Use of an inhibitor of endou in the manufacture of a medicament for treating airway epithelial inflammation

Abstract

This invention discloses the application of an ENDOU inhibitor in the preparation of drugs for treating airway epithelial inflammation, belonging to the field of biomedicine. This invention provides the application of a reagent for detecting ENDOU expression levels in the preparation of kits for diagnosing and / or detecting airway epithelial inflammation or bronchial asthma, and also provides the application of an ENDOU inhibitor in the preparation of drugs for treating airway epithelial inflammation or bronchial asthma. This invention confirms that ENDOU affects LPS-induced inflammatory responses by regulating the FOXO1 and IL-17A pathways, and is a key molecule regulating airway epithelial inflammation. ENDOU shows promise as a therapeutic target for airway inflammation, providing a theoretical basis for research on related drug targets, and also providing a new pathophysiological mechanism for the occurrence of bronchial asthma, which can be used for the prevention and treatment of bronchial asthma.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the use of an ENDOU inhibitor in the preparation of a medicament for treating airway epithelial inflammation. Background Technology

[0002] Bronchial asthma is a common chronic inflammatory respiratory disease characterized by airway inflammation, airway hyperresponsiveness, reversible airflow limitation, and mucus hypersecretion. Clinically, it manifests as recurrent episodes of wheezing, shortness of breath, chest tightness, and cough. The pathological mechanisms of asthma are complex, involving multiple inflammatory cells and mediators, leading to airway epithelial damage, smooth muscle contraction, and airway remodeling. Currently, asthma treatment mainly relies on corticosteroids (ICS), β2-receptor agonists (LABA), and leukotriene modifiers. While these therapies effectively control symptoms, they have significant limitations. Long-term use of corticosteroids may lead to local immunosuppression, oral candidiasis, and systemic side effects (such as osteoporosis and metabolic abnormalities). Biologics (such as anti-IgE monoclonal antibodies and anti-IL-5 / IL-4Rα antibodies), while targeting specific inflammatory pathways, are only suitable for patients with specific phenotypes and are expensive, limiting their clinical availability. Furthermore, some patients do not respond well to existing treatments, developing hormone resistance or dependence, necessitating the exploration of new therapeutic targets.

[0003] ENDOU (also known as Endonuclease U) is a conserved endonuclease, and early studies suggested its potential involvement in RNA metabolism and cellular stress response regulation. Existing literature indicates differential expression of ENDOU in various tissues, but its role in respiratory diseases remains unclear. Although some studies have reported that ENDOU may be associated with viral infection or DNA damage repair, research on its functional mechanisms in bronchial epithelial cells, particularly whether it affects epithelial homeostasis through non-inflammatory pathways, remains lacking, limiting its potential value as a therapeutic target. Against this backdrop, exploring the regulatory mechanisms of ENDOU in airway epithelial inflammation is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide an application of an ENDOU inhibitor in the preparation of a drug for treating airway epithelial inflammation, thereby addressing the problems existing in the prior art. This invention demonstrates that ENDOU, by regulating the FOXO1 and IL-17A pathways, affects LPS-induced inflammatory responses and is a key molecule in regulating airway epithelial inflammation; therefore, ENDOU holds promise as a therapeutic target for airway inflammation.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the application of a reagent for detecting ENDOU expression levels in the preparation of kits for diagnosing and / or detecting airway epithelial inflammation.

[0007] The present invention also provides the application of a reagent for detecting ENDOU expression levels in the preparation of kits for diagnosing and / or detecting bronchial asthma.

[0008] Furthermore, the reagent for detecting ENDOU expression level includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.7 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.8.

[0009] Furthermore, the downregulation of ENDOU expression levels reduces the expression of FOXO1 and IL-17A, thereby affecting the inflammatory response.

[0010] The present invention also provides the use of an ENDOU inhibitor in the preparation of a medicament for treating airway epithelial inflammation.

[0011] The present invention also provides the use of an ENDOU inhibitor in the preparation of a medicament for treating bronchial asthma.

[0012] Furthermore, the inhibitor includes siRNA that interferes with ENDOU, the siRNA comprising si-ENDOU 1 with positive and negative strand nucleotide sequences as shown in SEQ ID NO. 9-10, si-ENDOU 2 with positive and negative strand nucleotide sequences as shown in SEQ ID NO. 11-12, or si-ENDOU 3 with positive and negative strand nucleotide sequences as shown in SEQ ID NO. 13-14.

