tablets, capsules, granules, or powder.

[0018] In a preferred embodiment, the drug also contains other active ingredients for the treatment of asthma.

[0019] In a further preferred embodiment, the other active ingredients for asthma treatment are selected from inhaled corticosteroids, long-acting β2 receptor agonists, leukotriene receptor antagonists, and / or anti-IgE antibodies.

[0020] Based on the unique mechanism of action of Acevaltrate and the complementary and synergistic effects of existing standard asthma treatments, a combination formulation with synergistic therapeutic effects is formed. Specifically, Acevaltrate intervenes in macrophage M2 polarization at the source by targeting and inhibiting PCBP2, while the other drugs mentioned above exert their effects through different pathways such as anti-inflammation, bronchodilation, or blocking allergic mediators. The combination of the two can produce one or more of the following beneficial effects: (1) Synergistic enhancement of efficacy: It produces superimposed or synergistic effects that are superior to any single drug in inhibiting airway inflammation, reducing airway hyperresponsiveness, and improving airway remodeling; (2) Reduction of drug dosage and side effects: Under the premise of achieving the same or better efficacy, it may reduce the required dosage of one or more drugs (especially glucocorticoids) in the combination therapy, thereby reducing the systemic side effects caused by their long-term use; (3) Expanding the therapeutic window and addressing refractory asthma: It provides a new and effective treatment strategy for patients with refractory or severe asthma who do not respond well to existing single-drug therapy.

[0021] In addition, Acevaltrate can be used in combination with inhaled corticosteroids, long-acting β2 receptor agonists, leukotriene receptor antagonists and / or anti-IgE antibodies, as a single combination drug composition containing all active ingredients, or as a combination, sequential or alternating administration of a drug composition containing Acevaltrate with another drug composition containing the other asthma treatments in a treatment regimen.

[0022] In a further preferred embodiment, the inhaled corticosteroid is selected from budesonide, fluticasone, and / or ciroxone.

[0023] In a further preferred embodiment, the long-acting β2 receptor agonist is selected from salmeterol, formoterol, and vilanterol.

[0024] In a further preferred embodiment, the leukotriene receptor antagonist is selected from montelukast and / or zafirlukast.

[0025] In a further preferred embodiment, the anti-IgE antibody is selected from omalizumab.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. A novel therapeutic target and pathway: This invention, for the first time, reveals the crucial role of the RNA-binding protein PCBP2 in regulating macrophage M2 polarization in an allergic asthma model, and fully elucidates the novel action axis of "Acevaltrate (ACE) → Inhibition of PCBP2 → Blocking STAT6 signaling → Reversal of M2 polarization → Improvement of asthma pathology." This provides the field of asthma treatment with a previously completely unknown and clearly defined precise intervention target, overcoming the limitations of existing drugs that target multiple cell surface receptors or secreted factors.

[0027] 2. This invention represents a fundamental expansion and "drug repurposing" in the field of compound therapy: For the first time, this invention discovered and demonstrated that Acevaltrate can be used to treat allergic asthma, successfully transforming it from a known "anti-tumor ferroptosis inducer" into an "immunomodulatory anti-asthma drug." This transformation bridges the significant and often obscure pathological mechanistic gap between tumors and inflammatory immune diseases, endowing Acevaltrate with entirely new and greater medical value, and shortening the development cycle and risks of potential drugs.

[0028] 3. A mechanism-driven precision treatment strategy is provided: The treatment strategy of this invention directly targets the core link in the pathogenesis of asthma—"macrophage M2 polarization." By inhibiting the upstream key regulatory factor PCBP2, M2 macrophages and their mediated Th2 inflammation and tissue remodeling are suppressed at the source, which is expected to achieve more effective control of refractory asthma or airway remodeling, and may reduce dependence on broad-spectrum anti-inflammatory drugs (such as glucocorticoids) and their corresponding side effects.

[0029] 4. A complete R&D and evaluation system has been established: This invention not only provides new therapeutic uses and targets, but also establishes an in vitro screening model and method based on the "PCBP2-M2 polarization" axis, providing an efficient and specific tool for the continuous discovery and evaluation of candidate drugs with similar mechanisms of action, and has important methodological value.

[0030] 5. Clear clinical translation prospects and multiple development dimensions: The schemes protected by this invention cover everything from a single active ingredient (acevaltrate) to pharmaceutical compositions (including oral and inhaled formulations), and then to combination therapy strategies with existing drugs. This provides a clear path and flexible options for subsequent product development, allowing for both monotherapy development and enhancement of existing therapies, with broad application prospects.

