Application of palmitoylation modification inhibitor in preparation of medicine for treating or relieving allergic airway inflammation

Abstract

The invention relates to application of a palmitoylation modification inhibitor in preparation of a medicine for treating or relieving allergic airway inflammation. The palmitoylation modified inhibitor is applied to the field of treatment of allergic airway inflammatory diseases for the first time. Experimental results show that the inhibitor can significantly inhibit activation of a JAK1 / STAT6 signal channel in vitro and effectively inhibit secretion of Th2 type inflammatory factors such as IL-4 and IL-13 by eosinophilic granulocytes; in animal model research, the palmitoylation modification inhibitor can obviously relieve allergic airway inflammatory reaction of mice, and the allergic airway inflammatory reaction is specifically shown as reduction of the number of eosinophils in bronchoalveolar lavage fluid (BALF), reduction of expression and secretion levels of BALF and lung tissue Th2 type inflammatory factors and obvious relieving of lung inflammation pathological degree. The invention provides a theoretical basis and a potential target for clinically developing a novel therapeutic drug for allergic airway inflammatory diseases in the future.

Description

Technical Field

[0001] This invention relates to the field of respiratory disease treatment, and more specifically to the use of palmitoylation modification inhibitors in the preparation of drugs for treating or relieving allergic airway inflammation. Background Technology

[0002] Bronchial asthma (or simply asthma) is one of the most common chronic airway diseases worldwide, affecting a large population and experiencing a rapid increase in prevalence, making it a significant global public health issue. Therefore, elucidating the molecular basis of asthma pathogenesis and promoting precision medicine in clinical practice are crucial for achieving more effective treatment.

[0003] The pathogenesis of asthma is complex, and its exact mechanisms are not yet fully understood. Most of the immune-inflammatory mechanisms in asthma are characterized by type 2 immune responses mediated by inflammation. In airway inflammation in asthma, allergens first act on airway epithelial cells, leading to impaired airway mucosal barrier function and prompting the release of large amounts of alarm-like cytokines such as interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin. These cytokines not only activate type II innate lymphocytes but also participate in the differentiation and maturation of Th2 cells. Activated Th2 cells release type 2 cytokines, promoting the differentiation and maturation of eosinophils. Mature eosinophils are recruited and infiltrate the airways, rapidly activating under the influence of inflammatory factors in the lung microenvironment, further amplifying and maintaining the inflammatory state of asthma. Among these features, increased and abnormally activated airway eosinophils are the core pathological characteristics of asthma. Activated eosinophils can promote eosinophil differentiation, maturation, migration and survival by releasing cytokines such as IL-4, IL-5 and IL-13, thereby playing an immunomodulatory or pathological damaging role. At the same time, they can also release toxic granule proteins that interact with DNA fibers to form extracellular traps for eosinophils, directly activating pulmonary neuroendocrine cells, thereby enhancing and amplifying type 2 inflammatory responses.

[0004] Therefore, targeting and blocking key steps in eosinophil activation during airway inflammation in asthma, inhibiting the activation process, and intervening in the positive feedback loop of the asthma microenvironment have become important directions in asthma treatment research. Summary of the Invention

[0005] This invention aims to address the shortcomings of existing technologies by providing the application of palmitoylation modification inhibitors in the preparation of drugs for treating or alleviating allergic airway inflammation.

[0006] The objective of this invention is achieved through the following technical solution: This invention provides the use of palmitoylation modification inhibitors in the preparation of drugs for treating or relieving allergic airway inflammation, wherein the palmitoylation modification inhibitor is 2-bromohexadecanoic acid, CAS number 18263-25-7.

[0007] Furthermore, the palmitoylation modification inhibitor blocks the activation of eosinophils by inhibiting the activation of the JAK1 / STAT6 signaling pathway in eosinophils, thereby achieving the treatment or relief of allergic airway inflammation.

[0008] Furthermore, the inhibition of activation of the JAK1 / STAT6 signaling pathway in eosinophils specifically involves inhibiting phosphorylation of the JAK1 / STAT6 signaling pathway.

[0009] Furthermore, the blocking of eosinophil activation includes inhibiting the secretion and / or expression of Th2-type inflammatory factors by eosinophils.

[0010] Furthermore, the Th2 inflammatory factor is IL-4 and / or IL-13.

[0011] Furthermore, the allergic airway inflammation includes asthma, allergic rhinitis, or eosinophilic bronchitis.