[0013] Furthermore, the inhibitor alleviates the inflammatory response by inhibiting the expression of FOXO1 and IL-17A.

[0014] The present invention discloses the following technical effects:

[0015] The results of this invention show that ENDOU promotes LPS-induced inflammatory responses in bronchial epithelial cells BEAS-2B and 16HBE, and that ENDOU affects LPS-induced inflammatory responses by regulating the FOXO1 and IL-17A pathways. This invention confirms that ENDOU is a key molecule regulating airway epithelial inflammation, and that ENDOU holds promise as a therapeutic target for airway inflammation, providing a potential target for inhibiting human airway epithelial inflammation and offering a theoretical basis for research on related drug targets. The findings of this invention provide a novel pathophysiological mechanism for the development of bronchial asthma, which can be used for the prevention and treatment of bronchial asthma. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The effect of LPS on the expression of inflammatory factors and ENDOU mRNA in bronchial epithelial cells BEAS-2B(A) and 16HBE(B);

[0018] Figure 2 The effects of LPS on the expression of inflammatory factors and ENDOU protein in bronchial epithelial cells BEAS-2B and 16HBE are shown in Figure A. A shows the electrophoresis results of the proteins of inflammatory factors IL-6, IL-8, and ENDOU in the BEAS-2B bronchial epithelial cell line; B shows the statistical graph of the relative expression levels of the proteins of inflammatory factors IL-6, IL-8, and ENDOU in the BEAS-2B bronchial epithelial cell line; C shows the electrophoresis results of the proteins of inflammatory factors IL-6, IL-8, and ENDOU in the 16HBE bronchial epithelial cell line; and D shows the statistical graph of the relative expression levels of the proteins of inflammatory factors IL-6, IL-8, and ENDOU in the 16HBE bronchial epithelial cell line.

[0019] Figure 3 The expression of ENDOU in bronchial epithelial cells BEAS-2B(A) and 16HBE(B) 24 hours after transfection with si-ENDOU;

[0020] Figure 4 To investigate the effect of ENDOU on the expression of inflammatory factors in bronchial epithelial cells BEAS-2B and 16HBE, the following values ​​were used: A represents the mRNA expression level of inflammatory factor IL-6 in the bronchial epithelial cell line BEAS-2B after ENDOU interference; B represents the mRNA expression level of inflammatory factor IL-8 in the bronchial epithelial cell line BEAS-2B after ENDOU interference; C represents the mRNA expression level of inflammatory factor IL-6 in the bronchial epithelial cell line 16HBE after ENDOU interference; and D represents the mRNA expression level of inflammatory factor IL-8 in the bronchial epithelial cell line 16HBE after ENDOU interference.

[0021] Figure 5To investigate the effect of knocking down ENDOU on the expression levels of inflammatory factors and ENDOU protein in bronchial epithelial cells BEAS-2B and 16HBE, the following graphs were used: A shows the electrophoresis results of inflammatory factors IL-6, IL-8, and ENDOU in the bronchial epithelial cell line BEAS-2B after ENDOU interference; B shows the statistical graph of the relative expression levels of inflammatory factors IL-6, IL-8, and ENDOU in the bronchial epithelial cell line BEAS-2B after ENDOU interference; C shows the electrophoresis results of inflammatory factors IL-6, IL-8, and ENDOU in the bronchial epithelial cell line 16HBE after ENDOU interference; and D shows the statistical graph of the relative expression levels of inflammatory factors IL-6, IL-8, and ENDOU in the bronchial epithelial cell line 16HBE after ENDOU interference.

[0022] Figure 6 The binding and expression of ENDOU and IL-17A in bronchial epithelial cell lines;

[0023] Figure 7 To investigate the effect of knocking down ENDOU on the expression of ENDOU, FOXO1, and IL-17A proteins in bronchial epithelial cells BEAS-2B and 16HBE, the following graphs were used: A shows the electrophoresis results of ENDOU, FOXO1, and IL-17A proteins in the BEAS-2B bronchial epithelial cell line after ENDOU interference; B shows the statistical graph of the relative expression levels of ENDOU, FOXO1, and IL-17A proteins in the BEAS-2B bronchial epithelial cell line after ENDOU interference; C shows the electrophoresis results of ENDOU, FOXO1, and IL-17A proteins in the 16HBE bronchial epithelial cell line after ENDOU interference; and D shows the statistical graph of the relative expression levels of ENDOU, FOXO1, and IL-17A proteins in the 16HBE bronchial epithelial cell line after ENDOU interference.