[0031] In summary, this invention is the first to propose and validate a novel strategy for treating allergic asthma: Acevaltrate effectively blocks macrophage M2 polarization by targeting and inhibiting PCBP2. This discovery not only fills the functional gap in PCBP2's role in asthma immune regulation and expands the clinical application of Acevaltrate, but also provides a novel drug candidate with a clear mechanism of action and precise targeting for asthma treatment, demonstrating significant innovative value and broad application prospects. This invention not only possesses significant scientific innovation (new target, new mechanism, new use), but also demonstrates outstanding technological progress and practicality in its treatment strategy, drug development, and industrial application. Attached Figure Description

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 This is a graph showing how Acevaltrate inhibits macrophage M2 polarization by targeting PCBP2; among which, Figure 1 Figure A shows the Western Blot (WB) and quantitative PCR (qPCR) data after knocking down PCBP2 expression in macrophages using siRNA. Figure 1 Figure B shows the effect of PCBP2 knockdown on IL-4 / IL-13-induced M2 biomarker expression. Figure 1 C is a comparison of the basal expression levels of PCBP2 in peritoneal macrophages of wild-type (WT) and macrophage PCBP2 conditional knockout (Pcbp2-cKO) mice; Figure 1 D is a comparison of the expression levels of the M2 marker in peritoneal macrophages of WT and Pcbp2-cKO mice after stimulation with IL-4 / IL-13. Figure 1 Figure E shows the effect of different concentrations of Acevaltrate on macrophage viability (CCK-8 assay). Figure 1 F is a Western blotting plot showing the effect of Acevaltrate pretreatment on PCBP2 protein expression levels in macrophages. Figure 1 G is a graph showing the effect of Acevaltrate pretreatment on PCBP2 mRNA expression levels in macrophages (qPCR). Figure 1 H represents the effect of Acevaltrate pretreatment on the expression of IL-4 / IL-13-induced macrophage M2 markers; Figure 1 Figure I shows the Western blot results of IL-4 / IL-13-induced p-STAT6 protein expression at different time points (0 h, 3 h, 6 h) after PCBP2 knockdown. Figure 1 J is a WB diagram showing p-STAT6 expression in macrophages of WT and cKO mice at different time points (0 h, 3 h, 6 h, 9 h, 12 h) after stimulation with IL-4 / IL-13; Figure 1 K represents the Western blot results of IL-4 / IL-13-induced p-STAT6 protein expression at different time points after Acevaltrate treatment.

[0033] Figure 2 A graph showing data on PCBP2 conditional knockout (cKO) alleviating OVA-induced allergic asthma, in which... Figure 2 A is the experimental flowchart of the OVA asthma model in WT and Pcbp2-cKO mice; Figure 2 B is a comparison of the relative expression levels of M2 marker mRNA in the lung tissues of WT and Pcbp2-cKO mice after asthma provocation; Figure 2 C shows a comparison of staining of lung tissue pathological sections from WT and Pcbp2-cKO mice (HE staining, Masson's trichrome staining, and PAS staining). Figure 2 D is a comparison of serum total IgE and OVA-specific IgE levels in WT and Pcbp2-cKO mice; Figure 2 E shows the average fluorescence intensity of CD206 in the bronchoalveolar lavage fluid of WT and Pcbp2-cKO mice as detected by flow cytometry.

[0034] Figure 3 This is a graph showing the data on how Acevaltrate improved the disease phenotype and inhibited the PCBP2-M2 axis in the lungs in an OVA-induced allergic asthma mouse model; among which, Figure 3 A is a flowchart of the OVA-induced allergic asthma mouse model construction and Acevaltrate treatment experiment. Figure 3 B shows the results of serum total IgE levels in each group of mice; 3C shows the mean fluorescence intensity (MFI) of CD206 in bronchoalveolar lavage fluid of each group of mice detected by flow cytometry. Figure 3 D is a graph showing the relative expression levels of M2 polarization markers (Arg1, Mrc1, Fizz1) mRNA in the lung tissues of mice in each group; Figure 3 E shows the staining of lung tissue pathological sections (HE staining, Masson's trichrome staining, PAS staining). Detailed Implementation

[0035] The technical solution of the present invention will be further described and illustrated below through examples. All raw materials used in the examples are commercially available or prepared using conventional methods.