[0012] The beneficial effects of this invention are as follows: The palmitoylation modification inhibitor used in this invention is 2-bromohexadecanoic acid, which can effectively inhibit eosinophil activation and reduce the expression level of Th2 inflammatory factors, thereby alleviating allergic airway inflammation. It can serve as a new direction for the future development of drugs for the treatment or relief of allergic airway inflammation. Attached Figure Description

[0013] Figure 1 Figure 1 shows the secretion of Th2-related inflammatory factors in eosinophils after intervention with palmitoylation modification inhibitors. Figure 2 shows the statistical results of IL-4 content and Figure 3 shows the statistical results of IL-13 content. Figure 2 This diagram shows the activation status of the JAK1 / STAT6 signaling pathway after intervention with a palmitoylation modification inhibitor. Figure 3 The images show the secretion of Th2-related inflammatory factors in eosinophils after intervention with JAK1 / STAT6 signaling pathway inhibitors. Among them, (A) is a statistical chart of IL-4 content in eosinophils after intervention with JAK1 signaling pathway inhibitors, (B) is a statistical chart of IL-13 content in eosinophils after intervention with JAK1 signaling pathway inhibitors, (C) is a statistical chart of IL-4 content in eosinophils after intervention with STAT6 signaling pathway inhibitors, and (D) is a statistical chart of IL-13 content in eosinophils after intervention with STAT6 signaling pathway inhibitors.

[0014] Figure 4 A schematic diagram illustrating the construction of a classic mouse model of allergic airway inflammation induced by house dust mite (HDM); Figure 5 Figures show the BALF cell and eosinophil counts in mice after the application of palmitoylation modification inhibitors. Figure (A) shows the total number of BALF cells in mice, and Figure (B) shows the eosinophil count in mice. Figure 6 The images show the pathological images and scoring statistics of H&E staining in mouse lung tissue after the application of palmitoylation modification inhibitors. In the image, (A) is the pathological image of H&E staining in mouse lung tissue, and (B) is the scoring statistics of H&E staining in mouse tissue. Figure 7 The image shows the expression of Th2-related inflammatory factors in mouse lung tissue detected by real-time quantitative PCR after applying a palmitoylation modification inhibitor. Figure (A) shows the expression of Th2-related inflammatory factors in mouse lung tissue. Il4 Relative mRNA expression levels, (B) Figure shows the expression levels in mouse lung tissue. Il13 Relative mRNA expression levels, (C) Figure shows the expression levels in mouse lung tissue. Il25 Relative mRNA expression levels, (D) Figure shows the expression levels in mouse lung tissue. Il33 Relative mRNA expression level; Figure 8 Figure 1 shows the secretion of Th2-related inflammatory factors in the BALF supernatant of mice after the application of palmitoylation modification inhibitors. Figure 2(A) shows the statistical results of IL-4 content, and Figure 2(B) shows the statistical results of IL-13 content. Detailed Implementation

[0015] This invention proposes the application of palmitoylation modification inhibitors in the development of treatments for allergic airway inflammation. In this application, the palmitoylation modification inhibitors achieve the treatment or relief of allergic airway inflammation by inhibiting the activation of the eosinophil JAK1 / STAT6 signaling pathway and eosinophil activation.

[0016] Palmitoylation is a post-translational modification of proteins present in all eukaryotic cells, catalyzed by palmitoyltransferases, which covalently links a 16-carbon palmitic acid to a target protein. This modification affects protein structure, function, and intracellular localization, enhancing protein-cell membrane interactions and thus regulating various biological processes such as signal transduction and protein transport. The most commonly used palmitoylation inhibitor is 2-Bromopalmitate (2-BP), CAS number 18263-25-7, which broadly inhibits the activity of various palmitoyltransferases, thereby interfering with the activation of downstream signaling pathways.

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments; Example 1: In vitro experiments confirmed that palmitoylation modification inhibitors can effectively inhibit the activation of mouse eosinophils.

