A PLK1 inhibitor and its application

Abstract

The present invention discloses a PLK1 inhibitor and its application, belonging to the field of biomedicine technology. The present invention discloses the application of a PLK1 inhibitor in the preparation of a drug for treating pseudoallergic reactions, wherein the inhibitor includes volasertib. The present invention has confirmed through in vitro and in vivo cell experiments that the PLK1 inhibitor Volasertib can improve the morphological changes during degranulation of RBL-2H3 cells induced by C48 / 80, reduce cell apoptosis, and reduce the release of HA and β-HEX, and has a significant improvement effect on pseudoallergic reactions; in vivo experiments on a mouse PCA model also show that inhibiting PLK1 activity can alleviate local allergic reaction symptoms. The results show that PLK1 has a significant regulatory effect on pseudoallergic reactions, providing a theoretical basis and experimental basis for exploring the potential mechanism and treatment methods of pseudoallergic reactions.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a PLK1 inhibitor and application thereof. Background Art

[0002] With changes in people's living environments and lifestyles, allergic reactions induced by various factors have become a major threat to human health, severely impacting people's physical and mental health, and placing a huge burden on their quality of life and society as a whole. Allergic reactions are divided into immediate type I allergic reactions and delayed type II, III, and IV allergic reactions, all of which are related to human immune regulation. In addition to these allergic reactions mediated by the immune system, there is also a type of adverse reaction in clinical practice that has symptoms similar to type I allergic reactions but is not immune-mediated. Currently, the mechanism of this reaction has not been found to be related to immune pathways. This reaction is called pseudoallergic reactions, also known as non-immune hypersensitivity reactions.

[0003] Pseudoallergic reactions differ from IgE-mediated type I allergic reactions. While type I allergic reactions involve the participation of immune pathways, pseudoallergic reactions do not involve the immune system. They do not trigger an antigen-antibody reaction. Instead, they directly stimulate mast cells (MCs) to degranulate, prompting the release of inflammatory factors such as histamine, leading to allergic-like symptoms. MCs are the most critical effector cells in pseudoallergic reactions. When exposed to external stimuli, MCs first release stored cytokines and inflammatory mediators. As the disease progresses, they further secrete newly synthesized inflammatory mediators, inducing MC degranulation and causing allergic-like symptoms.

[0004] Polo-like kinase (PLK) is a serine / threonine protein kinase widely expressed in eukaryotic cells. The PLK family comprises five members: PLK1 (Polo-like kinase 1), PLK2, PLK3, PLK4, and PLK5. PLK family members differ primarily in the number of C-terminal Polo-box domains (PBDs), resulting in distinct functions. PLK1 plays a crucial role in regulating cell division, maintaining genomic stability, spindle assembly, mitosis, and the DNA damage response. Multiple studies have confirmed the role of PLK1 as an oncogene, with its high expression in various primary tumor tissues, including breast, gastric, liver, prostate, colorectal, and esophageal cancers. However, studies investigating PLK1 regulation of pseudoallergic reactions have been limited. Summary of the Invention

[0005] The purpose of the present invention is to provide a PLK1 inhibitor and its application to solve the problems existing in the above-mentioned prior art. The present invention confirms through in vitro and in vivo cell experiments that the PLK1 inhibitor Volasertib has a significant therapeutic effect on pseudoallergic reactions, providing a theoretical basis and experimental basis for exploring the potential mechanism and treatment methods of pseudoallergic reactions.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an application of a PLK1 inhibitor in preparing a drug for treating pseudoallergic reaction.

[0008] Furthermore, the inhibitor includes volacetidine.

[0009] Furthermore, the volacetide plays a role in treating pseudoallergic reaction by improving the morphological changes of mast cell degranulation, reducing cell apoptosis and lowering the release of histamine and β-hexosaminidase in cells.

[0010] Furthermore, the mast cell degranulation is C48 / 80-induced mast cell degranulation.

[0011] The present invention also provides a drug for preventing and treating pseudoallergic reaction, comprising volacetidine and a pharmaceutically acceptable carrier.

[0012] Furthermore, the carrier includes one or more of an excipient, an emulsifier and a surfactant.

[0013] Furthermore, the dosage form of the drug is tablets, capsules, granules, injections or sprays.

[0014] Furthermore, the drug is taken orally or parenterally.