[0036] Example 1 The verification that PCBP2 is a key regulator of macrophage M2 polarization and can be inhibited by Acevaltrate includes the following steps: S1. Isolation of primary mouse peritoneal macrophages: Healthy C57BL / 6J wild-type (WT) mice aged 6-8 weeks were injected intraperitoneally with 3 mL of sterile 3% mercaptoacetate medium. Four days later, the mice were sacrificed by cervical dislocation. Under sterile conditions, 5 mL of pre-cooled 1640 medium was injected into the peritoneal cavity, and the abdomen was gently massaged for 1 minute. The peritoneal lavage fluid was then collected using a syringe. The collected cell suspension was centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. The cells were seeded in cell culture dishes and incubated at 37 ℃ for 6 hours or overnight. The adherent cells were the primary peritoneal macrophages used in subsequent experiments. S2 and PCBP2 siRNA knockdown efficiency verification: S201. Targeting the mouse PCBP2 gene, specific small interfering RNA (siRNA) and a negative control (scramble siRNA) were designed and synthesized: Immortalized mouse bone marrow-derived macrophages (iBMDM) were seeded in 24-well plates and allowed to adhere overnight. Before transfection, the medium was replaced with antibiotic-free DMEM. Transfection was then performed according to the RNAiMAX transfection reagent instructions. Experimental group: 50 μL of Opti-MEM was gently mixed with 1 μL of RNAiMAX reagent (solution A), and another 50 μL of Opti-MEM was mixed with 1 μL of PCBP2 siRNA to a final concentration (solution B). The solutions A and B were mixed and incubated at room temperature for 20 minutes to form a transfection complex. 100 μL of the transfection complex was added evenly to the cell culture wells. 24 hours after transfection, cells were harvested for knockdown efficiency verification. Control group: 50 μL of Opti-MEM was gently mixed with 1 μL of RNAiMAX reagent (solution A), and another 50 μL of Opti-MEM was mixed with 1 μL of PCBP2 siRNA to a final concentration of 1 μL of RNAiMAX reagent (solution B). Mix μL of negative control siRNA (solution B), mix solution A with solution B, and incubate at room temperature for 20 minutes to form a transfection complex. Add 100 μL of the transfection complex evenly to the cell culture wells. After 24 hours of transfection, collect the cells as a control. S202, mRNA level verification: 500 μL of Trizol reagent was added directly to the cells collected in S201 after discarding the culture medium. After complete lysis, total RNA was extracted according to the instructions, and the RNA concentration and purity were measured. 2 μg of total RNA was taken and reverse transcribed to synthesize cDNA. Using the cDNA as a template, real-time quantitative PCR (qPCR) was performed using SYBR Green qPCR premix to detect the expression level of PCBP2 mRNA (results are shown below). Figure 1 As shown in A), β-Actin was used as an internal reference gene for normalization.