[0018] Eosinophils were isolated from the peripheral blood of NJ.1638 mice and cultured in vitro at 37°C in a 5% CO2 incubator using RPMI-1640 complete medium (containing 10% fetal bovine serum, 1% penicillin / streptomycin solution, and 10 ng / ml recombinant IL-5 protein). Pre-treatment with the palmitoylation modification inhibitor 2-BP (50 μmol / L) for 24 hours was performed. The DMSO group received an equal volume of the corresponding solvent, dimethyl sulfoxide (DMSO). Subsequently, IL-33 (20 ng / ml) was administered for 6 hours to induce eosinophil activation. The control group (CTL group) received an equal volume of the corresponding solvent, phosphate-buffered saline (PBS). After culture, the cell supernatant was collected by centrifugation, and the expression levels of IL-4 and IL-13 were detected using an enzyme-linked immunosorbent assay (ELISA) kit (Hangzhou Huaan Biotechnology Co., Ltd., catalog numbers EM0003 and EM0007) according to the manufacturer's instructions. Results are as follows: Figure 1 As shown, after treatment with 2-BP, the levels of Th2 inflammatory factors such as IL-4 and IL-13 secreted by eosinophils were significantly reduced, indicating that palmitoylation modification inhibitors can inhibit eosinophil activation.

[0019] Eosinophils were cultured in vitro under the conditions described above and treated with 2-BP and IL-33. Cells were collected by centrifugation, and protein lysis buffer was added to extract cellular proteins. Western blotting was used to detect the activation status of the classic inflammatory signaling pathway JAK1 / STAT6. After protein extraction, the proteins were subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, blocked with 5% skim milk powder, and sequentially bound with primary and secondary antibodies (p-JAK1: Cell signaling technology #3331; p-STAT6: Cell signaling technology #9361; ACTB: Hangzhou Huaan Biotechnology HA722023; HRP secondary antibody: Hangzhou Fude Biotechnology FDR007; ACTB was used as an internal control protein). The results are shown below. Figure 2 As shown, this inhibitor can suppress the phosphorylation activation of the JAK1 / STAT6 signaling pathway.

[0020] Eosinophils were cultured in vitro under the conditions described above. They were treated for 6 hours with JAK1 inhibitor (JAK1i, 20 μmol / L; MedChemExpress, catalog number HY-19569), STAT6 inhibitor (STAT6i, 20 μmol / L; MedChemExpress, catalog number HY-100614), and IL-33 (20 ng / ml), respectively. The DMSO group received an equal volume of DMSO to the corresponding inhibitor, while the CTL group received an equal volume of PBS to IL-33. Cell supernatants were collected, and the expression levels of IL-4 and IL-13 were detected using ELISA kits (Hangzhou Huaan Biotechnology, catalog numbers EM0003 and EM0007). Results are as follows: Figure 3 As shown, inhibiting the JAK1 / STAT6 signaling pathway can significantly reduce the secretion of Th2 inflammatory factors such as IL-4 and IL-13 in eosinophils, indicating that this inhibitor can inhibit eosinophil activation by blocking the activation of the JAK1 / STAT6 signaling pathway.

[0021] Example 2: A classic allergic airway disease model was constructed using wild-type mice. Palmitylation modification inhibitors were administered via intraperitoneal injection, and their regulatory effect on allergic airway inflammation in mice was observed.

[0022] Constructing a classic mouse model of HDM-induced allergic airway inflammation: such as Figure 4 As shown, 100 μg / animal (2 mg / ml, 50 μl / animal) of HDM was administered via airway infusion on days 0, 7, and 14, while the control group (NS group) received an equal volume of normal saline (NS) via airway infusion. Airway inflammation markers were measured on day 17.

[0023] Six-week-old male wild-type C57BL / 6 mice were randomly divided into groups to establish a classic allergic airway inflammation model. Following the last infusion of HDM, mice were intraperitoneally injected for three consecutive days on days 14, 15, and 16 with either a palmitoylation modification inhibitor (2-BP, 5 mg / kg) or an equal volume of the corresponding solvent, dimethyl sulfoxide (DMSO). The NS group served as a control group, receiving the same intraperitoneal injection treatment. Mice were sacrificed 72 hours after the last infusion, and specimens were collected. The right lung of each mouse was ligated, and the left lung was subjected to bronchoalveolar lavage with 0.4 mL of balanced salt solution (PBS) for each lavage, repeated three times. Approximately 1 mL of bronchoalveolar lavage fluid (BALF) was collected. 50 μL of BALF cell suspension was mixed with 50 μL of leukocyte counting solution, and 10 μL was added to a modified Neubauer hematology chamber. The total white blood cell count was performed under a light microscope. Centrifuge the remaining BALF (4℃, 6000 rpm, 10 min), and transfer the BALF supernatant to a new centrifuge tube for subsequent ELISA detection of cytokines. Resuspend the precipitated cells in PBS and mix thoroughly. Take an appropriate amount of cell suspension for cell smearing (850 rpm, 2 min), stain with Wright-Giemsa, and count the proportion of each cell type under a light microscope. Combine this with the total cell count to calculate the absolute number of eosinophils. Figure 5 As shown, intraperitoneal administration of 2-BP significantly reduced the total cell count and absolute number of eosinophils in BALF, suggesting that it has a relieving effect on allergic airway inflammation.