[0024] Figure 8This image shows the effect of AS1842856 on the expression of inflammatory factors (IL-6 and IL-8), IL-17A, and ATG5 proteins in bronchial epithelial cells BEAS-2B and 16HBE. Image A shows the electrophoresis results of inflammatory factors (IL-6 and IL-8), IL-17A, and ATG5 in the bronchial epithelial cell line BEAS-2B after the addition of the autophagy inhibitor AS1842856; image B shows the effect of AS1842856 on the expression of inflammatory factors (IL-6 and IL-8), IL-17A, and ATG5 in the bronchial epithelial cell line BEAS-2B after the addition of the autophagy inhibitor AS1842856. C is a statistical graph showing the relative expression levels of inflammatory factors (IL-6 and IL-8), IL-17A, and ATG5 in the bronchial epithelial cell line 16HBE after the addition of the autophagy inhibitor AS1842856; D is a statistical graph showing the relative expression levels of inflammatory factors (IL-6 and IL-8), IL-17A, and ATG5 in the bronchial epithelial cell line 16HBE after the addition of the autophagy inhibitor AS1842856. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] Example 1: Effects of LPS on inflammatory response and ENDOU expression in bronchial epithelial cells

[0031] In this embodiment, two different human bronchial epithelial cell lines (BEAS-2B and 16HBE) were selected. The BEAS-2B cell line was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and the 16HBE cell line was purchased from Applied Biological Materials (abm) of Canada.

[0032] I. Experimental Methods

[0033] 1. Cell pretreatment

[0034] Cell resuscitation: Frozen BEAS-2B and 16HBE cells were removed from liquid nitrogen and rapidly thawed in a 37°C water bath. After sterilization, they were transferred to centrifuge tubes containing complete culture medium and centrifuged at 800 rpm for 4 min. The supernatant was discarded, and the cells were resuspended and seeded into culture flasks. The cells were incubated at 37°C in a 5% CO2 incubator and observed periodically. The complete culture medium for BEAS-2B cells was RPMI-1640 complete medium containing 10% FBS, and the complete culture medium for 16HBE cells was DMEM complete medium containing 10% FBS.

[0035] Cell passage: When the cell confluence reaches 80%, wash with PBS, digest with 0.25% trypsin (30s for BEAS-2B cells, 4min for 16HBE cells), add serum-containing culture medium to stop digestion, pipette into a single-cell suspension, passage BEAS-2B at a ratio of 1:4, passage 16HBE at a ratio of 1:5, and continue culturing.

[0036] Cell cryopreservation: Select cells in the logarithmic growth phase, digest with trypsin, centrifuge, resuspend in cryopreservation solution, aliquot into cryovials, label with information, freeze using gradient cooling method, and then transfer to liquid nitrogen for long-term storage.

[0037] 2. Cell seeding plate

[0038] When the confluence of BEAS-2B and 16HBE cells reached 90%, the old culture medium was discarded, and the cells were washed with PBS, digested with trypsin, centrifuged at 800 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in DMEM or RPMI-1640 medium containing 10% FBS, respectively. 10 μL of the cell suspension was mixed with 90 μL of PBS and added to a counting chamber. Cells were counted and their density calculated under a microscope. The suspension concentration was adjusted to 5 × 10⁻⁶ cells / mL. 5Seed each cell / well in a 6-well plate, add 2 mL of complete culture medium to each well, shake well and incubate.

[0039] 3. Cell treatment

[0040] After washing cells with PBS, trypsin was added for thorough digestion, followed by pipetting and centrifugation. Cells were then resuspended in culture medium. Cell counting was completed, and cells were resuspended in culture medium. Cells were seeded into 6-well and 96-well plates and cultured overnight until cell adhesion was achieved. 10 μg / mL LPS was added as needed for the experiment.

[0041] 4. Experimental Grouping

[0042] BEAS-2B and 16HBE cells were divided into a control group and an LPS treatment group, respectively. The LPS treatment group was stimulated with 10 μg / mL LPS for 12 h, while the control group was treated with an equal volume of physiological saline.