[0037] S203, Protein Level Validation: Cells collected in S201 were discarded from the culture medium, washed once with PBS, and an appropriate amount of 1×SDS protein loading buffer (containing β-mercaptoethanol) was added directly to the culture wells. Cells were scraped off using a cell scraper and collected into centrifuge tubes. The samples were then boiled in a 100°C metal bath for 10 minutes to fully denature the proteins. After cooling, subsequent Western blotting analysis was performed (results are shown in Figure 1). Figure 1 (as shown in A); The effect of S3 and PCBP2 knockdown on M2 polarization: S301. Macrophages transfected with PCBP2 siRNA and negative control siRNA in step S201 were stimulated with IL-4 / IL-13 24 hours after transfection to induce M2 polarization. Cells were harvested after stimulation for 0 hours, 24 hours, 48 ​​hours and 3 hours and 6 hours, respectively. S302, qPCR detection: RNA was extracted from the cells collected in step S301 using the method described in step S201. The RNA was then reverse transcribed, and the mRNA expression levels of key M2 polarization markers Arg1, Fizz1, and Ym1 were detected, with Actin used as an internal control (results are shown in Figure 1). Figure 1 (as shown in B) S303, Western Blot Detection: The cells collected in step S301 were lysed using 1×SDS buffer and protein samples were prepared according to the method described in step S201. The expression changes of STAT6 signaling pathway proteins (phosphorylated STAT6, p-STAT6) were detected by Western Blot (results are shown below). Figure 1 (as shown in I) Comparison of macrophages from S4 wild-type and PCBP2 conditional knockout (cKO) mice: Primary peritoneal macrophages were isolated from littermates of WT mice and macrophage-specific Pcbp2-cKO mice according to the method described in step S1. WT and cKO macrophages were stimulated with IL-4 and IL-13. Cells were harvested at 0, 24, and 48 hours. The differences in the induced expression levels of Arg1, Fizz1, and Mrc1 between the two groups were detected and compared by qPCR (e.g., ...). Figure 1 (As shown in D) The expression level of PCBP2 in the 0-hour sample was individually measured to verify the knockout efficiency (e.g., Figure 1 Cells were harvested at 0, 3, 6, 9, and 12 hours (as shown in Figure C). They were lysed with 1×SDS buffer and protein samples were prepared according to the method described in step S203. The expression changes of STAT6 signaling pathway proteins (phosphorylated STAT6, p-STAT6) were detected by Western blotting (results shown in Figure J). The 0-hour samples were used to detect PCBP2 protein expression alone (results shown in Figure J). Figure 1 (as shown in C below) S5, Acevaltrate cytotoxicity assay (CCK-8 assay): Primary peritoneal macrophages obtained from S1 were seeded into 96-well plates and incubated overnight. Acevaltrate powder was dissolved in DMSO to prepare a 10 mM stock solution, which was then diluted to a series of working concentrations with DMEM complete medium (containing 10% FBS) for experiments. The old cell culture medium was discarded, and 100 μL of medium containing different concentrations of Acevaltrate was added, with 5 replicates for each concentration. A solvent control group containing only an equal volume of DMSO (final concentration ≤0.2%) was also included. After 24 and 48 hours of treatment, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 2–4 hours. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader. Cell viability (%) = [(OD(drug group) - OD(blank well)) / (OD(solvent control group) - OD(blank well))] × 100%. Based on the results, the safe concentration for subsequent experiments was determined (results are shown in the figure). Figure 1 (as shown in E) Inhibitory effects of S6 and Acevaltrate on PCBP2 expression and M2 polarization: Effect of S601 on PCBP2 expression: Primary peritoneal macrophages obtained from S1 were seeded into 24-well plates. After adhesion, the medium was replaced with DMEM containing safe concentrations of Acevaltrate (2 μM and 5 μM) and DMSO. After 2 hours of pretreatment, the cells were harvested. S602, mRNA level: RNA was extracted from the cells collected in step S601 according to the method in step S2 and qPCR was performed to detect the PCBP2 mRNA level (internal control: β-Actin) (results are shown in Figure 1). Figure 1 (as shown in G); S603, Protein Level: Prepare protein samples from the cells collected in step S601 using the method in step S2 and perform Western blotting to detect PCBP2 protein levels (internal control: GAPDH) (results are shown below). Figure 1 (as shown in F); S604, Inhibition of M2 polarization: After pretreatment with Acevaltrate (2 μM, 5 μM) or DMSO for 2 hours in step S601, without changing the culture medium, IL-4 + IL-13 were directly added to each well to induce M2 polarization. Cells were cultured further, and harvested at 0, 24, and 48 hours. The differences in the induced expression levels of Arg1, Fizz1, and Mrc1 between the two groups were detected and compared by qPCR (results are shown in Figure 1). Figure 1Cells were harvested at 0, 3, 6, and 9 hours (as shown in H). They were lysed with 1×SDS buffer and protein samples were prepared according to the method described in step S2. The expression changes of STAT6 signaling pathway proteins (phosphorylated STAT6, p-STAT6) were detected by Western blotting (results are shown in H). Figure 1 (As shown in K), to evaluate the inhibitory effect of Acevaltrate.

[0038] This embodiment is based on the appendix. Figure 1 The in vitro experimental results presented by (AK).

[0039] 1. Verification of the functional necessity of PCBP2: such as Figure 1 As shown in Figure A, specific siRNA can effectively knock down the mRNA and protein expression of PCBP2 in macrophages. Under these conditions, such as Figure 1 As shown in Figure B, stimulation with IL-4 and IL-13 significantly suppressed the upregulation of key M2 markers Arg1 and Fizz1 in macrophages. This genetically demonstrates that PCBP2 is essential for macrophage polarization towards the M2 phenotype.

[0040] 2. Validation of the gene knockout animal model: To validate the gene knockout model in a more physiologically similar manner, we isolated primary peritoneal macrophages from wild-type (WT) PCBP2 conditional knockout (cKO) mice. For example... Figure 1 As shown in Figure C, the basal expression level of PCBP2 in Pcbp2-cKO macrophages was significantly lower than that in the WT control. Correspondingly, as... Figure 1 As shown in Figure D, IL-4 / IL-13 stimulation significantly impaired the M2 polarization capacity of PCBP2-cKO macrophages, with M2 marker induction levels far lower than in WT cells. This further confirms the crucial role of PCBP2 in M2 polarization in primary cells.