[0015] The present invention discloses the following technical effects:

[0016] In vitro and in vivo cell experiments in this study confirmed that the PLK1 inhibitor Volasertib can improve morphological changes during C48 / 80-induced degranulation in RBL-2H3 cells, reduce cell apoptosis, and decrease the release of HA and β-HEX, significantly improving pseudoallergic reactions. In vivo experiments in a mouse PCA model also demonstrated that inhibiting PLK1 activity can alleviate localized allergic reaction symptoms. These results suggest that PLK1 significantly regulates pseudoallergic reactions, providing a theoretical and experimental basis for exploring the underlying mechanisms and treatments of pseudoallergic reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 is the effect of Volasertib on the proliferation activity of RBL-2H3 cells (n=3);

[0019] Figure 2 The results of neutral red staining showing the effect of Volasertib on degranulation of RBL-2H3 cells (bar = 100 μm);

[0020] Figure 3 is the effect of Volasertib on the release of β-Hex from RBL-2H3 cells (n=3);

[0021] Figure 4 Effects of Volasertib on histamine release from RBL-2H3 cells (n=3), compared with the normal group, **p<0.01; compared with the model group, ##p<0.01;

[0022] Figure 5 The effect of Volasertib on C48 / 80-induced apoptosis of RBL-2H3 cells (bar = 200 μm);

[0023] Figure 6 The statistical data of Volasertib on C48 / 80-induced apoptosis of RBL-2H3 cells (n=3) compared with the normal group, **p<0.01; compared with the model group, ##p<0.01;

[0024] Figure 7 The therapeutic effect of Volasertib on PCA reaction in the toes of mice (n=8); (a) Representative images of PCA in the toes; (b) Evans Blue exudation content in the toes; (c) Paw swelling degree compared with the normal group, **p<0.01; compared with the model group, #p<0.05, ##p<0.01;

[0025] Figure 8 The therapeutic effect of Volasertib on PCA response in the ears of mice (n=8); (a) Representative images of PCA in the ears; (b) Evans Blue exudation content in the ears; (c) Ear swelling degree (compared with the normal group, **p<0.01; compared with the model group, ##p<0.01);

[0026] Figure 9The therapeutic effect of Volasertib on PCA reaction in the dorsal skin of mice (n=8); (a) Representative images of PCA reaction in the dorsal skin; (b) Evans Blue exudation content in the dorsal skin compared with the normal group, **p<0.01; compared with the model group, ##p<0.01;

[0027] Figure 10 HE staining results of mouse ears (bar = 100 μm);

[0028] Figure 11 The results of toluidine blue staining of mouse ears (bar=100 μm). DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] The MRGPRX2 receptor on the surface of mast cells (MCs) has been found to be a key receptor inducing pseudoallergic reactions. MRGPRX2 is a low-selectivity receptor, enabling it to interact with a variety of ligands, including compounds such as C48 / 80, opioids, and neuroleptics. Compound C48 / 80, a synthetic condensation product of n-methyl-p-methoxyphenylethylamine and formaldehyde, exhibits degranulation activity in differentiated and mature MCs, LAD2 MCs, and MCs derived from CD341 cells, and is commonly used to study MC degranulation in humans and rodents. RBL-2H3 cells are rat basophils that mimic the physiological and pathological responses of MCs. Their cytoplasm contains abundant basophils that, when stimulated by antigens or non-antigens, release substances such as histamine, β-hexosaminidase, interleukins, and TNF-α. Therefore, RBL-2H3 cells have become a classic cellular model for studying degranulation reactions in surrogate MCs. In this study, compound C48 / 80 was used to induce RBL-2H3 cells to construct a cell degranulation model to simulate the process of mast cell degranulation and to study the mechanism of pseudoallergic reaction.