[0024] After left lung lavage, 0.4 mL of 4% formaldehyde solution was infused into the left lung via endotracheal intubation to inflate and fix the left lung (internal fixation). The trachea was then ligated, and the left lung was harvested using ophthalmic scissors and further fixed in 4% formaldehyde solution (external fixation) for 24-48 hours. The fixed lung tissue was dehydrated, embedded, and sectioned for H&E staining. The stained sections were observed for airway inflammation under an optical microscope (Olympus pathological imaging analysis system), and images were taken under the same conditions. A semi-quantitative score (0-3 points) was used based on the degree of inflammatory cell infiltration around the airway, with the following scoring criteria: 0 points, no inflammatory cell infiltration around the airway; 1 point, a few inflammatory cells infiltrating around the airway; 2 points, 1-5 layers of inflammatory cells infiltrating around most of the airway; 3 points, >5 layers of inflammatory cells infiltrating around most of the airway. The inflammation scoring was performed using a double-blind method, completed independently by two observers, and the final score for each sample was the average of the two scores. Figure 6 The H&E staining results showed that palmitoylation modification inhibitors could significantly reduce inflammatory cell infiltration around the airways and blood vessels in mouse lung tissue.

[0025] Right lung tissue samples from mice were collected and placed in imported 1.5 mL centrifuge tubes. 1 mL of Trizol was added to each tube, and the samples were homogenized thoroughly using a homogenizer for 15 seconds per homogenization, repeated three times. The homogenate was then incubated at room temperature for 10 minutes. RNA was extracted and purified using chloroform extraction, and cDNA was synthesized using reverse transcription (RT). Subsequently, PCR reaction systems were prepared and reaction programs were set according to the instructions using SYBR Green reagent (Hunan Aike Rui Biotechnology, catalog number AG11701). The mRNA expression levels of Th2-related inflammatory factors in mouse lung tissue were detected by real-time quantitative PCR. The primer sequences used are shown in Table 1. Results are as follows: Figure 7 As shown, palmitoylation modification inhibitors can significantly downregulate the levels of palmitoylation modification inhibitors in mouse lung tissue. Il4, Il13, Il25 and Il33 The expression of Th2-related inflammatory factors was reduced (P < 0.05).

[0026] Table 1 Primer sequences for real-time quantitative PCR Take the BALF supernatant and perform an ELISA experiment to detect the expression level of Th2-related inflammatory factors in the BALF supernatant, following the instructions of the mouse IL-4 and IL-13 ELISA kit (Hangzhou Huaan Biotechnology, catalog numbers EM0003 and EM0007). Figure 8 As shown, palmitoylation modification inhibitors can significantly inhibit the secretion of Th2-related inflammatory factors IL-4 and IL-13 in BALF supernatant (P < 0.0001), suggesting that they have a relieving effect on allergic airway inflammation in mice.

[0027] The above embodiments are used to explain the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. The application of palmitoylation modification inhibitors in the preparation of drugs for treating or alleviating allergic airway inflammation, characterized in that, The palmitoylation modification inhibitor is 2-bromohexadecanoic acid, CAS number 18263-25-7.

2. The application according to claim 1, characterized in that, The palmitoylation modification inhibitor blocks the activation of eosinophils by inhibiting the activation of the JAK1 / STAT6 signaling pathway in eosinophils, thereby treating or alleviating allergic airway inflammation.

3. The application according to claim 2, characterized in that, Specifically, inhibiting the activation of the JAK1 / STAT6 signaling pathway in eosinophils involves inhibiting phosphorylation of the JAK1 / STAT6 signaling pathway.

4. The application according to claim 2, characterized in that, The blocking of eosinophil activation includes inhibiting the secretion and / or expression of Th2-type inflammatory factors by eosinophils.

5. The application according to claim 4, characterized in that, The Th2 inflammatory factors are IL-4 and / or IL-13.

6. The application according to claim 1, characterized in that, The allergic airway inflammations include asthma, allergic rhinitis, or eosinophilic bronchitis.

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