[0041] 3. Pharmacological activity and safety assessment of Acevaltrate: To evaluate the application potential of Acevaltrate, we first conducted cytotoxicity tests. For example... Figure 1 As shown in E, the CCK-8 assay demonstrated that, at the selected concentration and duration of action, Acevaltrate had no significant effect on macrophage viability, thus establishing a safe dose and time window for subsequent experiments.

[0042] 4. Inhibitory effect of Acevaltrate on PCBP2 and M2 polarization: We investigated the pharmacological effects of Acevaltrate at safe concentrations. For example... Figure 1 F and Figure 1As shown in G, pretreatment of primary peritoneal macrophages from WT mice with Acevaltrate for 2 hours significantly reduced the mRNA and protein levels of PCBP2. More importantly, as Figure 1 As shown in Figure H, this pretreatment effectively antagonized the upregulation of M2 marker expression induced by subsequent IL-4 / IL-13 stimulation. These results collectively demonstrate that Acevaltrate can block the M2 polarization program in macrophages by inhibiting PCBP2 expression.

[0043] 5. Verification of the signal axis of PCBP2-STAT6-M2: (e.g.) Figure 1 As shown in Figure I, in macrophages with PCBP2 knocked down using siRNA, IL-4 / IL-13-induced STAT6 phosphorylation expression was suppressed. Meanwhile, as... Figure 1 As shown in Figure J, macrophages isolated from PCBP2 conditional knockout mice exhibited lower STAT6 phosphorylation expression than wild-type cells upon IL-4 / IL-13 stimulation. Correspondingly, as... Figure 1 As shown in K, pretreatment of macrophages with Acevaltrate inhibits IL-4 / IL-13-induced STAT6 phosphorylation. These parallel experimental results, from three levels—molecular intervention (knockdown), genetic deletion (knockout), and pharmacological blockade (inhibitor)—jointly confirm that PCBP2 is a key upstream node regulating STAT6 signaling activation and macrophage M2 polarization, elucidating the novel signaling axis of "PCBP2-STAT6-M2" that plays a central role in allergic asthma.

[0044] Example 2 Genetic knockout of PCBP2 can resist the development and progression of allergic asthma. The validation of the target's necessity includes the following steps: S1. Establishment of animal and asthma models, such as Figure 2 As shown in A: Six- to eight-week-old female macrophage-specific PCBP2 conditional knockout (Pcbp2-cKO) mice and their littermate wild-type (WT) control mice were used. Sensitization phase (days 0, 7, and 14): Mice were intraperitoneally injected with 200 mL of PBS suspension containing 20 μg OVA and 2 mg aluminum hydroxide adjuvant; Challenge phase (days 21-27): Mice were nebulized with 1% OVA solution for 30 minutes daily. S2, Sample Collection: S201, 24 hours after the last challenge and administration (day 28), mice were anesthetized by intraperitoneal injection of afodin (30 uL / g); S202. Serum collection: Blood was collected by enucleation and placed in centrifuge tubes. The blood was allowed to stand at room temperature for half an hour, then centrifuged at 4000 rpm for 15 minutes. The supernatant serum was carefully aspirated, aliquoted into EP tubes, and stored at -80 ℃ for subsequent IgE detection. S203. Collection and processing of bronchoalveolar lavage fluid (BALF): Endotracheal intubation was performed, and 0.5 mL of pre-cooled PBS was slowly injected into the lungs using a 1 mL syringe. Gentle aspiration was performed, and the lavage was repeated three times, maintaining a recovery rate of over 80%. The recovered BALF was centrifuged at 4000 rpm for 10 minutes at 4°C. The supernatant was transferred to a new EP tube and stored at -80°C for later use. The cell pellet was resuspended in 100 μL of PBS for subsequent experiments. S204. Lung tissue collection: Open the thoracic cavity and completely separate the lungs; take the left lung lobe, immerse it in 4% paraformaldehyde solution, fix it for more than 24 hours, and use it for subsequent paraffin embedding and sectioning; take the right lower lobe, rinse it with pre-cooled PBS, blot the surface liquid with sterile filter paper, quickly immerse it in liquid nitrogen for flash freezing, and then transfer it to a -80 ℃ freezer for storage for RNA extraction; the remaining right lung is flash frozen and stored at -80 ℃. S3. Asthma phenotype assessment: S301. Serum IgE Level Detection: Using a commercially available mouse total IgE ELISA kit and an OVA-specific IgE ELISA kit, the procedure was strictly followed according to the instructions. The concentrations of total IgE and OVA-specific IgE in the serum of each group of mice collected in step S202 were measured (results are shown in Figure 1). Figure 2 (as shown in D).