[0035] In the early stages of this study, transcriptome sequencing was used to screen for genes associated with pseudoallergic reactions. Degranulation of RBL-2H3 cells was induced by C48 / 80, and RNA was extracted for transcriptome sequencing to obtain differentially expressed genes. GO and KEGG enrichment analysis were performed to screen for significantly enriched signal pathways. By screening for differential fold changes in genes associated with the enriched pathways and combining them with literature data, PLK1 was ultimately selected as the target gene for research. PLK1 expression was verified using qPCR and protein immunoblotting techniques. It was found that PLK1 expression levels were significantly increased in C48 / 80-induced RBL-2H3 cells, indicating that PLK1 is closely related to pseudoallergic reactions. This study was conducted to verify the role of PLK1 in regulating pseudoallergic reactions, as follows:

[0036] Example 1 Effect of PLK1 Inhibitors on Pseudoallergic Reactions

[0037] 1 Materials and Methods

[0038] 1.1 Experimental reagents and instruments

[0039] Experimental animals: SPF-grade female Kunming mice aged 6-7 weeks were purchased from Weitonglihua Laboratory Animal Technology Co., Ltd. [Production license number is SCXK (Beijing) 2016-0011].

[0040] The main experimental reagents: 4% paraformaldehyde was purchased from Biyuntian Biotechnology Co., Ltd., formamide was purchased from Shenggong Bioengineering Co., Ltd., Volasertib was purchased from TOPSCIENCE, dexamethasone was purchased from Meilun Biotechnology Co., Ltd., toluidine blue dye was purchased from Aladdin, and HE staining kit was purchased from Solarbio.

[0041] 1.2 Experimental methods

[0042] 1.2.1 Effect of PLK1 inhibitors on the proliferation activity of RBL-2H3 cells

[0043] RBL-2H3 cells in logarithmic growth phase were seeded into 96-well plates, with 10 cells per well. 4 Cells were cultured in 100 μL / well of MEM complete medium at 37°C and 5% CO2. A normal group was set up: cells were inoculated; a drug-treated group was set up: cells were inoculated and treated with different concentrations of Volasertib (0.1, 0.5, 1, 5, 10, 25, 50 / 100 nM); a blank group was set up: no treatment; each group had 6 replicate wells. After 24 hours of culture, complete medium was added to the normal group, and complete medium containing different concentrations of Volasertib was added to the drug-treated group. After 12 hours, 10 μL of CCK-8 reagent was added to the 96-well plate and allowed to act for 1 hour. The OD value was checked at 450 nm. The calculation method is as follows:

[0044] Cell survival rate (%) = (OD value of experimental group - OD value of blank group) / (OD value of normal group - OD value of blank group) × 100%

[0045] Effects of PLK1 inhibitors on RBL-2H3 cell morphology

[0046] RBL-2H3 cells in the logarithmic growth phase were seeded in 24-well plates at 8 × 10 4 Cells were incubated with 1 mL of complete MEM medium per well. The cells were divided into five groups: normal group (DMSO added); model group (C48 / 80 added); volasertib group (C48 / 80 added at concentrations of 0.1 and 0.5 nM); and positive control group (dexamethasone added at a concentration of 10 μM). Each group had three replicates. After 24 hours of culture, the culture medium was discarded. The volasertib and dexamethasone groups were treated with 1 mL of complete medium diluted in the drug, while the remaining groups were treated with an equal volume of complete medium. One hour later, 200 μL of complete medium containing 25 μg / mL C48 / 80 was added to each group except the normal group. The normal group was treated with complete medium containing an equal volume of DMSO. After 30 minutes, cells were observed under a microscope and the reaction was terminated by incubating on ice for 10 minutes. Neutral red stain was added to each well and incubated at room temperature for 10 minutes. The wells were then rinsed twice with distilled water for 2 minutes each and observed and photographed under a microscope.

[0047] 1.2.3 Effect of PLK1 inhibitors on β-Hex release from RBL-2H3 cells

[0048] Cell culture was performed as described in 1.2.2. Cells were divided into five groups: normal group (DMSO added); model group (C48 / 80 added); volasertib group (C48 / 80 added at concentrations of 0.1 and 0.5 nM); and positive control group (dexamethasone added at a concentration of 10 μM). Each group was cultured in triplicate. After 24 hours of culture, the culture medium was discarded, and the volasertib and dexamethasone groups were each treated with 1 mL of complete culture medium diluted with the drug. One hour later, the culture medium was discarded, and 200 μL of complete culture medium containing 25 μg / mL C48 / 80 was added to each group, except for the normal group, which was treated with complete culture medium containing an equal volume of DMSO. After 30 minutes, cells were observed under a microscope and the reaction was terminated by incubating on ice for 10 minutes. The supernatant was aspirated into a centrifuge tube and centrifuged at 4°C, 2500 rpm, for 6 minutes. For each group, 50 μL of supernatant was added to a 96-well plate. 50 μL of colorimetric solution was added and the plate was allowed to stand for 1 hour. 200 μL of stop solution was added and the OD value was measured at 405 nm and recorded. The β-Hex release rate was calculated as follows:

[0049] β-Hex release rate (%) = (OD value of the supernatant of the experimental group - OD value of the supernatant of the blank well) / (OD value of the total enzyme well - OD value of the supernatant of the blank well) × 100%.