[0043] 5. Real-time quantitative PCR (qPCR)

[0044] Total RNA was extracted from cells in each group, and cDNA was obtained by reverse transcription. Following the instructions of the ChamQ Universal SYBR qPCR MasterMix kit (catalog number Q711), a 20 μL PCR reaction mixture was prepared on ice. The specific steps are as follows: To prepare the 20 μL qPCR reaction mixture, add the following components: 10 μL 2×ChamQ Universal SYBR qPCR premix, 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM), 2 μL cDNA template, and 7.2 μL RNase-free water. All components must be handled on ice to ensure the stability of the reaction mixture.

[0045] After thoroughly mixing the prepared reaction mixture, briefly centrifuge. Add the samples to a 96-well plate and amplify using a real-time quantitative PCR instrument. The reaction program is as follows: pre-denaturation phase: 95℃ for 30 s; amplification cycling phase: 95℃ for 10 s, 60℃ for 30 s, for a total of 40 cycles; melting curve analysis phase: 95℃ for 15 s, 60℃ for 60 s, 95℃ for 15 s. After the experiment, 2... -ΔΔCt Data analysis was performed using β-actin as an internal reference gene to calculate the relative expression level of the target gene.

[0046] The primer sequence information for each target gene is shown in Table 1.

[0047] Table 1

[0048]

[0049] 6. Western Blot Assay

[0050] After washing cells with pre-chilled PBS, lysis buffer containing PMSF and a phosphatase inhibitor (RIPA) was added and the cells were lysed on ice for 5 min. Cells were scraped and the lysate was collected and centrifuged at 12,000 rpm at 4°C for 15 min. The supernatant was mixed with 5× protein loading buffer, boiled to denature, and then stored at -20°C to obtain total cellular protein. The obtained total cellular protein sample was analyzed by Western blotting.

[0051] II. Experimental Results

[0052] RNA and protein samples were collected from the experimental and control groups of BEAS-2B and 16HBE cells for analysis. First, the mRNA expression levels of inflammation-related factors IL-6, IL-8, and ENDOU were detected using qRT-PCR. The experimental results are shown below. Figure 1 As shown, compared with the control group, LPS stimulation significantly upregulated the transcriptional levels of IL-6, IL-8, and ENDOU (P<0.05). To further verify the above results, Western blot was used to detect the expression of the corresponding proteins, and the experimental results are shown below. Figure 2 As shown, the expression levels of inflammatory factors and ENDOU protein in the LPS group were significantly higher than those in the control group (P<0.05). In summary, this embodiment successfully constructed an inflammation model in BEAS-2B and 16HBE bronchial epithelial cells using LPS.

[0053] Example 2: Study on the role of ENDOU in the regulation of bronchial epithelial cell inflammation

[0054] I. Experimental Methods

[0055] 1. Experimental Grouping

[0056] (1) Control group: Add PBS solution to make it equal in volume to the LPS added to the experimental group.

[0057] (2) LPS treatment group: 10 μg / mL LPS was added for stimulation for 24 h.

[0058] (3) LPS+si-NC group: si-NC was transfected for 10 h and then stimulated with 10 μg / mL LPS for 24 h.

[0059] (4) LPS+si-ENDOU group: si-ENDOU was transfected for 10 h and then stimulated with 10 μg / mL LPS for 24 h.

[0060] 2. Cell transfection

[0061] Transfection complex preparation: In a clean bench, take two enzyme-free EP tubes, and add 125 μL of Opti-MEM to tube A.TM Culture medium and 5 μL of Lipofectamine TM 3000 reagents; add 125 μL of Opti-MEM to tube B. TM Mix the culture medium with 4 μL of si-ENDOU / si-NC, and let it stand at room temperature for 5 min. Add the liquid from tube B to tube A, mix well, and incubate at room temperature for 10-15 min.

[0062] Cell preparation: Observe the cell status in the 6-well plate. When the density is ≥60%, remove the culture medium, wash once with PBS, and add 1.75 mL of fresh culture medium to each well.

[0063] Transfection: Add the transfection complex to a 6-well plate, shake gently to mix, sterilize, and incubate for 10 min. Discard the transfection medium and starve the plate for 12 h in preparation for subsequent LPS stimulation experiments.