[0045] S302, Lung tissue pathological analysis: The left lung tissue fixed in step S204 was sent to a professional company for routine paraffin embedding, sectioning, and analysis (results are as follows). Figure 2 (as shown in C): (1) Hematoxylin-eosin (H&E) staining: to observe the infiltration of inflammatory cells around the airways and blood vessels; (2) Periodic acid Schiff (PAS) staining: to observe the proliferation of goblet cells and mucus secretion in the airways; (3) Masson's trichrome staining: Observe the collagen deposition (blue) around the airway wall and blood vessels; S303. Flow cytometry analysis of immune cell infiltration in lung tissue: The bronchoalveolar lavage fluid from step S203 was centrifuged to obtain cell pellet. First, the obtained cell pellet was blocked with FC Block solution containing CD16 / CD32 antibody to block non-specific binding. Then, a mixture containing FVD510 (inactivated / deactivated dye), CD45, F4 / 80, CD11b, CD11c, and CD170 antibodies was added, and surface marker staining was performed on ice in the dark to identify the macrophage population. Intracellular staining was then performed using the BD permeabilization kit. The cells were first fixed with fixative, then treated with permeabilization agent, and finally stained with CD206 antibody to detect specific markers of M2 macrophages. S304. Flow Cytometry Detection and Analysis: After staining in step S303, the cells were resuspended and analyzed using a flow cytometer. FlowJo software was used for analysis, with FVD510 negative cells identified as viable and CD45 cells identified as viable. + Leukocyte population, further analysis of CD170 + CD11c + F4 / 80 + The expression level and mean fluorescence intensity of CD206 in macrophages (results are shown in Figure 1) Figure 2 (as shown in E) S4. Detection of targets and downstream molecules in lung tissue: Approximately 30 mg of right lower lobe lung tissue, frozen at -80 ℃ in S204, was used for qPCR detection: 500 μL Trizol reagent was added, the tissue was ground, thoroughly homogenized, and lysed. Total RNA was extracted, and after determining the RNA concentration, 2 μg of RNA was used for reverse transcription to synthesize cDNA. Using the cDNA as a template, qPCR was performed using the SYBR Green method to detect the mRNA expression levels of PCBP2 and M2 polarization-related markers (Arg1, Mrc1, Fizz1). β-Actin was used as an internal control gene (results are shown in the figure). Figure 2 As shown in B).

[0046] This embodiment is based on the appendix. Figure 2 The in vivo genetic evidence presented.

[0047] 1. Animal models: such as Figure 2 As shown in Figure A, we used PCBP2 conditional knockout (Pcbp2-cKO) mice and their littermate wild-type (WT) controls to induce an allergic asthma model using the same OVA protocol.

[0048] 2. Analysis of M2 polarization state in lung tissue: After asthma provocation, we first examined the M2 polarization state of lung tissue. For example... Figure 2As shown in Figure B, compared with WT asthmatic mice, the mRNA expression levels of several key M2 markers in the lung tissue of Pcbp2-cKO asthmatic mice were significantly reduced. This indicates that at the whole animal level, PCBP2 deficiency inhibits the M2 polarization process in the asthmatic lung environment.

[0049] 3. Pathological evaluation of lung tissue: such as Figure 2 As shown in Figure C, lung tissue section staining provides direct evidence. H&E staining revealed significantly reduced peri-airway inflammatory cell infiltration in PCBP2-cKO mice compared to WT mice; Masson's trichrome staining showed decreased airway collagen deposition; and PAS staining showed suppressed goblet cell proliferation and mucus secretion. This combined indicates that PCBP2 deficiency can significantly improve the core pathological features of asthma.

[0050] 4. Systemic Allergic Reaction Assessment: We further tested systemic allergy markers, such as... Figure 2 As shown in Figure D, the serum total IgE and OVA-specific IgE levels in Pcbp2-cKO asthmatic mice were significantly lower than those in WT asthmatic mice. This indicates that PCBP2 deficiency not only improves local lung inflammation but also weakens the systemic Th2 immune response.

[0051] 5. Analysis of M2 macrophage infiltration in the lungs: such as Figure 2 As shown in Figure E, flow cytometry was used to directly quantify intrapulmonary immune cells, revealing that the average fluorescence intensity of CD206 macrophages in the lung tissue of Pcbp2-cKO asthmatic mice was significantly lower than that in WT asthmatic mice. This is corroborated by the mRNA and pathological results.