[0050] 1.2.4 Effect of PLK1 inhibitors on histamine release from RBL-2H3 cells

[0051] Cell culture and grouping were performed as described in 1.2.2. After 24 hours of culture, the original culture medium was discarded, and the drug group was treated with 1 mL of complete culture medium diluted in the drug-treated group, while the normal and model groups were treated with an equal volume of complete culture medium. One hour later, 200 μL of complete culture medium (C48 / 80 at a concentration of 25 μg / mL) was added to each group except the normal group. The normal group was treated with complete culture medium containing an equal volume of DMSO. After 30 minutes, the cells were observed under a microscope and the reaction was terminated by incubating on ice for 10 minutes. The supernatant from each group was collected and centrifuged at 12,000 rpm at 4°C for 5 minutes. The histamine ELISA kit instructions were followed.

[0052] 1.2.5 Effects of PLK1 inhibitors on apoptosis in RBL-2H3 cells

[0053] Cell culture and grouping were the same as in 1.2.2. After 24 hours of culture, the original culture medium was discarded, and the drug group was diluted with 1 mL of complete culture medium, while the normal and model groups were added with an equal amount of complete culture medium. One hour later, 200 μL of complete culture medium was added to each group except the normal group, with a C48 / 80 concentration of 25 μg / mL. The normal group was added with complete culture medium containing an equal volume of DMSO. After 30 minutes, the cell state was observed under a microscope, and the reaction was terminated by ice bathing for 10 minutes. According to the instructions of the AO / EB staining kit, the cells were observed under a fluorescence microscope, and 5 random images of the cells were taken. The number of cells of different colors was quantitatively analyzed using ImageJ software, and the cell apoptosis rate was analyzed and calculated using the following formula.

[0054]

[0055] 1.2.6 Effects of PLK1 inhibitors on passive cutaneous hypersensitivity in mice

[0056] 1.2.6.1 Experimental Animal Grouping

[0057] Forty-eight Kunming mice were randomly divided into six groups: normal group, model group, positive control drug dexamethasone 10 mg / kg group, Volasertib 5 mg / kg group, Volasertib 10 mg / kg group, and Volasertib 20 mg / kg group, with 8 mice in each group.

[0058] 1.2.6.2 Drug administration to experimental animals

[0059] One hour before administration of C48 / 80 to mice, the dexamethasone group and the Volasertib group were intraperitoneally injected with 200 μL of the corresponding dose of drug according to body weight, and the other groups were injected with an equal amount of normal saline.

[0060] 1.2.6.3 Sensitization of experimental animals

[0061] Mice were anesthetized with sodium pentobarbital. 20 μL of 1 mg / mL C48 / 80 was injected intradermally into the right ear, back, and right toe of the mice, respectively, except for the normal group. The normal group mice were injected with an equal volume of normal saline at the corresponding locations.

[0062] 1.2.6.4 Experimental Animal Handling

[0063] One hour after administration, mice except the normal group were injected with 200 μL of 1% Evans blue via tail vein. Evans blue extravasation in mice was observed 30 minutes after stimulation, and mice were killed by cervical dislocation.

[0064] The swelling of the mouse toes and ears and the exudation of Evans blue were observed and recorded, and photos were taken. The mouse toes and ears were cut off with scissors, and the thickness of the ears was measured with a vernier caliper to calculate the swelling. The ears were cut into pieces and immersed in 500 μL formamide, in a 65°C water bath overnight, centrifuged at 7500 rpm for 10 min, and the supernatant was taken for OD measurement at 620 nm; the mouse toes were cut off and weighed, and the swelling of the mouse toes was calculated. The skin of the same position and size of the mouse toes was cut into pieces and immersed in 500 μL formamide, in a 65°C water bath overnight, centrifuged at 7500 rpm for 10 min, and the supernatant was taken for absorbance measurement at 620 nm.