[0064] II. Experimental Results

[0065] 1. Verification of the efficiency of interfering with ENDOU expression in bronchial epithelial cell lines

[0066] This example investigates the role of ENDOU in bronchial epithelial cells (BEAS-2B and 16HBE). A custom-designed small interfering RNA (si-ENDOU) was used, and quantitative PCR was employed to detect gene expression efficiency after transfection. The siRNA sequences used are as follows:

[0067] si-ENDOU 1: Positive chain UAAAUUUCUCAUUACAUCG (SEQ ID NO.9), negative chain AUUUAAAGAGUAAUGUAGC (SEQ ID NO.10);

[0068] si-ENDOU 2: positive chain UUCUCCUCCAGGUAGAAGC (SEQ ID NO.11), negative chain AAGAGGAGGUCCAUCUUCG (SEQ ID NO.12);

[0069] si-ENDOU3: Positive chain UAAAUUUCUCAUUACAUCG (SEQ ID NO.13), negative chain AUUUAAAGAGUAAUGUAGC (SEQ ID NO.14);

[0070] si-NC: positive chain AAGAGGCUUGCACAGUGCA (SEQ ID NO. 15), negative chain UUCUCCGAACGUGUCACGU (SEQ ID NO. 16).

[0071] like Figure 3As shown, 24 hours after transfection with si-ENDOU in the bronchial epithelial cell lines BEAS-2B and 16HBE, the expression level of ENDOU in both cell lines was significantly reduced compared with si-NC, with si-ENDOU3 showing the most significant interference effect on ENDOU (P<0.001). Based on these results, si-ENDOU3 was selected for subsequent experiments.

[0072] 2. Interfering with ENDOU expression can reduce the inflammatory response of bronchial epithelial cells.

[0073] si-ENDOU3, which has the best gene knockdown effect, was selected for this experiment. After interfering with ENDOU expression in the BEAS-2B and 16HBE cell lines, the mRNA expression levels of IL-6 and IL-8 were further examined. Figure 4 This indicates that after LPS stimulation, the expression levels of inflammatory factors IL-6 and IL-8 showed a significant upward trend compared to the control group, while the difference between the si-NC group and the LPS-treated group was not significant. Notably, the expression levels of inflammatory factors in the si-ENDOU group were significantly lower than those in the si-NC group. Furthermore, the results of Western blotting and qPCR experiments showed the same trend (…). Figure 5 The above results indicate that knocking down ENDOU can alleviate LPS-induced inflammatory responses in bronchial epithelial cells.

[0074] Example 3: Study on the mechanism of ENDOU's involvement in LPS-induced bronchial epithelial cell inflammatory response

[0075] I. Experimental Methods

[0076] 1. Experimental Grouping

[0077] Interference with ENDOU inhibits the expression of IL-17A and FOXO1:

[0078] (1) Control group: Add PBS solution to make it equal in volume to the LPS added to the experimental group.

[0079] (2) LPS treatment group: 10 μg / mL LPS was added for stimulation for 24 h.

[0080] (3) LPS+si-NC group: si-NC was transfected for 10 h and then stimulated with 10 μg / mL LPS for 24 h.

[0081] (4) LPS+si-ENDOU group: si-ENDOU was transfected for 10 h and then stimulated with 10 μg / mL LPS for 24 h.

[0082] AS1842856 inhibits the IL-17 signaling pathway, alleviating inflammatory responses and suppressing autophagy-related proteins.

[0083] (1) Control group: Add PBS solution to make it equal in volume to the LPS added to the experimental group.

[0084] (2) LPS treatment group: 10 μg / mL of LPS was added for stimulation for 24 h.

[0085] (3) LPS+AS1842856 group: First, treat with 1 mL of 10 μmol / LAS1842856 for 1 h, starve the cells for 12 h, and then stimulate with 10 μg / mL LPS for 24 h.

[0086] 2. Co-immunoprecipitation

[0087] (1) Lysis and preparation of cell samples

[0088] Place the 6-well plate on ice, aspirate the culture medium, and wash the cells once with PBS buffer to thoroughly remove any residual liquid. Then, add 150 μL of pre-prepared lysis working solution containing protease inhibitors to each well of the 6-well plate, ensuring the lysis buffer evenly covers the cell surface and that the cells are in full contact with the lysis buffer. Centrifuge at 12000g for 3 minutes in a centrifuge pre-cooled to 4°C, and collect the supernatant for subsequent immunoprecipitation experiments.

[0089] (2) Binding of antibody to Protein A+G magnetic beads

[0090] Antibody binding procedure: The pretreated magnetic beads were placed on a magnetic separator for solid-liquid separation, and the supernatant was removed. Then, 500 μL of IL-17A specific antibody working solution and 500 μL of normal IgG control working solution were added, respectively. After resuspending, the tubes were inverted on a test tube mixer and incubated at room temperature for 30 min.