[0052] Example 3 The validation of the therapeutic effect of Acevaltrate in an allergic asthma model by inhibiting the PCBP2-M2 axis in the lungs includes the following steps: S1. Animal grouping and asthma model establishment, such as Figure 3 As shown in A: Six- to eight-week-old female C57BL / 6J wild-type (WT) mice were randomly divided into four groups (n=6-8 per group): ① Normal control group (Control); ② Asthma model group; ③ Low-dose Acevaltrate treatment group (Low-ACE, 20 mg / kg); ④ High-dose Acevaltrate treatment group (High-ACE, 50 mg / kg); Sensitization phase: On days 0, 7, and 14, mice in groups ②, ③, and ④ were intraperitoneally injected with a suspension (containing 20 μg ovalbumin (OVA) and 2 mg aluminum hydroxide adjuvant, dissolved in 200 μL PBS); mice in group ① were intraperitoneally injected with an equal volume of PBS. Challenge phase: From day 21 to day 27, mice in groups ②, ③, and ④ were placed in a self-made plexiglass nebulizer and nebulized with 1% OVA solution (dissolved in PBS) for 30 minutes each time; mice in group ① inhaled nebulized PBS. Dosing regimen: During the challenge phase (days 21-27), mice in groups ③ and ④ were administered the drug by gavage 30 minutes before daily nebulization, with a dosage of 10 μL / g body weight; Acevaltrate was dissolved in 0.5% CMC-Na; mice in groups ① and ② were administered the same volume of the above solvent by gavage.

[0053] S2, Sample Collection: Twenty-four hours after the last challenge and drug administration (day 28), mice were anesthetized by intraperitoneal injection of afodin (30 uL / g); S201. Serum collection: Blood was collected by enucleation and placed in a centrifuge tube. The blood was left to stand at room temperature for half an hour. Then, the blood was centrifuged at 4000 rpm for 15 minutes. The supernatant serum was carefully aspirated, aliquoted into EP tubes, and stored at -80℃ for subsequent IgE detection. S202. Bronchoalveolar lavage fluid (BALF) collection and processing: Endotracheal intubation was performed, and 0.5 mL of pre-cooled PBS was slowly injected into the lungs using a 1 mL syringe. Gentle aspiration was performed, and the lavage was repeated three times, maintaining a recovery rate of over 80%. The recovered BALF was centrifuged at 4000 rpm for 10 minutes at 4°C. The supernatant was transferred to a new EP tube and stored at -80°C for later use; the cell pellet was used for subsequent experiments. S203. Lung Tissue Collection: Open the thoracic cavity and completely separate the lungs. Take the left lung lobe, immerse it in 4% paraformaldehyde solution, and fix for at least 24 hours for subsequent paraffin embedding and sectioning. Take the lower lobe of the right lung, rinse with pre-cooled PBS, blot dry with sterile filter paper, and quickly freeze in liquid nitrogen. Then transfer to a -80°C freezer for RNA extraction. The remaining right lung lobe is stored at -80°C for later use. S3. Asthma phenotype assessment: S301. Serum IgE Level Detection: Using a commercially available mouse total IgE ELISA kit, strictly following the instructions, the concentration of total IgE in the serum of each group of mice collected in step S201 was measured (results are shown in Figure 1). Figure 3 (as shown in B) S302. Lung tissue pathological analysis: The fixed left lung tissue was sent to a professional company for routine paraffin embedding, sectioning, and analysis (results are as follows). Figure 3(as shown in E) (1) Hematoxylin-eosin (H&E) staining: to observe the infiltration of inflammatory cells around the airways and blood vessels; (2) Periodic acid Schiff (PAS) staining: to observe the proliferation of goblet cells and mucus secretion in the airways; (3) Masson's trichrome staining: Observe the collagen deposition (blue) around the airway wall and blood vessels; S303. Flow cytometry analysis of immune cell infiltration in lung tissue: The cell pellet obtained by centrifugation of bronchoalveolar lavage fluid from step S202 was first blocked with FC Block solution containing CD16 / CD32 antibody to block non-specific binding. Subsequently, a mixture containing antibodies against FVD510 (live / dead dye), CD45, F4 / 80, CD11b, CD11c, and CD170 was added, and surface marker staining was performed on ice in the dark to identify the macrophage population. Then, intracellular staining was performed using the BD permeabilization kit. The cells were first fixed with fixative, then treated with permeabilization agent, and finally stained with CD206 antibody to detect specific markers of M2 macrophages. S304. Flow Cytometry Detection and Analysis: Resuspend the stained cells from step S303 above and perform flow cytometry analysis. Use FlowJo software for analysis, identifying viable cells by FVD510 negative markers and then identifying CD45 cells. + Leukocyte population, further analysis of CD170 + CD11c + F4 / 80 + The mean fluorescence intensity of CD206 in macrophages (results as follows) Figure 3 (as shown in C) S4. Detection of targets and downstream molecules in lung tissue: Approximately 30 mg of right lower lobe lung tissue, frozen at -80 ℃ in S204, was used for qPCR detection: 500 μL Trizol reagent was added, the tissue was ground, homogenized thoroughly, and total RNA was extracted. After determining the RNA concentration, 2 μg of RNA was used for reverse transcription to synthesize cDNA. Using the cDNA as a template, qPCR was performed using the SYBR Green method to detect the mRNA expression levels of PCBP2 and M2 polarization-related markers (Arg1, Mrc1, Fizz1), with β-Actin as an internal reference gene (results are shown in the figure). Figure 3 (as shown in D).