[0065] Obtain skin from the back of the mouse and observe the size of the blue spots within the skin. Take photos and record them. Obtain skin with blue spots of the same size and location at the same location. Place the skin in 800 μL of formamide, incubate in a 65°C water bath overnight, centrifuge at 7500 rpm for 10 minutes, and measure the absorbance of the supernatant at 620 nm.

[0066] Ear swelling (%) = (right ear piece thickness - left ear piece thickness) / left ear piece thickness × 100% Toe swelling (%) = (right toe weight - left toe weight) / left toe weight × 100%.

[0067] 1.2.6.5 Histological analysis of mouse ears

[0068] Mouse ear tissues were collected for HE and toluidine blue staining.

[0069] 1.2.6.5.1 Preparation of paraffin sections: The right ear tissue of the mouse was excised and fixed in neutral formalin fixative for 30 min. After fixation, 5 μm paraffin sections were prepared by rinsing, dehydration, clearing, paraffinization, embedding, and sectioning.

[0070] 1.2.6.5.2 HE staining:

[0071] (1) Dewax the sections in xylene, soak them in anhydrous ethanol for 5 min, anhydrous ethanol for 5 min, 95% alcohol for 5 min, 90% alcohol for 5 min, 80% alcohol for 5 min, and 70% alcohol for 5 min, and then wash with distilled water;

[0072] (2) The hydrated sections were washed with PBS three times for 5 minutes each time, stained with hematoxylin for 10 minutes, and then washed with distilled water to remove excess stain. The sections were differentiated with 1% hydrochloric acid and ethanol, rinsed with double-distilled water, and blued with 0.6% ammonia solution. After the staining, the sections were rinsed with clean water.

[0073] (3) Stain the slices from the previous step with eosin solution for 3 minutes, dehydrate with 80% ethanol for 5 seconds, 95% ethanol for 2 minutes, and anhydrous ethanol for 2 minutes. After dehydration, soak in xylene twice, each for 4 minutes. After drying, slices were sealed with neutral gum.

[0074] (4) Observe under a microscope and take photos.

[0075] 1.2.6.5.3 Toluidine blue staining

[0076] The tissue sections prepared in the previous step were stained with 0.5% toluidine blue solution for 30 minutes, washed with water to remove excess stain, dehydrated with graded alcohol, transparentized with xylene, sealed with neutral gum, and observed and photographed under a microscope.

[0077] 1.2.7 Statistical analysis

[0078] SPSS22.0 software was used for statistical analysis. The data were expressed as mean ± standard deviation. One-way analysis of variance was performed for pairwise comparisons of multiple samples. P < 0.05 was considered statistically significant.

[0079] 1.3 Experimental Results

[0080] 1.3.1 Effect of PLK1 inhibitors on the proliferation activity of RBL-2H3 cells

[0081] Effects of different concentrations of Volasertib on cell proliferation activity Figure 1 As shown in the results, when Volasertib concentrations were 0.1 and 0.5 nM, cell proliferation activity was barely affected. However, when Volasertib concentration reached 1 nM, the proliferation activity of RBL-2H3 cells decreased significantly. This indicates that Volasertib has an inhibitory effect on RBL-2H3 cell proliferation above 1 nM, and the inhibitory effect increases with increasing concentration. Therefore, 0.1 nM and 0.5 nM Volasertib concentrations were ultimately selected for subsequent experiments.

[0082] Effects of PLK1 inhibitors on RBL-2H3 cell morphology

[0083] Neutral red staining results Figure 2 As shown in the figure, neutral red staining was used to observe the cell morphology and intracellular granule distribution. The normal group RBL-2H3 cells had smooth edges, relatively complete morphology, and evenly distributed intracellular granules. However, after C48 / 80 induction, the cell morphology of the model group cells changed significantly, the cells were obviously swollen and rounded, and the intracellular granules were unevenly distributed. Compared with the model group, the degree of cell rounding and swelling in the Volasertib group and the positive control group was alleviated to varying degrees, the cell integrity and cell membrane smoothness were also improved, and the uneven coloring of intracellular granules was significantly reduced, indicating that Volasertib has a significant inhibitory effect on C48 / 80-induced RBL-2H3 cell morphological changes and degranulation.