[0091] Washing: Remove the test tubes from the tube mixer and place the samples in a magnetic separator for 10 seconds for solid-liquid separation. Carefully remove the supernatant with a pipette. Then add 500 μL TBS buffer to each test tube and gently aspirate to resuspend the magnetic beads. Perform magnetic separation again for 10 seconds and discard the supernatant. Repeat this washing process three times to ensure thorough removal of unbound material.

[0092] (3) Immunoprecipitation

[0093] Based on a ratio of 20 μL magnetic bead suspension to 500 μL protein sample, Protein A / G magnetic beads conjugated with specific antibodies and normal IgG were added separately. The mixture was incubated in a 4°C incubator for 12–16 h. After incubation, the sample was transferred to a magnetic separator for 10 s solid-liquid separation, and the supernatant was discarded. Then, 0.5 mL of lysis buffer containing protease inhibitors was added, and the magnetic beads were gently resuspended by pipetting. The magnetic separation was repeated for 10 s, and this washing process was repeated three times to remove non-specific bindings.

[0094] (4) Washing

[0095] Add 100 μL of SDS-PAGE Sample Loading Buffer to each test tube and heat in a 95°C water bath for 5 min. Then transfer to a magnetic separator for 10 s solid-liquid separation, and pipette the supernatant for subsequent Western blot analysis.

[0096] II. Experimental Results

[0097] 1. Verify the binding of ENDOU to IL-17A using immunoprecipitation.

[0098] Previous bioinformatics analysis results showed that ENDOU was enriched in the IL-17 signaling pathway. To verify this result, this embodiment used immunoprecipitation to verify whether ENDOU and IL-17A bind. Figure 6 The results show that the Input group exhibits a band of the ENDOU gene, indicating that ENDOU is indeed expressed on bronchial epithelial cells. The IgG group serves as a control group to remove non-specific binding. The Anti-IL-17A group shows a significant difference in band depth compared to the Input group, indicating that IL-17A and ENDOU bind sufficiently on bronchial epithelial cells.

[0099] 2. Interference with ENDOU inhibits the expression of IL-17A and FOXO1 in human bronchial epithelial cells.

[0100] The above immunoprecipitation experiment only demonstrates that ENDOU binds fully to IL-17A; the specific mechanism still needs further investigation. ENDOU should be interfered with in BEAS-2B and 16HBE bronchial epithelial cell lines, and the protein expression of FOXO1 and IL-17A should be observed, respectively. Figure 7As shown, when LPS stimulation was added, the expression levels of ENDOU, FOXO1, and IL-17A proteins increased compared to the control group. There was no significant difference in protein levels between the si-NC group and the LPS group. However, the expression levels of ENDOU, FOXO1, and IL-17A proteins in the si-ENDOU group were significantly lower than those in the si-NC group. These results indicate that knocking down ENDOU in bronchial epithelial cell lines leads to a decrease in the expression levels of FOXO1 and IL-17A proteins.

[0101] 3. Inhibition of autophagy-related proteins

[0102] To investigate the role of autophagy in LPS-induced inflammatory responses, BEAS-2B and 16HBE cells were pretreated with the autophagy inhibitor AS1842856. Western blotting and bar chart analysis were used to detect the expression levels of IL-17A, IL-6, IL-8, ATG5, and the internal reference protein β-actin in different treatment groups. The experiment included a control group, an LPS treatment group, and a combined LPS and AS1842856 treatment group. Results are as follows: Figure 8 As shown, compared with the control group, the expression of IL-17A, IL-6, IL-8, and ATG5 was significantly increased in the LPS-treated group (p<0.05), while the expression of these proteins was significantly decreased in the LPS+AS1842856-treated group (p<0.05). β-actin, as an internal reference protein, maintained a stable expression level in all groups. Based on these results, it can be concluded that AS1842856 can effectively inhibit LPS-induced inflammatory responses and significantly reduce the expression of IL-17A, autophagy-related protein ATG5, and inflammatory factors IL-6 and IL-8.

[0103] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of an ENDOU inhibitor in the preparation of a medicament for treating airway epithelial inflammation, characterized in that, The inhibitor is an siRNA that interferes with ENDOU, and the siRNA is si-ENDOU 2 with positive and negative strand nucleotide sequences as shown in SEQ ID NO.11-12, respectively.

Images