[0054] This embodiment is based on the appendix. Figure 3 (AE) presents the in vivo experimental results.

[0055] 1. Animal models and treatment: such as Figure 3As shown in Figure A, we established a classic OVA-induced mouse model of allergic asthma. Mice were randomly divided into five groups: a normal control group, an asthma model group, a low-dose Acevaltrate treatment group (20 mg / kg), and a high-dose Acevaltrate treatment group (50 mg / kg). During the challenge phase, the mice were administered the medication by gavage daily.

[0056] 2. Systemic and Local Inflammation Assessment: Following the final provocation, we assessed multiple aspects of the disease. For example... Figure 3 As shown in Figure B, serum IgE levels were significantly elevated in the asthma model group mice, while Acevaltrate treatment dose-dependently reduced IgE levels. Figure 3 As shown in Figure C, flow cytometry analysis revealed a significant increase in the mean fluorescence intensity of CD206 in macrophages in the lungs of asthmatic mice, and Acevaltrate treatment significantly reduced the expression of this biomarker.

[0057] 3. Validation of the target and downstream pathways in lung tissue: To directly validate the mechanism of action in the target organ of the disease, we examined lung tissue samples. For example... Figure 3 As shown in Figure D, the mRNA levels of M2 markers were significantly elevated in the lung tissue of asthmatic mice, while Acevaltrate treatment effectively downregulated the expression of these genes. This directly indicates that Acevaltrate also works in vivo by inhibiting the "PCBP2-M2 axis".

[0058] 4. Improvement in pathological morphology: Lung tissue pathology is the gold standard for evaluating treatment efficacy. For example... Figure 3 As shown in Figure E, HE staining revealed that Acevaltrate treatment significantly reduced peri-airway inflammatory cell infiltration; Masson staining showed a reduction in collagen deposition in the airway walls, suggesting improved airway remodeling; and PAS staining showed inhibition of airway goblet cell proliferation and excessive mucus secretion. These results collectively demonstrate that Acevaltrate can effectively improve the core pathological features of allergic asthma by targeting the PCBP2-M2 axis.

[0059] It should be understood that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Application of Acevaltrate, an inhibitor targeting PCBP2, in the preparation of drugs for treating allergic asthma.

2. The application according to claim 1, characterized in that, The drug uses PCBP2 as a molecular target for the treatment of allergic asthma.

3. The application according to claim 1, characterized in that, The drug uses Acevaltrate or its pharmaceutically acceptable salts, solvates, prodrugs, or isomers with the same therapeutic activity as its active ingredient; wherein, a solvate refers to a complex of Acevaltrate and a solvent molecule; and a prodrug refers to a derivative that can be converted into Acevaltrate in vivo.

4. The application according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

5. The application according to claim 4, characterized in that, The carrier includes excipients, diluents, disintegrants, adhesives, lubricants, or stabilizers.

6. The application according to claim 1, characterized in that, The drug is administered orally.

7. The application according to claim 1, characterized in that, The dosage form of the drug is tablets, capsules, granules or powder.

8. The application according to claim 1, characterized in that, The medication also contains other active ingredients for treating asthma.

9. The application according to claim 8, characterized in that, The other active ingredients for asthma treatment mentioned above are selected from inhaled corticosteroids, long-acting β2 receptor agonists, leukotriene receptor antagonists, and / or anti-IgE antibodies.

10. The application according to claim 9, characterized in that, The inhaled corticosteroid is selected from budesonide, fluticasone, and / or cyclosporine; the long-acting β2 receptor agonist is selected from salmeterol, formoterol, and vilanterol; the leukotriene receptor antagonist is selected from montelukast and / or zafirlukast; and the anti-IgE antibody is selected from omalizumab.

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