[0084] Effects of PLK1 inhibitors on β-Hex release from RBL-2H3 cells

[0085] β-Hex release rate Figure 3 As shown in the figure, compared with the normal group, the β-Hex release rate of the model group cells was significantly enhanced, which was significantly different from the normal group (p<0.01); after Volasertib 0.1nM and 0.5nM acted on the cells, the β-Hex release rate was significantly decreased, which was significantly lower than that of the model group. The β-Hex release rate of the positive control group was also significantly decreased compared with the model group, with significant differences (p<0.01).

[0086] 1.3.4 Effect of PLK1 inhibitors on histamine release from RBL-2H3 cells

[0087] Histamine release Figure 4 As shown in the figure, compared with the normal group, the histamine release of the cells in the C48 / 80-induced model group was significantly increased, with a significant difference (p<0.01); while after the cells in the drug group were treated with Volasertib 0.1nM and 0.5nM, the histamine release was significantly reduced compared with the model group. After the cells in the positive control group were treated with dexamethasone, the histamine release of the cells was significantly reduced compared with the model group, with a significant difference (p<0.01).

[0088] Effects of PLK1 inhibitors on apoptosis in RBL-2H3 cells

[0089] AO / EB staining results are as follows Figure 5 As shown in the figure, after staining, the cells in the normal group emitted green fluorescence and had intact structure without rupture or shrinkage under a fluorescence microscope. After C48 / 80 induced pseudo-allergy, the cells in the model group showed obvious apoptosis, with multiple cells emitting red fluorescence and abnormal structure. The apoptosis rate was significantly higher than that in the normal group, with a significant difference (p<0.01). Figure 6 As shown in the figure, after the action of Volasertib 0.1nM and 0.5nM, the apoptosis of cells was significantly improved compared with the model group, and the apoptosis rate was significantly decreased. The apoptosis rate of cells in the positive control group was also significantly decreased after dexamethasone, showing a significant difference (p<0.01).

[0090] Effects of PLK1 inhibitors on C48 / 80-induced PCA in mice

[0091] 1.3.6.1 Effects of PLK1 Inhibitors on C48 / 80-Induced PCA Responses in Mouse Paws

[0092] The results are as follows Figure 7As shown, the paws of mice in the normal group showed normal morphology, no swelling, and no Evans blue exudation, while the paws of mice in the model group showed significant swelling and Evans blue exudation. After treatment with the PLK1 inhibitor Volasertib, C48 / 80-induced paw swelling and Evans blue exudation in mice were alleviated with increasing doses. After treatment with 20 mg / kg of Volasertib, the degree of Evans blue exudation in mice was even less than that in the positive control group, demonstrating a significant therapeutic effect on allergic symptoms. Quantitative analysis of the degree of paw swelling and Evans blue exudation in mice showed that the paw swelling and Evans blue OD values ​​in the normal group were significantly lower than those in the model group, showing a significant difference. After Volasertib treatment, the paw swelling and Evans blue exudation in mice decreased with increasing Volasertib concentration. The paw swelling and Evans blue OD values ​​of mice in the 10 mg / kg and 20 mg / kg groups were significantly reduced, showing a significant difference from the model group.

[0093] 1.3.6.2 Effects of PLK1 Inhibitors on C48 / 80-Induced PCA Responses in Mouse Ears

[0094] The results are as follows Figure 8 As shown, the ears of mice in the normal group showed no Evans blue exudation, and the thickness of the ears did not change significantly. Compared with the normal group, the ears of mice in the model group showed obvious blue spots and significant ear thickening. After administration of Volasertib, the size of the blue spots in the ears of mice was significantly reduced, and the degree of ear thickening was significantly reduced in a concentration-dependent manner.

[0095] Measurements of mouse ear thickness and Evans Blue OD values ​​revealed that the normal group had significantly lower ear thickness and Evans Blue OD values ​​than the model group. Volasertib treatment significantly decreased ear thickness and Evans Blue OD values, significantly different from the model group. The 20 mg / kg group exhibited even greater inhibitory effects on ear swelling and Evans Blue exudation than the positive control drug.

[0096] 1.3.6.3 Effects of PLK1 Inhibitors on C48 / 80-Induced PCA Responses in Mice

[0097] The results are as follows Figure 9 As shown, there were no blue spots on the back skin of mice in the normal group, while large and obvious blue spots appeared on the back skin of mice in the model group. After administration of 10 and 20 mg / kg Volasertib, the size of the blue spots on the back skin of mice decreased significantly, showing significant differences from the model group.

[0098] The OD value of Evans blue on the back skin of mice was measured. The results showed that the OD value of Evans blue on the back skin of mice in the model group was much higher than that in the normal group, while the OD value of Evans blue in the Volasertib group was significantly lower than that in the model group, showing concentration dependence.

[0099] 1.3.6.4 HE staining of mouse ears

[0100] HE staining results of mouse ears Figure 10 As shown, there was no inflammatory cell infiltration and ear swelling in the normal group; compared with the normal group, the ear tissue of the mice in the model group was significantly swollen, with a large number of inflammatory cells infiltrating, and the blood vessels in the ear were significantly dilated. After treatment with Volasertib 10mg / kg and 20mg / kg, the swelling of the mice's ears was significantly reduced, the number of inflammatory cell infiltrations was significantly reduced, the degree of vascular dilation was reduced, and the degree of pathological improvement increased with increasing concentration, which was dose-dependent.

[0101] 1.3.6.5 Toluidine blue staining of mouse ears

[0102] Toluidine blue is a commonly used cell dye that can dye the nucleus of mast cells blue and the granules in the contents of mast cells purple-red, which can well identify mast cells. Figure 11 As shown, toluidine blue staining did not show obvious mast cell staining in the normal group mice, while a large number of inflammatory cell infiltrations appeared in the ears of the model group mice, and the number of mast cells increased significantly. After treatment with Volasertib 10mg / kg and 20mg / kg, the number of mast cells in the ears of the mice decreased significantly, the degranulation phenomenon was significantly improved, and the swelling was significantly reduced, indicating that Volasertib has a certain therapeutic effect on the allergic symptoms of mice.

[0103] 1.4 Results Analysis

[0104] Volasertib is a dihydropteridinone derivative, also known as Volasertib in Chinese. It can be used as a small molecule kinase inhibitor of Plk1 and is a derivative of another PLK1 inhibitor, BI2536. While inhibiting PLK1, BI2536 also acts on PLK2 and PLK3, which are highly homologous to PLK1. Its IC 50 The values ​​were 0.83, 3.5 and 9.0 nmol / L, respectively, and Volasertib’s IC 50The specificity of Volasertib is significantly higher than that of BI2536, which targets the ATP-active pocket of PLK1, thereby inhibiting its activity. Volasertib is primarily used for research on its therapeutic potential in oncology, but its use in allergic reactions is currently unknown. Given its high selectivity and potent inhibitory effect on PLK1, Volasertib was selected as a PLK1 inhibitor for this study.

[0105] In this experiment, the CCK-8 method was used to detect the effect of drugs on cell proliferation activity, thereby screening the appropriate concentration of Volasertib for RBL-2H3 cells. According to the test results, when the concentrations were 0.1nM and 0.5nM, the cell proliferation activity was less affected than that of the normal group. When the concentration reached 1nM, the cell proliferation activity began to decline significantly, and the degree of decline increased with the increase of Volasertib administration concentration, indicating that high concentrations of Volasertib are more toxic to RBL-2H3 cells.

[0106] Dexamethasone is a glucocorticoid used clinically to treat various allergic diseases and inflammation. It can inhibit the activation of the NF-κB pathway and reduce the expression of inflammatory mediators. Studies have shown that dexamethasone can effectively reduce the release of inflammatory factors and inhibit mast cell degranulation induced by C48 / 80. Therefore, it is often used as a positive control drug in pseudoallergic reaction studies. Based on relevant references, 10μM was selected as the concentration used in this experiment.

[0107] During an allergic reaction, RBL-2H3 cells undergo morphological changes such as rounding and swelling, and a large number of intracellular granules are released, which can be clearly visualized using neutral red staining. Staining results showed that treatment with different concentrations of Volasertib significantly ameliorated C48 / 80-induced cell morphological changes, indicating that PLK1 inhibition has a certain inhibitory effect on C48 / 80-induced cellular allergic symptoms. β-Hex and histamine are markers of mast cell degranulation. Only when mast cells degranulate are they released in large quantities into the bloodstream, causing symptoms such as vasodilation and local tissue swelling. This study found that Volasertib effectively inhibited C48 / 80-induced cell degranulation and significantly reduced the release of β-Hex and histamine, indicating that PLK1 inhibition can effectively reduce the release of inflammatory factors induced by C48 / 80. In addition, AO / EB staining results showed that Volasertib could reduce the number of apoptotic cells induced by C48 / 80 and alleviate the effect of C48 / 80 on apoptosis, further confirming that PLK1 has a certain regulatory role in pseudoallergic reactions.

[0108] The passive cutaneous anaphylaxis (PCA) model in mice is a classic animal model for studying allergic reactions. Allergens stimulate mast cells in the paws, ears, and back of mice, inducing mast cell activation and the release of inflammatory factors, leading to symptoms such as vasodilation and tissue edema. This model has high credibility. In this study, C48 / 80 was selected as the stimulatory agent to establish the PCA model. C48 / 80 is a mixed polymer of p-methoxy-N-methylphenylethylamine cross-linked with formaldehyde. It is widely used as a mast cell activator and can effectively induce mast cell degranulation. C48 / 80 can induce mast cell activation in mice, triggering the release of inflammatory factors and inducing inflammation, making it a classic inducer of allergic reactions in animals. C48 / 80 has a short duration of action. Based on literature review and preliminary experiments, we selected a 20 μg C48 / 80 sensitization condition for half an hour. Injection of 20 μg C48 / 80 into the paws, ears, and back of mice resulted in an allergic reaction, causing localized swelling, within half an hour. Evans blue is injected into the tail vein, and it can extravasate at the sensitized site to form blue spots. The severity of the allergy is expressed by observing the size of the blue spots and measuring the OD value of Evans blue extravasation, providing visualization and quantitative analysis for observing allergic reactions.

[0109] Based on literature review and preliminary experiments, this study selected 5mg / kg, 10mg / kg, and 20mg / kg of Volasertib as the low, medium, and high dose groups, and administered them to mice via intraperitoneal injection. The results showed that the swelling of the mice's toes and ears, the size of the blue spots on the back skin, and the Evans blue exudation values ​​all decreased to varying degrees. The degree of reduction was positively correlated with the Volasertib dose and was dose-dependent.

[0110] In this study, HE and toluidine blue staining were performed on the sensitized areas of the mouse ears. HE staining can be used to observe morphological and structural changes in pathological tissue and the accumulation of inflammatory cells, while toluidine blue staining can be used to observe changes in the number of mast cells and degranulation. HE and toluidine blue staining results showed that the ears of the model group mice showed significant swelling and inflammatory cell infiltration, dilation of ear blood vessels, and a significant increase in the number of mast cells and degranulation. Volasertib effectively improved C48 / 80-induced ear swelling and inflammatory cell infiltration in mice, reduced mast cell aggregation and degranulation at the sensitized areas, and the degree of improvement increased with increasing concentration. Pathological tissue staining results further confirmed that the PLK1 inhibitor Volasertib can effectively ameliorate the symptoms of the C48 / 80-induced PCA mouse model.

[0111] In summary, the present invention demonstrates, through in vitro and in vivo cell experiments, that the PLK1 inhibitor Volasertib can ameliorate morphological changes during C48 / 80-induced degranulation in RBL-2H3 cells, reduce apoptosis, and decrease the release of HA and β-HEX, significantly improving pseudoallergic reactions. In vivo experiments in a mouse PCA model also demonstrated that inhibiting PLK1 activity can alleviate localized allergic reaction symptoms. These results suggest that PLK1 significantly regulates pseudoallergic reactions, providing a theoretical and experimental basis for exploring the underlying mechanisms and treatments of pseudoallergic reactions.

[0112] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of a PLK1 inhibitor in the preparation of a drug for treating pseudoallergic reaction, characterized in that: The inhibitor is volacetidine.

2. The use according to claim 1, characterized in that The volacetide plays a role in treating pseudoallergic reaction by improving the morphological changes of mast cell degranulation, reducing cell apoptosis and lowering the release of histamine and β-hexosaminidase in cells.

3. The use according to claim 2, characterized in that The mast cell degranulation is C48 / 80-induced mast cell degranulation.

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